The gut metabolite indole-3 propionate promotes nerve regeneration and repair.

The regenerative potential of mammalian peripheral nervous system neurons after injury is critically limited by their slow axonal regenerative rate1. Regenerative ability is influenced by both injury-dependent and injury-independent mechanisms2. Among the latter, environmental factors such as exercise and environmental enrichment have been shown to affect signalling pathways that promote axonal regeneration3. Several of these pathways, including modifications in gene transcription and protein synthesis, mitochondrial metabolism and the release of neurotrophins, can be activated by intermittent fasting (IF)4,5. However, whether IF influences the axonal regenerative ability remains to be investigated. Here we show that IF promotes axonal regeneration after sciatic nerve crush in mice through an unexpected mechanism that relies on the gram-positive gut microbiome and an increase in the gut bacteria-derived metabolite indole-3-propionic acid (IPA) in the serum. IPA production by Clostridium sporogenes is required for efficient axonal regeneration, and delivery of IPA after sciatic injury significantly enhances axonal regeneration, accelerating the recovery of sensory function. Mechanistically, RNA sequencing analysis from sciatic dorsal root ganglia suggested a role for neutrophil chemotaxis in the IPA-dependent regenerative phenotype, which was confirmed by inhibition of neutrophil chemotaxis. Our results demonstrate the ability of a microbiome-derived metabolite, such as IPA, to facilitate regeneration and functional recovery of sensory axons through an immune-mediated mechanism.

A versatile ratiometric fluorescence nanoprobe for the determination of clonazepam in patients' plasma samples.

Despite the necessity of the study of therapeutic drug monitoring of clonazepam (CLZ), there are only a few fast detection methods available for determining CLZ in biological media. This study aims to develop a cost-effective and ratiometric probe for the quantification of CLZ in plasma samples. Fluorescent polydopamine nanoparticles were produced through a self-polymerization process at a pH of 8.5. Rhodamine B molecules were employed as a fluorescent reference material, emitting stable fluorescence in the visible range. The fabricated probe exhibited a specific detection capability for CLZ. The fluorescence emission of the probe was enhanced in two concentration ranges: from 50 ng/mL to 1.0 µg/mL and from 1.0 to 15.0 µg/mL with a lower limit of quantification of 50 ng/mL, indicating the sensitivity of the probe for detecting CLZ plasma levels. The accuracy of the probe is favorable which could be recommended for CLZ monitoring in the biological media. Furthermore, this probe is highly specific towards CLZ in the presence of various interfering agents which is mainly caused by its ratiometric nature. The developed platform showed high reliability in quantifying CLZ concentrations in patients' plasma samples. Hence, the fabricated probe could be recommended as a reliable method for the routine detection of CLZ in clinical settings.

A gut bacterial pathway metabolizes aromatic amino acids into nine circulating metabolites.

The human gut microbiota produces dozens of metabolites that accumulate in the bloodstream, where they can have systemic effects on the host. Although these small molecules commonly reach concentrations similar to those achieved by pharmaceutical agents, remarkably little is known about the microbial metabolic pathways that produce them. Here we use a combination of genetics and metabolic profiling to characterize a pathway from the gut symbiont Clostridium sporogenes that generates aromatic amino acid metabolites. Our results reveal that this pathway produces twelve compounds, nine of which are known to accumulate in host serum. All three aromatic amino acids (tryptophan, phenylalanine and tyrosine) serve as substrates for the pathway, and it involves branching and alternative reductases for specific intermediates. By genetically manipulating C. sporogenes, we modulate serum levels of these metabolites in gnotobiotic mice, and show that in turn this affects intestinal permeability and systemic immunity. This work has the potential to provide the basis of a systematic effort to engineer the molecular output of the gut bacterial community.

Pharmacokinetic comparison of four arbidol hydrochloride preparations in beagle dogs.

This study aimed to compare the pharmacokinetic properties of four preparations (dispersible tablets, ordinary tablets, capsules and granules) of arbidol hydrochloride, a broad-spectrum antiviral drug, in beagle dogs. Briefly, a single dose of 100 mg of the four preparations of arbidol hydrochloride was orally administered to dogs; blood was then collected from the veins of the foreleg at different times after administration to prepare plasma samples. The plasma concentration of arbidol hydrochloride was measured using a validated liquid chromatography-tandem mass spectrometry (LC-MS/MS). The results showed that when orally administered with dispersible tablets, ordinary tablets, capsules and granules suspended with water, there were no significant differences in the pharmacokinetic parameters (including peak time, peak concentration, elimination half-life, area under the curve (AUC0-t ), and mean retention time) of arbidol hydrochloride. However, in the case of the dispersible tablets, the pharmacokinetics of arbidol hydrochloride was significantly affected by the mode of administration. Compared with direct feeding, peak time [0.50 (0.13, 0.50) vs. 1.00 (0.50, 2.00)] was significantly shortened (P = 0.033) and the AUC0-48 h (8726.5 ± 2509.3 vs. 3650.8 ± 1536.9 ng h/ml) was significantly increased (P = 0.012) when the dispersible tablets were orally administered as water dispersion. In conclusion, the pharmacokinetics of four preparations of arbidol hydrochloride were not significant different in beagle dogs. However, compared with direct feeding, the absorption of arbidol hydrochloride was faster and the bioavailability was better when the dispersible tablets were orally administered as water dispersion.

A Simple LC-MS/MS Method for the Quantification of PDA-66 in Human Plasma.

The treatment of cancer is one of the most important pharmacotherapeutic challenges. To this end, chemotherapy has for some time been complemented by targeted therapies against specific structures. PDA-66, a structural analogue of the inhibitor of serine-threonine kinase glycogen synthase kinase 3ß SB216763, has shown preclinical antitumour effects in various cell lines, with the key pathways of its anticancer activity being cell cycle modulation, DNA replication and p53 signalling. For the monitoring of anticancer drug treatment in the context of therapeutic drug monitoring, the determination of plasma concentrations is essential, for which an LC-MS/MS method is particularly suitable. In the present study, a sensitive LC-MS/MS method for the quantification of the potential anticancer drug PDA-66 in human plasma with a lower limit of quantification of 2.5 nM is presented. The method was successfully validated and tested for the determination of PDA-66 in mouse plasma and sera.

Understanding Exposure-Receptor Occupancy Relationships for Metabotropic Glutamate Receptor 5 Negative Allosteric Modulators across a Range of Preclinical and Clinical Studies.

The metabotropic glutamate receptor 5 (mGlu5) is a recognized central nervous system therapeutic target for which several negative allosteric modulator (NAM) drug candidates have or are continuing to be investigated for various disease indications in clinical development. Direct measurement of target receptor occupancy (RO) is extremely useful to help design and interpret efficacy and safety in nonclinical and clinical studies. In the mGlu5 field, this has been successfully achieved by monitoring displacement of radiolabeled ligands, specifically binding to the mGlu5 receptor, in the presence of an mGlu5 NAM using in vivo and ex vivo binding in rodents and positron emission tomography imaging in cynomolgus monkeys and humans. The aim of this study was to measure the RO of the mGlu5 NAM HTL0014242 in rodents and cynomolgus monkeys and to compare its plasma and brain exposure-RO relationships with those of clinically tested mGlu5 NAMs dipraglurant, mavoglurant, and basimglurant. Potential sources of variability that may contribute to these relationships were explored. Distinct plasma exposure-response relationships were found for each mGlu5 NAM, with >100-fold difference in plasma exposure for a given level of RO. However, a unified exposure-response relationship was observed when both unbound brain concentration and mGlu5 affinity were considered. This relationship showed <10-fold overall difference, was fitted with a Hill slope that was not significantly different from 1, and appeared consistent with a simple Emax model. This is the first time this type of comparison has been conducted, demonstrating a unified brain exposure-RO relationship across several species and mGlu5 NAMs with diverse properties. SIGNIFICANCE STATEMENT: Despite the long history of mGlu5 as a therapeutic target and progression of multiple compounds to the clinic, no formal comparison of exposure-receptor occupancy relationships has been conducted. The data from this study indicate for the first time that a consistent, unified relationship can be observed between exposure and mGlu5 receptor occupancy when unbound brain concentration and receptor affinity are taken into account across a range of species for a diverse set of mGlu5 negative allosteric modulators, including a new drug candidate, HTL0014242.

Indole Alleviates Diet-Induced Hepatic Steatosis and Inflammation in a Manner Involving Myeloid Cell 6-Phosphofructo-2-Kinase/Fructose-2,6-Biphosphatase 3.

BACKGROUND AND AIMS: Indole is a microbiota metabolite that exerts anti-inflammatory responses. However, the relevance of indole to human non-alcoholic fatty liver disease (NAFLD) is not clear. It also remains largely unknown whether and how indole acts to protect against NAFLD. The present study sought to examine the association between the circulating levels of indole and liver fat content in human subjects and explore the mechanisms underlying indole actions in mice with diet-induced NAFLD. APPROACH AND RESULTS: In a cohort of 137 subjects, the circulating levels of indole were reversely correlated with body mass index. In addition, the circulating levels of indole in obese subjects were significantly lower than those in lean subjects and were accompanied with increased liver fat content. At the whole-animal level, treatment of high-fat diet (HFD)-fed C57BL/6J mice with indole caused significant decreases in the severity of hepatic steatosis and inflammation. In cultured cells, indole treatment stimulated the expression of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), a master regulatory gene of glycolysis, and suppressed macrophage proinflammatory activation in a PFKFB3-dependent manner. Moreover, myeloid cell-specific PFKFB3 disruption exacerbated the severity of HFD-induced hepatic steatosis and inflammation and blunted the effect of indole on alleviating diet-induced NAFLD phenotype. CONCLUSIONS: Taken together, our results demonstrate that indole is relevant to human NAFLD and capable of alleviating diet-induced NAFLD phenotypes in mice in a myeloid cell PFKFB3-dependent manner. Therefore, indole mimetic and/or macrophage-specific PFKFB3 activation may be the viable preventive and/or therapeutic approaches for inflammation-associated diseases including NAFLD.

3,3'-Diindolylmethane Exhibits Significant Metabolism after Oral Dosing in Humans.

3,3'-Diindolylmethane (DIM), a major phytochemical derived from ingestion of cruciferous vegetables, is also a dietary supplement. In preclinical models, DIM is an effective cancer chemopreventive agent and has been studied in a number of clinical trials. Previous pharmacokinetic studies in preclinical and clinical models have not reported DIM metabolites in plasma or urine after oral dosing, and the pharmacological actions of DIM on target tissues is assumed to be solely via the parent compound. Seven subjects (6 males and 1 female) ranging from 26-65 years of age, on a cruciferous vegetable-restricted diet prior to and during the study, took 2 BioResponse DIM 150-mg capsules (45.3 mg DIM/capsule) every evening for one week with a final dose the morning of the first blood draw. A complete time course was performed with plasma and urine collected over 48 hours and analyzed by UPLC-MS/MS. In addition to parent DIM, two monohydroxylated metabolites and 1 dihydroxylated metabolite, along with their sulfate and glucuronide conjugates, were present in both plasma and urine. Results reported here are indicative of significant phase 1 and phase 2 metabolism and differ from previous pharmacokinetic studies in rodents and humans, which reported only parent DIM present after oral administration. 3-((1H-indole-3-yl)methyl)indolin-2-one, identified as one of the monohydroxylated products, exhibited greater potency and efficacy as an aryl hydrocarbon receptor agonist when tested in a xenobiotic response element-luciferase reporter assay using Hepa1 cells. In addition to competitive phytochemical-drug adverse reactions, additional metabolites may exhibit pharmacological activity highlighting the importance of further characterization of DIM metabolism in humans. SIGNIFICANCE STATEMENT: 3,3'-Diindolylmethane (DIM), derived from indole-3-carbinol in cruciferous vegetables, is an effective cancer chemopreventive agent in preclinical models and a popular dietary supplement currently in clinical trials. Pharmacokinetic studies to date have found little or no metabolites of DIM in plasma or urine. In marked contrast, we demonstrate rapid appearance of mono- and dihydroxylated metabolites in human plasma and urine as well as their sulfate and glucuronide conjugates. The 3-((1H-indole-3-yl)methyl)indolin-2-one metabolite exhibited significant aryl hydrocarbon receptor agonist activity, emphasizing the need for further characterization of the pharmacological properties of DIM metabolites.

A novel method for the quantification of anlotinib in human plasma using two-dimensional liquid chromatography.

A simple, efficient, and stable detection method of two-dimensional liquid chromatography (2D-LC) was established and validated to determine anlotinib in the human plasma. The 2D-LC system comprises a first-dimensional column (LC1), an intermediate transfer column, and a second-dimensional column (LC2). With simple protein precipitation treatment, the samples were processed directly for detection. The analysis cycle time was completed within 9.50 min. For the anlotinib concentrations, the calibration curve was linear over the 5.00-320.00 ng/mL range. The intra-day and inter-day precision ranges were 0.77-6.22% and 1.92-4.26%, respectively, for anlotinib concentrations. The recoveries were in the range of 97.85-102.50%. A total of 135 plasma samples from 94 patients were analyzed by our method. The plasma concentrations of patients were in the range of 5.17-106.38 ng/mL, in which the female had a higher plasma concentration (6.44-106.38 ng/mL). The simultaneous application of dexamethasone can increase the anlotinib concentration in the plasma. In our clinical application, we found that the factors that affect the plasma concentration include the time and dose of the medication, gender, and drug interactions. The method appears to be sensitive, precise, selective, and suitable for determining the concentration of anlotinib in the plasma sample.

Phase I study of the indoleamine 2,3-dioxygenase 1 inhibitor navoximod (GDC-0919) as monotherapy and in combination with the PD-L1 inhibitor atezolizumab in Japanese patients with advanced solid tumours.

Navoximod (GDC-0919) is a small molecule inhibitor of indoleamine-2,3-dioxygenase 1. This study investigated the safety, tolerability and pharmacokinetics of navoximod alone and in combination with atezolizumab in Japanese patients with advanced solid tumours. This was a phase I, open-label, dose-escalation study. Patients received monotherapy with navoximod 400 mg, 600 mg or 1000 mg orally twice daily (BID) in Stage 1 and navoximod 200 mg, 400 mg, 600 mg or 1000 mg orally BID plus atezolizumab 1200 mg intravenously every 21 days in Stage 2. Objectives included safety, tolerability, efficacy and pharmacokinetic outcomes.Overall, 20 patients were enrolled (Stage 1: n = 10; Stage 2: n = 10). No dose-limiting toxicities were observed. In Stage 1, treatment-related adverse events (TRAEs) of any grade that occurred in ≥20% of patients were chromaturia (50%) and maculopapular rash (20%). Grade ≥ 3 TRAEs were reported in two patients (20%; maculopapular rash and lipase increased). In Stage 2, TRAEs that occurred in ≥30% of patients were chromaturia (60%) and, decreased appetite (40%). Grade ≥ 3 TRAEs were reported in three patients (30%; hyponatraemia, aspartate aminotransferase increased, alanine aminotransferase increased, lymphopaenia and neutropaenia). Stable disease was observed in five patients (50%) in Stage 1 and eight patients (80%) in Stage 2. Navoximod showed linear pharmacokinetics. The recommended dose of navoximod monotherapy was determined as 1000 mg orally BID, and could be considered 1000 mg orally BID in combination with atezolizumab. Navoximod as monotherapy and in combination with atezolizumab was well tolerated in Japanese patients with advanced solid tumours.

Ingestion of resistant starch by mice markedly increases microbiome-derived metabolites.

Recent research has shown significant health benefits deriving from high-dietary fiber or microbiome-accessible carbohydrate consumption. Compared with native starch (NS), dietary resistant starch (RS) is a high microbiome-accessible carbohydrate that significantly alters the gut microbiome. The aim of this study was to determine the systemic metabolic effects of high microbiome-accessible carbohydrate. Male C57BL/6 mice were divided into 2 groups and fed either NS or RS for 18 wk (n = 20/group). Metabolomic analyses revealed that plasma levels of numerous metabolites were significantly different between the RS-fed and NS-fed mice, many of which are microbiome-derived. Most strikingly, we observed a 22-fold increase in gut microbiome-derived tryptophan metabolite indole-3-propionate (IPA), which was positively correlated with several gut microbiota, including Allobaculum, Bifidobacterium, and Lachnospiraceae, with Allobaculum having the most consistently increased abundance of all the IPA-associated taxa across all RS-fed mice. In addition, major changes were observed for metabolites solely or primarily metabolized in the gut (e.g., trimethylamine-N-oxide), metabolites that have a significant entero-hepatic circulation (i.e., bile acids), lipid metabolites (e.g., cholesterol sulfate), metabolites indicating increased energy turnover (e.g., tricarboxylic acid cycle intermediates and ketone bodies), and increased antioxidants such as reduced glutathione. Our findings reveal potentially novel mediators of high microbiome-accessible carbohydrate-derived health benefits.-Koay,Y. C., Wali. J. A., Luk, A. W. S., Macia, L., Cogger, V. C., Pulpitel, T. J., Wahl, D., Solon-Biet, S. M., Holmes, A., Simpson, S. J., O'Sullivan, J. F. Ingestion of resistant starch by mice markedly increases microbiome-derived metabolites.

Plasma metabolite biomarkers related to secondary hyperparathyroidism and parathyroid hormone.

BACKGROUND: Hyperphosphatemia, hypocalcemia, and elevation of parathyroid hormone (PTH) are typical abnormalities of uremic patients with Secondary hyperparathyroidism (SHPT). However, metabolic imbalance associated with SHPT is not well understood. METHODS: A total of 15 SHPT patients with an intact parathyroid hormone (iPTH) level > 600 pg/mL were set as preoperative (PR) group, 15 age- and gender-matched controls who had undergone parathyroidectomy plus forearm transplantation because of hyperparathyroidism and achieved an iPTH level <150 pg/mL were set as postoperative (PO) group. Metabolite profiling of these 30 uremic patients and five healthy controls (HC) was performed using ultra performance liquid chromatography-mass spectrometry. RESULTS: Five differential metabolites, including allyl isothiocyanate, L-phenylalanine, D-Aspartic acid, indoleacetaldehyde, and D-galactose correlated with PTH were identified in this study. Taking them as a biomarker signature, PR group can be distinguished from HC group with an area under the curve (AUC) of 0.947 (95% CI, 0.76-1) and PO group with an AUC of 0.6 (95% CI, 0.38-0.807). CONCLUSIONS: The serum metabolome correlated with PTH is successfully demonstrated for a better understanding of the pathogenesis of SHPT.

Relationship between Pharmacokinetics and Pharmacodynamic Responses in Healthy Smokers Informs a Once-Daily Dosing Regimen for Nemiralisib.

Nemiralisib (GSK2269557), a potent inhaled inhibitor of phosphoinositide 3-kinase δ (PI3Kδ), is being developed for the treatment of respiratory disorders including chronic obstructive pulmonary disease. Determining the pharmacokinetic (PK) and pharmacodynamic (PD) responses of inhaled drugs early during drug development is key to informing the appropriate dose and preferred dose regimen in patients. We set out to measure PD changes in induced sputum in combination with drug concentrations in plasma and bronchoalveolar lavage (BAL) taken from healthy smokers (n = 56) treated for up to 14 days with increasing doses of inhaled nemiralisib (0.1-6.4 mg). Induced sputum analysis demonstrated a dose-dependent reduction in phosphatidylinositol-(4,5)-trisphosphate (PIP3, the product of PI3K activation), with a maximum placebo-corrected reduction of 23% (90% confidence interval [CI], 11%-34%) and 36% (90% CI, 11%-64%) after a single dose or after 14 days of treatment with nemiralisib, respectively (2 mg, once daily). Plasma analysis suggested a linear PK relationship with an observed accumulation of ∼3- to 4.5-fold (peak vs. trough) in plasma exposure after 14 days of nemiralisib treatment. The BAL analysis at trough confirmed higher levels of the drug in the lungs versus plasma (32-fold in the BAL fluid component, and 214-fold in the BAL cellular fraction). A comparison of the drug levels in plasma and the reductions in sputum PIP3 showed a direct relationship between exposure and PIP3 reduction. These results demonstrated target engagement upon treatment with inhaled nemiralisib and provide confidence for a once-daily dosing regimen.

Quantitative Profiling of Platelet-Rich Plasma Indole Markers by Direct-Matrix Derivatization Combined with LC-MS/MS in Patients with Neuroendocrine Tumors.

BACKGROUND: Currently, several indole markers are measured separately to support diagnosis and follow-up of patients with neuroendocrine tumors (NETs). We have developed a sensitive mass spectrometry method that simultaneously quantifies all relevant tryptophan-related indoles (tryptophan, 5-hydroxytryptophan, serotonin, 5-hydroxyindoleacetic acid) in platelet-rich plasma. Direct-matrix derivatization was used to make the chemical properties of the indoles uniform and to improve the analytical sensitivity and specificity of the assay. METHODS: In situ derivatization was performed directly in platelet-rich plasma with propionic anhydride at an ambient temperature. The derivatized indoles were extracted by online solid-phase extraction and eluted to the analytical column for separation followed by mass spectrometric detection. The method was validated according to international guidelines. Platelet-rich plasma samples from 68 healthy individuals and 40 NET patients were analyzed for tryptophan, 5-hydroxytryptophan, serotonin, and 5-hydroxyindoleacetic acid. RESULTS: The method reproducibly quantified relevant indoles in 8.5 min, including online sample cleanup. Intra- and interassay imprecision, evaluated at 3 different concentrations, ranged from 2.0% to 12% and 1.9% to 13%, respectively. The limit of quantification was sufficient to measure endogenous concentrations of all 4 indoles. Healthy individuals and NET patients had different concentrations of 5-hydroxytryptophan, serotonin, and 5-hydroxyindoleacetic acid, but tryptophan concentrations were the same. CONCLUSIONS: Direct-matrix derivatization in combination with LC-MS/MS is a powerful tool for the simultaneous quantification of all tryptophan-related indoles in platelet-rich plasma. Simultaneous profiling of relevant indoles improves the biochemical characterization and follow-up of NETs.

An Innovative Approach to Characterize Clinical ADME and Pharmacokinetics of the Inhaled Drug Nemiralisib Using an Intravenous Microtracer Combined with an Inhaled Dose and an Oral Radiolabel Dose in Healthy Male Subjects.

An innovative open-label, crossover clinical study was used to investigate the excretion balance, pharmacokinetics, and metabolism of nemiralisib-an inhaled phosphoinositide 3-kinase delta inhibitor being developed for respiratory diseases. Six healthy men received a single intravenous microtracer of 10 µg [14C]nemiralisib with a concomitant inhaled nonradiolabeled 1000 µg dose followed by an oral 800 µg dose of [14C]nemiralisib 14 days later. Complementary methods including accelerator mass spectrometry allowed characterization of a range of parameters including oral absorption (Fabs), proportion of nemiralisib escaping gut wall metabolism (Fg), hepatic extraction (Eh), fraction of dose absorbed from inhaled dose (Flung), and renal clearance. Intravenous pharmacokinetics of nemiralisib were characterized by low blood clearance (10.0 l/h), long terminal half-life (55 hours), and high volume of distribution at steady state (728 l). Nemiralisib exhibited moderate inhaled and oral bioavailability (38% and 35%) while Flung was 29%. Absorption and first-pass parameters were corrected for blood renal clearance and compared with values without correction. Any swallowed nemiralisib was relatively well absorbed (Fabs, 0.48) with a high fraction escaping gut wall metabolism and low extraction by the liver (Fg and Eh being 0.83 and 0.10, respectively). There were no major human plasma metabolites requiring further qualification in animal studies. Both unchanged nemiralisib and its oxidative/conjugative metabolites were secreted in bile, with nemiralisib likely subject to further metabolism through enterohepatic recirculation. Direct renal clearance and metabolism followed by renal clearance were lesser routes of elimination. SIGNIFICANCE STATEMENT: A number of innovative features have been combined into one small clinical study enabling a comprehensive description of the human pharmacokinetics and metabolism of an inhaled molecule. Design elements included an intravenous 14C tracer administration concomitant with an inhalation dose that enabled derivation of parameters such as fraction absorbed (Fabs), the proportion of drug escaping first-pass extraction through the gut wall and liver (Fg and Fh) and hepatic extraction (Eh). Entero-test bile sampling enabled characterization of biliary elimination pathways.

Brain Distributional Kinetics of a Novel MDM2 Inhibitor SAR405838: Implications for Use in Brain Tumor Therapy.

Achieving an effective drug concentration in the brain is as important as targeting the right pathway when developing targeted agents for brain tumors. SAR405838 is a novel molecularly targeted agent that is in clinical trials for various solid tumors. Its application for tumors in the brain has not yet been examined, even though the target, the MDM2-p53 interaction, is attractive for tumors that could occur in the brain, including glioblastoma and brain metastases. In vitro and in vivo studies indicate that SAR405838 is a substrate of P-glycoprotein (P-gp). P-gp mediated active efflux at the blood-brain barrier plays a dominant role in limiting SAR405838 brain distribution. Even though the absence of P-gp significantly increases the drug exposure in the brain, the systemic exposure, including absorption and clearance processes, were unaffected by P-gp deletion. Model-based parameters of SAR405838 distribution across the blood-brain barrier indicate the CLout of the brain was approximately 40-fold greater than the CLin The free fraction of SAR405838 in plasma and brain were found to be low, and subsequent Kpuu values were less than unity, even in P-gp/Bcrp knockout mice. These results indicate additional efflux transporters other than P-gp and Bcrp may be limiting distribution of SAR405838 to the brain. Concomitant administration of elacridar significantly increased brain exposure, also without affecting the systemic exposure. This study characterized the brain distributional kinetics of SAR405838, a novel MDM2 inhibitor, to evaluate its potential in the treatment of primary and metastatic brain tumors. SIGNIFICANCE STATEMENT: This paper examined the brain distributional kinetics of a novel MDM2-p53 targeted agent, SAR405838, to see its possible application for brain tumors by using in vitro, in vivo, and in silico approaches. SAR405838 is found to be a substrate of P-glycoprotein (P-gp), which limits its distribution to the brain. Based on the findings in the paper, manipulation of the function of P-gp can significantly increase the brain exposure of SAR405838, which may give an insight on its potential benefit as a treatment for primary and metastatic brain cancer.

A selective and robust UPLC-MS/MS method for the simultaneous quantitative determination of anlotinib, ceritinib and ibrutinib in rat plasma and its application to a pharmacokinetic study.

A selective and robust UPLC-MS/MS method has been firstly developed for simultaneous determination of three anti-tumor tyrosine kinase inhibitors (anlotinib, ANL; ceritinib, CER; ibrutinib, IBR) in rat plasma using cost-effective protein precipitation extraction. LC separation was achieved on Waters XBrige C18 column (50 mm × 2.1 mm, 3.5 µm) under gradient conditions in a run time of 5 min. ESI+ was involved through mass spectrometry. Multiple reaction monitoring transitions were at m/z 408.2 → 339.2 for ANL, 558.2 → 433.2 for CER, 441.0 → 138.0 for IBR, 285.0 → 193.1 for diazepam (internal standard), respectively. The optimized method was validated based on US FDA guideline, EMEA guideline as well as Pharmacopoeia of the People's Republic of China. The assay was linear in the range of 0.1-20 ng mL-1 for ANL, 2-1000 ng mL-1 for CER, 1-500 ng mL-1 for IBR. Intra- and inter-day accuracy and precision for all analytes were ≦13.84% and ≦12.56%, respectively. ANL, CER and IBR were sufficiently stable under most investigated conditions. The optimized method was successfully applied for a pharmacokinetic study after single oral gavage administration of mixture (ANL, CER and IBR) at dose of 6 mg kg-1, 25 mg kg-1 and 10 mg kg-1.

Effect of the selective 5-HT2A receptor antagonist EMD-281,014 on L-DOPA-induced abnormal involuntary movements in the 6-OHDA-lesioned rat.

L-3,4-Dihydroxyphenylalanine (L-DOPA) is the most effective therapy for motor symptoms of Parkinson's disease (PD); however, with repeated administration, as many as 94% of PD patients develop complications such as L-DOPA-induced dyskinesia. We previously demonstrated that EMD-281,014, a highly selective serotonin 2A (5-HT2A) receptor antagonist, reduces the severity of dyskinesia in the parkinsonian marmoset, without interfering with L-DOPA anti-parkinsonian benefit. Here, we assessed the effects of EMD-281,014 on L-DOPA-induced abnormal involuntary movements (AIMs) in the 6-hydroxydopamine (6-OHDA)-lesioned rat. We first determined the pharmacokinetic profile of EMD-281,014, to administer doses leading to clinically relevant plasma levels in the behavioural experiments. Dyskinetic 6-OHDA-lesioned rats were then administered EMD-281,014 (0.01, 0.03 and 0.1 mg/kg) or vehicle in combination with L-DOPA and AIMs severity was evaluated. We also assessed the effect of EMD-281,014 on L-DOPA anti-parkinsonian action with the cylinder test. We found that the addition of EMD-281,014 (0.01, 0.03 and 0.1 mg/kg) to L-DOPA did not reduce AIMs severity (P > 0.05), when compared to vehicle. EMD-281,014 did not compromise L-DOPA anti-parkinsonian action. Our results suggest that the highly selective 5-HT2A receptor antagonist EMD-281,014 is well-tolerated by parkinsonian rats, but does not attenuate L-DOPA-induced AIMs. Our results highlight differences between rodent and primate models of PD when it comes to determining the anti-dyskinetic action of 5-HT2A receptor antagonists.

Meprin ß metalloproteases associated with differential metabolite profiles in the plasma and urine of mice with type 1 diabetes and diabetic nephropathy.

BACKGROUND: Meprin metalloproteases are abundantly expressed in the brush border membranes of kidney proximal tubules and small intestines. Meprins are also expressed in podocytes and leukocytes (monocytes and macrophages). Meprins are implicated in the pathophysiology of diabetic nephropathy (DN) but underlying mechanisms are not fully understood. Single nucleotide polymophisms (SNPs) in the meprin ß gene were associated with DKD in human subjects. Furthermore, meprin α and ß double deficiency resulted in more severe kidney injury and higher mortality rates in mice with Streptozotocin (STZ)-induced type 1 diabetes. Identification of meprin substrates has provided insights on how meprins could modulate kidney injury. Meprin targets in the kidney include extracellular matrix (ECM) proteins, modulators of inflammation, and proteins involved in the protein kinase A (PKA) and PKC signaling pathways. The current study used a global metabolomics approach to determine how meprin ß expression impacts the metabolite milieu in diabetes and DKD. METHODS: Low dose STZ was used to induce type 1 diabetes in 8-week old wild-type (WT) and meprin ß knockout (ßKO) mice. Blood and urine samples were obtained at 4 and 8 weeks post-STZ injection. Assays for albumin, creatinine, neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule - 1 (KIM-1), and cystatin C were used for biochemical assessment of kidney injury. Data for biomarkers of kidney injury utilized two-way ANOVA. Metabolomics data analysis utilized UPLC-QTOF MS and multivariate statistics. RESULTS: The number of metabolites with diabetes-associated changes in levels were significantly higher in the WT mice when compared to meprin ßKO counterparts. Annotated meprin ß expression-associated metabolites with strong variable importance in projection (VIP) scores play roles in lipid metabolism (LysoPC(16:1(9Z)), taurocholic acid), amino acid metabolism (indoxyl sulfate, hippuric acid), and neurotransmitter/stress hormone synthesis (cortisol, 3-methoxy-4-hydroxyphenylethylene glycolsulfate, homovanillic acid sulfate). Metabolites that associated with meprin ß deficiency include; 3,5-dihydroxy-3',4'-dimethoxy-6,7-methylenedioxyflavone 3-glucuronide, pantothenic acid, and indoxyl glucuronide (all decreased in plasma). CONCLUSION: Taken together, the annotated metabolites suggest that meprin ß impacts complications of diabetes such as DKD by altering distinct metabolite profiles.

Plasma metabolites and lipids associate with kidney function and kidney volume in hypertensive ADPKD patients early in the disease course.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD) is the most common hereditary kidney disease and is characterized by gradual cyst growth and expansion, increase in kidney volume with an ultimate decline in kidney function leading to end stage renal disease (ESRD). Given the decades long period of stable kidney function while cyst growth occurs, it is important to identify those patients who will progress to ESRD. Recent data from our and other laboratories have demonstrated that metabolic reprogramming may play a key role in cystic epithelial proliferation resulting in cyst growth in ADPKD. Height corrected total kidney volume (ht-TKV) accurately reflects cyst burden and predicts future loss of kidney function. We hypothesize that specific plasma metabolites will correlate with eGFR and ht-TKV early in ADPKD, both predictors of disease progression, potentially indicative of early physiologic derangements of renal disease severity. METHODS: To investigate the predictive role of plasma metabolites on eGFR and/or ht-TKV, we used a non-targeted GC-TOF/MS-based metabolomics approach on hypertensive ADPKD patients in the early course of their disease. Patient data was obtained from the HALT-A randomized clinical trial at baseline including estimated glomerular filtration rate (eGFR) and measured ht-TKV. To identify individual metabolites whose intensities are significantly correlated with eGFR and ht-TKV, association analyses were performed using linear regression with each metabolite signal level as the primary predictor variable and baseline eGFR and ht-TKV as the continuous outcomes of interest, while adjusting for covariates. Significance was determined by Storey's false discovery rate (FDR) q-values to correct for multiple testing. RESULTS: Twelve metabolites significantly correlated with eGFR and two triglycerides significantly correlated with baseline ht-TKV at FDR q-value < 0.05. Specific significant metabolites, including pseudo-uridine, indole-3-lactate, uric acid, isothreonic acid, and creatinine, have been previously shown to accumulate in plasma and/or urine in both diabetic and cystic renal diseases with advanced renal insufficiency. CONCLUSIONS: This study identifies metabolic derangements in early ADPKD which may be prognostic for ADPKD disease progression. CLINICAL TRIAL: HALT Progression of Polycystic Kidney Disease (HALT PKD) Study A; Clinical www.clinicaltrials.gov identifier: NCT00283686; first posted January 30, 2006, last update posted March 19, 2015.