Small-molecule IKKß activation modulator (IKAM) targets MAP3K1 and inhibits pancreatic tumor growth.

Activation of inhibitor of nuclear factor NF-κB kinase subunit-ß (IKKß), characterized by phosphorylation of activation loop serine residues 177 and 181, has been implicated in the early onset of cancer. On the other hand, tissue-specific IKKß knockout in Kras mutation-driven mouse models stalled the disease in the precancerous stage. In this study, we used cell line models, tumor growth studies, and patient samples to assess the role of IKKß and its activation in cancer. We also conducted a hit-to-lead optimization study that led to the identification of 39-100 as a selective mitogen-activated protein kinase kinase kinase (MAP3K) 1 inhibitor. We show that IKKß is not required for growth of Kras mutant pancreatic cancer (PC) cells but is critical for PC tumor growth in mice. We also observed elevated basal levels of activated IKKß in PC cell lines, PC patient-derived tumors, and liver metastases, implicating it in disease onset and progression. Optimization of an ATP noncompetitive IKKß inhibitor resulted in the identification of 39-100, an orally bioavailable inhibitor with improved potency and pharmacokinetic properties. The compound 39-100 did not inhibit IKKß but inhibited the IKKß kinase MAP3K1 with low-micromolar potency. MAP3K1-mediated IKKß phosphorylation was inhibited by 39-100, thus we termed it IKKß activation modulator (IKAM) 1. In PC models, IKAM-1 reduced activated IKKß levels, inhibited tumor growth, and reduced metastasis. Our findings suggests that MAP3K1-mediated IKKß activation contributes to KRAS mutation-associated PC growth and IKAM-1 is a viable pretherapeutic lead that targets this pathway.

Crosstalk of hepatocyte nuclear factor 4a and glucocorticoid receptor in the regulation of lipid metabolism in mice fed a high-fat-high-sugar diet.

BACKGROUND: Hepatocyte nuclear factor 4α (HNF4α) and glucocorticoid receptor (GR), master regulators of liver metabolism, are down-regulated in fatty liver diseases. The present study aimed to elucidate the role of down-regulation of HNF4α and GR in fatty liver and hyperlipidemia. METHODS: Adult mice with liver-specific heterozygote (HET) and knockout (KO) of HNF4α or GR were fed a high-fat-high-sugar diet (HFHS) for 15 days. Alterations in hepatic and circulating lipids were determined with analytical kits, and changes in hepatic mRNA and protein expression in these mice were quantified by real-time PCR and Western blotting. Serum and hepatic levels of bile acids were quantified by LC-MS/MS. The roles of HNF4α and GR in regulating hepatic gene expression were determined using luciferase reporter assays. RESULTS: Compared to HFHS-fed wildtype mice, HNF4α HET mice had down-regulation of lipid catabolic genes, induction of lipogenic genes, and increased hepatic and blood levels of lipids, whereas HNF4α KO mice had fatty liver but mild hypolipidemia, down-regulation of lipid-efflux genes, and induction of genes for uptake, synthesis, and storage of lipids. Serum levels of chenodeoxycholic acid and deoxycholic acid tended to be decreased in the HNF4α HET mice but dramatically increased in the HNF4α KO mice, which was associated with marked down-regulation of cytochrome P450 7a1, the rate-limiting enzyme for bile acid synthesis. Hepatic mRNA and protein expression of sterol-regulatory-element-binding protein-1 (SREBP-1), a master lipogenic regulator, was induced in HFHS-fed HNF4α HET mice. In reporter assays, HNF4α cooperated with the corepressor small heterodimer partner to potently inhibit the transactivation of mouse and human SREBP-1C promoter by liver X receptor. Hepatic nuclear GR proteins tended to be decreased in the HNF4α KO mice. HFHS-fed mice with liver-specific KO of GR had increased hepatic lipids and induction of SREBP-1C and PPARγ, which was associated with a marked decrease in hepatic levels of HNF4α proteins in these mice. In reporter assays, GR and HNF4α synergistically/additively induced lipid catabolic genes. CONCLUSIONS: induction of lipid catabolic genes and suppression of lipogenic genes by HNF4α and GR may mediate the early resistance to HFHS-induced fatty liver and hyperlipidemia.

Intramuscular and subcutaneous administration of antiretroviral drugs, compared with oral, enhances delivery to lymphoid tissues in BALB/c mice.

BACKGROUND: Multiple tissue reservoirs are established soon after HIV infection, and some tissues may also be pharmacological sanctuaries. Parenteral administration of antiretroviral (ARV) drugs for treatment and prevention of HIV infection is an active area of drug development. The influence of route of administration on ARV tissue pharmacokinetics is not known. OBJECTIVES: To investigate ARV pharmacokinetics in lymphatic and select non-lymphatic tissues (e.g. brain and testes) after intramuscular and subcutaneous administration compared with oral in BALB/c mice. METHODS: Tissue concentrations of cobicistat, efavirenz, elvitegravir, maraviroc, rilpivirine, tenofovir alafenamide and tenofovir disoproxil fumarate were determined. The tissue penetration ratio (TPR) was the primary measure for comparison; a change in TPR arises from factors affecting tissue distribution controlling for changes in systemic bioavailability. RESULTS: Intramuscular and subcutaneous delivery increased TPRs in the lymph node and spleen for 27 of 28 (96%) drug administration events. Decreased TPRs, however, were found in some tissues such as the brain and testes. CONCLUSIONS: These results demonstrate a change in route of drug administration from oral to intramuscular or subcutaneous can change tissue uptake. This has implications for HIV pharmacotherapy. For example, HIV persists in lymphoid tissues despite long-term oral ARV therapy, and low ARV concentrations have been found in lymphoid tissues. The improved ARV lymphatic tissue bioavailability with intramuscular and subcutaneous administration allows future studies to investigate these routes of drug administration as a therapeutic manoeuvre to limit viral persistence and eliminate viral sanctuaries in the lymphatic tissues, which is a prerequisite for eradication of HIV.

A year-long extended release nanoformulated cabotegravir prodrug.

Long-acting cabotegravir (CAB) extends antiretroviral drug administration from daily to monthly. However, dosing volumes, injection site reactions and health-care oversight are obstacles towards a broad usage. The creation of poloxamer-coated hydrophobic and lipophilic CAB prodrugs with controlled hydrolysis and tissue penetrance can overcome these obstacles. To such ends, fatty acid ester CAB nanocrystal prodrugs with 14, 18 and 22 added carbon chains were encased in biocompatible surfactants named NMCAB, NM2CAB and NM3CAB and tested for drug release, activation, cytotoxicity, antiretroviral activities, pharmacokinetics and biodistribution. Pharmacokinetics studies, performed in mice and rhesus macaques, with the lead 18-carbon ester chain NM2CAB, showed plasma CAB levels above the protein-adjusted 90% inhibitory concentration for up to a year. NM2CAB, compared with NMCAB and NM3CAB, demonstrated a prolonged drug release, plasma circulation time and tissue drug concentrations after a single 45 mg per kg body weight intramuscular injection. These prodrug modifications could substantially improve CAB's effectiveness.

Preliminary preclinical study of Chol-DsiRNA polyplexes formed with PLL[30]-PEG[5K] for the RNAi-based therapy of breast cancer.

RNA interference molecules have tremendous potential for cancer therapy but are limited by insufficient potency after i.v. administration. We previously found that Chol-DsiRNA polyplexes formed between cholesterol-modified dicer-substrate siRNA (Chol-DsiRNA) and the cationic diblock copolymer PLL[30]-PEG[5K] greatly increase the activity of Chol-DsiRNA against a stably expressed reporter mRNA in primary murine syngeneic breast tumors after daily i.v. dosing. Here, we provide a more thorough preliminary preclinical study of Chol-DsiRNA polyplexes against the therapeutically relevant target protein, STAT3. We found that Chol-DsiSTAT3 polyplexes greatly increase plasma exposure, distribution, potency, and therapeutic activity of Chol-DsiSTAT3 in primary murine syngeneic 4T1 breast tumors after i.v. administration. Furthermore, inactive Chol-DsiCTRL polyplexes are well tolerated by healthy female BALB/c mice after chronic i.v. administration at 50 mg Chol-DsiCTRL/kg over 28 days. Thus, Chol-DsiRNA polyplexes may be a good candidate for Phase I clinical trials to improve the treatment of breast cancer and other solid tumors.

Synthesis and Characterization of Long-Acting Darunavir Prodrugs.

Antiretroviral therapy (ART) has improved the quality of life in patients infected with HIV-1. However, complete viral suppression within anatomical compartments remains unattainable. This is complicated by adverse side effects and poor adherence to lifelong therapy leading to the emergence of viral drug resistance. Thus, there is an immediate need for cellular and tissue-targeted long-acting (LA) ART formulations. Herein, we describe two LA prodrug formulations of darunavir (DRV), a potent antiretroviral protease inhibitor. Two classes of DRV prodrugs, M1DRV and M2DRV, were synthesized as lipophilic and hydrophobic prodrugs and stabilized into aqueous suspensions designated NM1DRV and NM2DRV. The formulations demonstrated enhanced intracellular prodrug levels with sustained drug retention and antiretroviral activities for 15 and 30 days compared to native DRV formulation in human monocyte-derived macrophages. Pharmacokinetics tests of NM1DRV and NM2DRV administered to mice demonstrated sustained drug levels in blood and tissues for 30 days. These data, taken together, support the idea that LA DRV with sustained antiretroviral responses through prodrug nanoformulations is achievable.

Head-to-head comparative pharmacokinetic and biodistribution (PK/BD) study of two dexamethasone prodrug nanomedicines on lupus-prone NZB/WF1 mice.

HPMA copolymer-based dexamethasone prodrug (P-Dex) and PEG-based dexamethasone prodrug (PEG-Dex, ZSJ-0228) were previously found to passively target the inflamed kidney and provide potent and sustained resolution of nephritis in NZB/WF1 lupus-prone mice. While both prodrug nanomedicines effectively ameliorate lupus nephritis, they have demonstrated distinctively different safety profiles. To explore the underlining mechanisms of these differences, we conducted a head-to-head comparative PK/BD study of P-Dex and PEG-Dex on NZB/WF1 mice. Overall, the systemic organ/tissue exposures to P-Dex and Dex released from P-Dex were found to be significantly higher than those of PEG-Dex. The high prodrug concentrations were sustained in kidney for only 24 h, which cannot explain their lasting therapeutic efficacy (>1 month). P-Dex showed sustained presence in liver, spleen and adrenal gland, while the presence of PEG-Dex in these organs was transient. This difference in PK/BD profiles may explain PEG-Dex' superior safety than P-Dex.

Assessing the lymphoid tissue bioavailability of antiretrovirals in human primary lymphoid endothelial cells and in mice.

BACKGROUND: The secondary lymphoid tissues (LTs), lymph nodes (LNs) and gut-associated lymphoid tissue (GALT) are considered reservoirs for HIV. Antiretrovirals (ARVs) have lower penetration into LT. In vitro models predictive of ARV LT penetration have not been established. OBJECTIVES: To develop an in vitro model of LT bioavailability using human lymphoid endothelial cells (HLECs) and investigate its predictability with in vivo pharmacokinetic (PK) studies in mice. METHODS: ARV bioavailability in HLECs was evaluated at the maximum plasma concentration (Cmax) observed in HIV-infected patients. ARVs were: abacavir, atazanavir, darunavir, dolutegravir, efavirenz, elvitegravir, emtricitabine, maraviroc, raltegravir, rilpivirine, ritonavir, tenofovir disoproxil fumarate and the PK booster cobicistat. The LT PK of representative drugs showing high (efavirenz), intermediate (dolutegravir) and low (emtricitabine) HLEC bioavailability was investigated in BALB/c mice given 50/10/30 mg/kg efavirenz/dolutegravir/emtricitabine orally, daily for 3 days. The concordance of in vitro and in vivo ARV bioavailability was examined. RESULTS: ARVs showed high (>67th percentile; rilpivirine, efavirenz, elvitegravir and cobicistat), intermediate (67th-33rd percentile; ritonavir, tenofovir disoproxil fumarate, dolutegravir and maraviroc) and low (<33rd percentile; atazanavir, darunavir, raltegravir, emtricitabine and abacavir) HLEC bioavailability. The hierarchy of efavirenz, dolutegravir and emtricitabine bioavailability in LN, gut and brain tissues of mice was: efavirenz>dolutegravir>emtricitabine. CONCLUSIONS: ARVs displayed distinct HLEC penetration patterns. PK studies of representative ARVs in LT of mice were concordant with HLEC bioavailability. These findings support further development of this approach and its translational predictability in humans.

Modulation of the intestinal bile acid/farnesoid X receptor/fibroblast growth factor 15 axis improves alcoholic liver disease in mice.

Alcoholic liver disease (ALD) is associated with changes in the intestinal microbiota. Functional consequences of alcohol-associated dysbiosis are largely unknown. The aim of this study was to identify a mechanism of how changes in the intestinal microbiota contribute to ALD. Metagenomic sequencing of intestinal contents demonstrated that chronic ethanol feeding in mice is associated with an over-representation of bacterial genomic DNA encoding choloylglycine hydrolase, which deconjugates bile acids in the intestine. Bile acid analysis confirmed an increased amount of unconjugated bile acids in the small intestine after ethanol administration. Mediated by a lower farnesoid X receptor (FXR) activity in enterocytes, lower fibroblast growth factor (FGF)-15 protein secretion was associated with increased hepatic cytochrome P450 enzyme (Cyp)-7a1 protein expression and circulating bile acid levels. Depletion of the commensal microbiota with nonabsorbable antibiotics attenuated hepatic Cyp7a1 expression and reduced ALD in mice, suggesting that increased bile acid synthesis is dependent on gut bacteria. To restore intestinal FXR activity, we used a pharmacological intervention with the intestine-restricted FXR agonist fexaramine, which protected mice from ethanol-induced liver injury. Whereas bile acid metabolism was only minimally altered, fexaramine treatment stabilized the gut barrier and significantly modulated hepatic genes involved in lipid metabolism. To link the beneficial metabolic effect to FGF15, a nontumorigenic FGF19 variant-a human FGF15 ortholog-was overexpressed in mice using adeno-associated viruses. FGF19 treatment showed similarly beneficial metabolic effects and ameliorated alcoholic steatohepatitis. CONCLUSION: Taken together, alcohol-associated metagenomic changes result in alterations of bile acid profiles. Targeted interventions improve bile acid-FXR-FGF15 signaling by modulation of hepatic Cyp7a1 and lipid metabolism, and reduce ethanol-induced liver disease in mice. (Hepatology 2018;67:2150-2166).

Discovery, synthesis and characterization of a series of (1-alkyl-3-methyl-1H-pyrazol-5-yl)-2-(5-aryl-2H-tetrazol-2-yl)acetamides as novel GIRK1/2 potassium channel activators.

The present study describes the discovery and characterization of a series of 5-aryl-2H-tetrazol-3-ylacetamides as G protein-gated inwardly-rectifying potassium (GIRK) channels activators. Working from an initial hit discovered during a high-throughput screening campaign, we identified a tetrazole scaffold that shifts away from the previously reported urea-based scaffolds while remaining effective GIRK1/2 channel activators. In addition, we evaluated the compounds in Tier 1 DMPK assays and have identified a (3-methyl-1H-pyrazol-1-yl)tetrahydrothiophene-1,1-dioxide head group that imparts interesting and unexpected microsomal stability compared to previously-reported pyrazole head groups.

Pharmacokinetics of a Long-Acting Nanoformulated Dolutegravir Prodrug in Rhesus Macaques.

A nanoformulated myristoylated dolutegravir prodrug (NMDTG) was prepared using good laboratory practice protocols. Intramuscular injection of NMDTG (118 ± 8 mg/ml, 25.5 mg of DTG equivalents/kg of body weight) to three rhesus macaques led to plasma DTG levels of 86 ± 12 and 28 ± 1 ng/ml on days 35 and 91, respectively. The NMDTG platform showed no significant adverse events. Further modification may further extend the drug's apparent half-life for human use.

Increased glycine-amidated hyocholic acid correlates to improved early weight loss after sleeve gastrectomy.

BACKGROUND: Bile acids (BAs) are post-prandial hormones that play an important role in glucose and lipid homeostasis as well as energy expenditure. Total and glycine-amidated BAs increase after sleeve gastrectomy (SG) and correlate to improved metabolic disease. No specific bile acid subtype has been shown conclusively to mediate the weight loss effect. Therefore, the objective of this study was to prospectively evaluate the comprehensive changes in meal-stimulated BAs after SG and determine if a specific change in the BA profile correlates to the early weight loss response. METHODS: Patients were prospectively enrolled at the University of Nebraska Medical Center who were undergoing a SG for treatment of morbid obesity. Primary and secondary plasma bile acids and their amidated (glycine, G-, or taurine, T-) subtypes were measured at fasting, 30 and 60 min after a liquid meal performed pre-op, and at 6 and 12 weeks post-op. Area under the curve (AUC) was calculated for the hour meal test for each bile acid subtype. BAs that were significantly increased post-op were correlated to body mass index (BMI) loss. RESULTS: Total BA AUC was significantly increased at 6 (p < 0.01) and 12 weeks post-op (p < 0.01) compared to pre-operative values. The increase in total BA AUC was due to a statistically significant increase in G-BAs. Nine different BA AUC subtypes were significantly increased at both 6 and 12 weeks post-op. Increased total and G-chenodeoxycholic acid AUC was significantly correlated to the 6 week BMI loss (p = 0.03). Increased G-hyocholic acid was significantly correlated to increased weight loss at both 6 (p = 0.05) and 12 weeks (p = 0.006). CONCLUSIONS: SG induced an early and persistent post-prandial surge in multiple bile acid subtypes. Increased G-hyocholic consistently correlated with greater early BMI loss. This study provides evidence for a role of BAs in the surgical weight loss response after SG.

Species differences in bile acids II. Bile acid metabolism.

One of the mechanisms of drug-induced liver injury (DILI) involves alterations in bile acid (BA) homeostasis and elimination, which encompass several metabolic pathways including hydroxylation, amidation, sulfation, glucuronidation and glutathione conjugation. Species differences in BA metabolism may play a major role in the failure of currently used in vitro and in vivo models to predict reliably the DILI during the early stages of drug discovery and development. We developed an in vitro cofactor-fortified liver S9 fraction model to compare the metabolic profiles of the four major BAs (cholic acid, chenodeoxycholic acid, lithocholic acid and ursodeoxycholic acid) between humans and several animal species. High- and low-resolution liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance imaging were used for the qualitative and quantitative analysis of BAs and their metabolites. Major species differences were found in the metabolism of BAs. Sulfation into 3-O-sulfates was a major pathway in human and chimpanzee (4.8%-52%) and it was a minor pathway in all other species (0.02%-14%). Amidation was primarily with glycine (62%-95%) in minipig and rabbit and it was primarily with taurine (43%-81%) in human, chimpanzee, dog, hamster, rat and mice. Hydroxylation was highest (13%-80%) in rat and mice followed by hamster, while it was lowest (1.6%-22%) in human, chimpanzee and minipig. C6-ß hydroxylation was predominant (65%-95%) in rat and mice, while it was at C6-α position in minipig (36%-97%). Glucuronidation was highest in dog (10%-56%), while it was a minor pathway in all other species (<12%). The relative contribution of the various pathways involved in BA metabolism in vitro were in agreement with the observed plasma and urinary BA profiles in vivo and were able to predict and quantify the species differences in BA metabolism. In general, overall, BA metabolism in chimpanzee is most similar to human, while BA metabolism in rats and mice is most dissimilar from human.

Species differences in bile acids I. Plasma and urine bile acid composition.

Maintenance of bile acid (BA) homeostasis is essential to achieve their physiologic functions and avoid their toxic effects. The marked differences in BA composition between preclinical safety models and humans may play a major role in the poor prediction of drug-induced liver injury using preclinical models. We compared the composition of plasma and urinary BAs and their metabolites between humans and several animal species. Total BA pools and their composition varied widely among different species. Highest sulfation of BAs was observed in human and chimpanzee. Glycine amidation was predominant in human, minipig, hamster and rabbit, while taurine amidation was predominant in mice, rat and dogs. BA profiles consisted primarily of tri-OH BAs in hamster, rat, dog and mice, di-OH BAs in human, rabbit and minipig, and mono-OH BA in chimpanzee. BA profiles comprised primarily hydrophilic and less toxic BAs in mice, rat, pig and hamster, while it primarily comprised hydrophobic and more toxic BAs in human, rabbit and chimpanzee. Therefore, the hydrophobicity index was lowest in minipig and mice, while it was highest in rabbit, monkey and human. Glucuronidation and glutathione conjugation were low in all species across all BAs. Total concentration of BAs in urine was up to 10× higher and more hydrophilic than plasma in most species. This was due to the presence of more tri-OH, amidated, sulfated and primary BAs, in urine compared to plasma. In general, BA profiles of chimpanzee and monkeys were most similar to human, while minipig, rat and mice were most dissimilar to human.

Simultaneous LC-MS/MS analysis of eicosanoids and related metabolites in human serum, sputum and BALF.

The differences among individual eicosanoids in eliciting different physiological and pathological responses are largely unknown because of the lack of valid and simple analytical methods for the quantification of individual eicosanoids and their metabolites in serum, sputum and bronchial alveolar lavage fluid (BALF). Therefore, a simple and sensitive LC-MS/MS method for the simultaneous quantification of 34 eicosanoids in human serum, sputum and BALF was developed and validated. This method is valid and sensitive with a limit of quantification ranging from 0.2 to 3 ng/mL for the various analytes, and has a large dynamic range (500 ng/mL) and a short run time (25 min). The intra- and inter-day accuracy and precision values met the acceptance criteria according to US Food and Drug Administration guidelines. Using this method, detailed eicosanoid profiles were quantified in serum, sputum and BALF from a pilot human study. In summary, a reliable and simple LC-MS/MS method to quantify major eicosanoids and their metabolites was developed and applied to quantify eicosanoids in human various fluids, demonstrating its suitability to assess eicosanoid biomarkers in human clinical trials.

Harmonizing lipidomics: NIST interlaboratory comparison exercise for lipidomics using SRM 1950-Metabolites in Frozen Human Plasma.

As the lipidomics field continues to advance, self-evaluation within the community is critical. Here, we performed an interlaboratory comparison exercise for lipidomics using Standard Reference Material (SRM) 1950-Metabolites in Frozen Human Plasma, a commercially available reference material. The interlaboratory study comprised 31 diverse laboratories, with each laboratory using a different lipidomics workflow. A total of 1,527 unique lipids were measured across all laboratories and consensus location estimates and associated uncertainties were determined for 339 of these lipids measured at the sum composition level by five or more participating laboratories. These evaluated lipids detected in SRM 1950 serve as community-wide benchmarks for intra- and interlaboratory quality control and method validation. These analyses were performed using nonstandardized laboratory-independent workflows. The consensus locations were also compared with a previous examination of SRM 1950 by the LIPID MAPS consortium. While the central theme of the interlaboratory study was to provide values to help harmonize lipids, lipid mediators, and precursor measurements across the community, it was also initiated to stimulate a discussion regarding areas in need of improvement.

Leveraging of Rifampicin-Dosed Cynomolgus Monkeys to Identify Bile Acid 3-O-Sulfate Conjugates as Potential Novel Biomarkers for Organic Anion-Transporting Polypeptides.

In the search for novel bile acid (BA) biomarkers of liver organic anion-transporting polypeptides (OATPs), cynomolgus monkeys received oral rifampicin (RIF) at four dose levels (1, 3, 10, and 30 mg/kg) that generated plasma-free Cmax values (0.06, 0.66, 2.57, and 7.79 µM, respectively) spanning the reported in vitro IC50 values for OATP1B1 and OATP1B3 (≤1.7 µM). As expected, the area under the plasma concentration-time curve (AUC) of an OATP probe drug (i.v. 2H4-pitavastatin, 0.2 mg/kg) was increased 1.2-, 2.4-, 3.8-, and 4.5-fold, respectively. Plasma of RIF-dosed cynomolgus monkeys was subjected to a liquid chromatography-tandem mass spectrometry method that supported the analysis of 30 different BAs. Monkey urine was profiled, and we also determined that the impact of RIF on BA renal clearance was minimal. Although sulfated BAs comprised only 1% of the plasma BA pool, a robust RIF dose response (maximal ≥50-fold increase in plasma AUC) was observed for the sulfates of five BAs [glycodeoxycholate (GDCA-S), glycochenodeoxycholate (GCDCA-S), taurochenodeoxycholate, deoxycholate (DCA-S), and taurodeoxycholate (TDCA-S)]. In vitro, RIF (≤100 µM) did not inhibit cynomolgus monkey liver cytosol-catalyzed BA sulfation and cynomolgus monkey hepatocyte-mediated uptake of representative sulfated BAs (GDCA-S, GCDCA-S, DCA-S, and TDCA-S) was sodium-independent and inhibited (≥70%) by RIF (5 µM); uptake of taurocholic acid was sensitive to sodium removal (74% decrease) and relatively refractory to RIF (≤21% inhibition). We concluded that sulfated BAs may serve as sensitive biomarkers of cynomolgus monkey OATPs and that exploration of their utility as circulating human OATP biomarkers is warranted.

Pharmacokinetic and Biodistribution Studies of HPMA Copolymer Conjugates in an Aseptic Implant Loosening Mouse Model.

N-(2-Hydroxypropyl) methacrylamide (HPMA) copolymers were previously found to represent a versatile delivery platform for the early detection and intervention of orthopedic implant loosening. In this article, we evaluated the impact of different structural parameters of the HPMA copolymeric system (e.g., molecular weight (MW), drug content) to its pharmacokinetics and biodistribution (PK/BD) profile. Using 125I, Alexa Fluor 488, and IRDye 800 CW-labeled HPMA copolymer-dexamethasone (P-Dex) conjugates with different MW and dexamethasone (Dex) contents, we found the MW to be the predominant impact factor on the PK/BD profiles of P-Dex, with Dex content as a secondary impact factor. In gamma counter-based PK/BD studies, increased MW of P-Dex reduced elimination, leading to lower clearance, longer half-life, and higher systemic exposure (AUC and MRT). In the semiquantitative live animal optical imaging evaluation, the distribution of P-Dex to the peri-implant inflammatory lesion increased when MW was increased. This result was further confirmed by FACS analyses of cells isolated from peri-implant regions after systemic administration of Alexa Fluor 488-labeled P-Dex. Since the in vitro cell culture study suggested that the internalization of P-Dex by macrophages is generally independent of P-Dex's MW and Dex content, the impact of the MW and Dex content on its PK/BD profile was most likely exerted at physiological and pathophysiological levels rather than at the cellular level. In both gamma counter-based PK/BD analyses and semiquantitative optical imaging analyses, P-Dex with 6 wt % Dex content showed fast clearance. Dynamic light scattering analyses unexpectedly revealed significant molecular aggregation of P-Dex at this Dex content level. The underlining mechanisms of the aggregation and fast in vivo clearance of the P-Dex warrant further investigation.

Impaired synaptic development in a maternal immune activation mouse model of neurodevelopmental disorders.

Both genetic and environmental factors are thought to contribute to neurodevelopmental and neuropsychiatric disorders with maternal immune activation (MIA) being a risk factor for both autism spectrum disorders and schizophrenia. Although MIA mouse offspring exhibit behavioral impairments, the synaptic alterations in vivo that mediate these behaviors are not known. Here we employed in vivo multiphoton imaging to determine that in the cortex of young MIA offspring there is a reduction in number and turnover rates of dendritic spines, sites of majority of excitatory synaptic inputs. Significantly, spine impairments persisted into adulthood and correlated with increased repetitive behavior, an ASD relevant behavioral phenotype. Structural analysis of synaptic inputs revealed a reorganization of presynaptic inputs with a larger proportion of spines being contacted by both excitatory and inhibitory presynaptic terminals. These structural impairments were accompanied by altered excitatory and inhibitory synaptic transmission. Finally, we report that a postnatal treatment of MIA offspring with the anti-inflammatory drug ibudilast, prevented both synaptic and behavioral impairments. Our results suggest that a possible altered inflammatory state associated with maternal immune activation results in impaired synaptic development that persists into adulthood but which can be prevented with early anti-inflammatory treatment.

Irreversible binding of an anticancer compound (BI-94) to plasma proteins.

1. We investigated the mechanisms responsible for the in vivo instability of a benzofurazan compound BI-94 (NSC228148) with potent anti-cancer activity. 2. BI-94 was stable in MeOH, water, and in various buffers at pHs 2.5-5, regardless of the buffer composition. In contrast, BI-94 was unstable in NaOH and at pHs 7-9, regardless of the buffer composition. BI-94 disappeared immediately after spiking into mice, rat, monkey, and human plasma. BI-94 stability in plasma can be only partially restored by acidifying it, which indicated other mechanisms in addition to pH for BI-94 instability in plasma. 3. BI-94 formed adducts with the trapping agents, glutathione (GSH) and N-acetylcysteine (NAC), in vivo and in vitro via nucleophilic aromatic substitution reaction. The kinetics of adduct formation showed that neutral or physiological pHs enhanced and accelerated GSH and NAC adduct formation with BI-94, whereas acidic pHs prevented it. Therefore, physiological pHs not only altered BI-94 chemical stability but also enhanced adduct formation with endogenous nucleophiles. In addition, adduct formation with human serum albumin-peptide 3 (HSA-T3) at the Cys34 position was demonstrated. 4. In conclusion, BI-94 was unstable at physiological conditions due to chemical instability and irreversible binding to plasma proteins.