Dong

Metabolite elucidation of the Hsp90 inhibitor SNX-2112 using ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS)

Abstract 1. The novel heat-shock protein 90 inhibitor SNX-2112 is a promising drug candidate for treating various types of cancers. Here we aim to determine the metabolic pathways of SNX-2112 in rats in vivo and in humans in vitro. 2. Metabolite identification was performed using ultraperformance liquid chromatography/quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS) method. In vitro metabolism studies were performed using liver and intestine microsomes, as well as recombinant human cytochrome P450 (CYP) enzymes. 3. Analysis of rat plasma, urine, and feces revealed a total of eight metabolites, one reductive metabolite (M1), one structurally unknown metabolite (M2), and six mono-oxidative metabolites (M3-1, M3-2, M3-3, M3-4, M3-5, and M3-6). The reduction, M2, and mono-oxidation pathways were responsible for 0.8 ± 0.3 %, 18.3 ± 9.1 %, and 39.4% ± 6.1 of SNX-2112 clearance from rats, respectively. 4. SNX-2112 was subjected to the same types of metabolism in human liver and intestine microsomes. Reaction phenotyping showed that CYP3A4, 3A5, 2D6, and 1A1 were mainly responsible for SNX-2112 metabolism. 5. In conclusion, we have elucidated the metabolic pathways of SNX-2112 and highlighted that metabolism was the predominant pathway for its clearance. Better understanding of SNX-2112 metabolism should facilitate the drug development of this promising anti-cancer agent.

CCR2 and CCR2 corneal macrophages exhibit distinct characteristics and balance inflammatory responses after epithelial abrasion.

Macrophages are distributed throughout the body and are crucial for the restoration of damaged tissues. However, their characteristics in the cornea and roles in the repair of corneal injures are unclear. Here we show that corneal macrophages can be classified as CCR2− macrophages, which already exist in the cornea at embryonic day 12.5 (E12.5) and are similar to yolk sac-derived macrophages, microglia, in phenotype and gene expression, and CCR2+ macrophages, which do not appear in the cornea until E17.5. At a steady state, CCR2− corneal macrophages have local proliferation capacity and are rarely affected by monocytes; however, following corneal epithelial abrasion, most CCR2− corneal macrophages are replaced by monocytes. In contrast, CCR2+ macrophages are repopulated by monocytes under both a steady-state condition and following corneal wounding. Depletion of CCR2+ macrophages decreases corneal inflammation after epithelial abrasion, whereas depletion of CCR2− macrophages increases inflammation of the injured cornea. Loss of either cell type results in a delay in corneal healing. These data indicate that there are two unique macrophage populations present in the cornea, both of which participate in corneal wound healing by balancing the inflammatory response.

Cremophor EL-based nanoemulsion enhances transcellular permeation of emodin through glucuronidation reduction in UGT1A1-overexpressing MDCKII cells.

Oral emodin, a natural anthraquinone and active component of many herbal medicines, is poorly bioavailable because of extensive first-pass glucuronidation. Here we aimed to prepare emodin nanoemulsion (EMO-NE) containing cremophor EL, and to assess its potential for enhancing transcellular absorption of emodin using UGT1A1-overexpressing MDCKII cells (or MDCK1A1 cells). EMO-NE was prepared using a modified emulsification technique and subsequently characterized by particle size, morphology, stability, and drug release. MDCKII cells were stably transfected with UGT1A1 using the lentiviral transfection approach. Emodin transport and metabolism were evaluated in Transwell-cultured MDCK1A1 cells after apical dosing of EMO-NE or control solution. The obtained EMO-NE (116 ± 6.5 nm) was spherical and stable for at least 2 months. Emodin release in vitro was a passive diffusion-driven process. EMO-NE administration increased the apparent permeability of emodin by a 2.3-fold (p<0.001) compared to the pure emodin solution (1.2 × 10(-5) cm/s vs 5.3 × 10(-6) cm/s). Further, both apical and basolateral excretion of emodin glucuronide (EMO-G) were significantly decreased (≥56.5%, p<0.001) in EMO-NE group. This was accompanied by a marked reduction (57.4%, p<0.001) in total emodin glucuronidation. It was found that the reduced glucuronidation was due to inhibition of cellular metabolism by cremophor EL. Cremophor EL inhibited UGT1A1-mediated glucuronidation of emodin using the mixed-type inhibition mechanism. In conclusion, cremophor EL-based nanoemulsion greatly enhanced transcellular permeation of emodin through inhibition of UGT metabolism. This cremophor EL-based nanoformulation may be a promising strategy to improve the oral bioavailability of emodin.

Arylsulfatase B Mediates the Sulfonation-Transport Interplay in Human Embryonic Kidney 293 Cells Overexpressing Sulfotransferase 1A3.

Elucidating the intricate relationships between metabolic and transport pathways contributes to improved predictions of in vivo drug disposition and drug-drug interactions. Here we reported that inhibited excretion of conjugative metabolites [i.e., hesperetin 3′-O-sulfate (H3′S) and hesperetin 7-O-sulfate (H7S)] by MK-571 led to reduced metabolism of hesperetin (a maximal 78% reduction) in human embryonic kidney 293 cells overexpressing sulfotransferase 1A3 (named SULT293 cells). The strong dependence of cellular sulfonation on the efflux transport of generated sulfated metabolites revealed an interplay of sulfonation metabolism with efflux transport (or sulfonation-transport interplay). Polymerase chain reaction (PCR) and Western blot analyses demonstrated that SULT293 cells expressed multiple sulfatases such as arylsulfatase A (ARSA), ARSB, and ARSC. Of these three desulfonation enzymes, only ARSB showed significant activities toward hesperetin sulfates. The intrinsic clearance values for the hydrolysis of H3′S and H7S were estimated at 0.6 and 0.5 μl/h/mg, respectively. Furthermore, knockdown of ARSB attenuated the regulatory effect of efflux transporter on cellular sulfonation, whereas overexpression of ABSB enhanced the transporter effect. Taken together, the results indicated that ARSB mediated the sulfonation-transport interplay in SULT293 cells.

Metabolism elucidation of BJ-B11 (a heat shock protein 90 inhibitor) by human liver microsomes: identification of main contributing enzymes.

Objective: The aim of this article is to elucidate the metabolic pathways of BJ-B11, a heat shock protein 90 inhibitor, in human liver microsomes (HLM) and determine the main enzymes responsible for formation of each metabolite. Methods: Metabolites of BJ-B11 were identified using the ultra performance liquid chromatography- quadrupole time-of-flight/mass spectrometry (UPLC-QTOF/MS) method. Esterase contributing to the hydrolysis of BJ-B11 was identified by chemical inhibition and activity correlation assays. Reaction phenotyping and kinetic studies using expressed cytochrome P450 (CYP) enzymes were performed to determine the contributions of CYP isozymes to BJ-B11 metabolism. Results: BJ-B11 was rapidly hydrolyzed to generate a deacetylated product M1-1. M1-1 was subsequently metabolized to form eight metabolites. Hydrolysis of BJ-B11 was markedly inhibited by vinblastine (a dual inhibitor of arylacetamide deacetylase and carboxylesterase 2). By contrast, digitonin and telmisartan (the specific inhibitors for carboxylesterase 1 and carboxylesterase 2, respectively) did not inhibit BJ-B11 hydrolysis at all. Further, BJ-B11 hydrolysis was significantly correlated with hydrolysis of phenacetin (an activity marker of arylacetamide deacetylase). Moreover, reaction phenotyping revealed that metabolism of M1-1 in HLM was attributable to several CYP enzymes, including CYP1A1, 1B1, 3A4 and 3A5. Conclusion: BJ-B11 was subjected to efficient metabolism in the liver, generating nine metabolites. BJ-B11 metabolism was contributed mainly by arylacetamide deacetylase and multiple CYP enzymes.

Identification of UDP-glucuronosyltransferases 1A1, 1A3 and 2B15 as the main contributors to glucuronidation of bakuchiol, a natural biologically active compound

Abstract 1. Bakuchiol, one of the main active compounds of Psoralea corylifolia, possesses a variety of pharmacological activities such as anti-tumor and anti-aging effects. Here, we aimed to characterize the glucuronidation of bakuchiol using human liver microsomes (HLM) and expressed UDP-glucuronosyltransferase (UGT) enzymes. 2. The glucuronide of bakuchiol was confirmed by liquid chromatography–mass spectrometry (LC-MS) and β-glucuronidase hydrolysis assay. Glucuronidation rates and kinetic parameters were derived by enzymatic incubation and model fitting. Activity correlation analyses were performed to identify the main UGT isoforms contributing to hepatic metabolism of bakuchiol. 3. Among the three UGT enzymes (i.e., UGT1A1, UGT1A3 and UGT2B15) capable of catalyzing bakuchiol glucuronidation, UGT2B15 showed the highest activity with a CLint value of 100 μl/min/nmol. Bakuchiol glucuronidation was strongly correlated with glucuronidation of 5-hydroxyrofecoxib (r = 0.933; p < 0.001), 3-O-glucuronidation of β-estradiol (r = 0.719; p < 0.01) and significantly correlated with 24-O-glucuronidation of CDCA (r = 0.594; p < 0.05). In addition, a marked species difference existed in hepatic glucuronidation of bakuchiol. 4. In conclusion, UGT1A1, UGT1A3 and UGT2B15 were identified as the main contributors to glucuronidation of bakuchiol.

Modulation of Circadian Rhythms Affects Corneal Epithelium Renewal and Repair in Mice

Purpose In mammalian corneal epithelium, mitosis shows a distinct circadian pattern. However, how this circadian pattern is maintained, and how it or its disruption influence renewal and regeneration remain unclear. Methods C57BL/6 mice were maintained under 12-hour light/12-hour dark (LD), 12-hour light/12-hour light (LL), 12-hour dark/12-hour dark (DD), or reversed LD (DL, 12-hour dark/12-hour light; jet-lag defined as a shift of 12 hours) conditions. Mitotic cells in corneal epithelium were enumerated and analyzed via immunofluorescence at different zeitgeber times (ZTs). Expression of core clock genes (Clock, Bmal1, Period2, Cry1, and Rev-erbα) was qualified via quantitative RT-PCR. The rate and quality of healing at different ZT times and after administration of two small-molecule modifiers of the circadian clock, KL001 and SR8278, was evaluated. Results In this study, photic cues were found to influence the 24-hour rhythm of corneal clock gene expression and epithelial cell mitosis in mice. Disruption of the circadian clock by exposure to constant light, constant dark, or jet-lag conditions modified the normal 24-hour patterns of corneal epithelial mitosis and corneal clock gene expression. The time of day of wound occurrence affected the rate and quality of corneal healing, with both of these parameters peaking during the more mitotically active hours of the morning. The two small-molecule modifiers of the circadian clock, KL001 and SR8278, had negative and positive effects on corneal wound healing, respectively. Conclusions Circadian rhythms significantly influence corneal epithelium renewal and repair in mice. Our findings reveal possible opportunities for biological rhythm-based interventional strategies to control corneal healing and restore corneal homeostasis.

A validated ultra-performance liquid chromatography-tandem mass spectrometry method to identify the pharmacokinetics of SR8278 in normal and streptozotocin-induced diabetic rats.

There is a relationship between circadian rhythm and metabolic disorders. The active agent, SR8278, could competitively bind to and inhibit the nuclear receptor, Rev-erb (a major modulator of mammalian circadian clock system), to regulate the metabolism in organisms. However, we had limited knowledge of the pharmacokinetic (PK) characteristics of SR8278. Here, we describe a sensitive and reproducible ultra-performance liquid chromatography-tandem mass spectrometric (UPLC-MS/MS) method to quantify SR8278 in vivo. The linearity range and the limit of quantification (LOQ) for SR8278 were 30-3000 ng/mL and 6 ng/mL, respectively. The inter-day and intra-day variability were within 10%. This UPLC-MS/MS method was successfully used to characterize the PK behaviors of SR8278 in normal and diabetic rats after intravenous (i.v.) injection at a dosage of 2mg/kg. No significant differences were observed in the PK parameters of SR8278 in normal and diabetic rats. Specifically, the values of areas under plasma concentration time curves (AUC), initial plasma concentrations (C0), elimination half-lives (t1/2), and clearances (CL) were 608.33 ± 295.25 vs. 598.59 ± 276.92 ng·h/mL, 2410.25 ± 202.36 vs. 3742.11 ± 1300.21 ng/mL, 0.17 ± 0.08 vs. 0.11 ± 0.04 h, 3330.83 ± 1609.48 vs. 3364.81 ± 1111.38 mL/kg·h for SR8278 in normal rats vs. diabetic rats, respectively. In conclusion, a UPLC-MS/MS method was successfully developed and validated for the first time, with a wide linearity range, low LOQ, small sample volume (10 μL), rapid analysis (4 min) and excellent recoveries (>80%). It was also used to clarify the PK characteristics of SR8378 in rats. The same PK behaviors of SR8278 in normal and diabetic rats showed that diabetes may have little or no effect on the disposition, metabolism and/or elimination in vivo, which may be of great importance for future clinical studies.

Identification of UGTs and BCRP as potential pharmacokinetic determinants of the natural flavonoid alpinetin

Abstract Alpinetin is a natural flavonoid showing a variety of pharmacological effects such as anti-inflammatory, anti-tumor and hypolipidemic activities. Here, we aim to determine the roles of UDP-glucuronosyltransferases (UGTs) and breast cancer resistance protein (BCRP) in disposition of alpinetin. Glucuronidation potential of alpinetin was evaluated using pooled human liver microsomes (pHLM), pooled human intestine microsomes (pHIM) and expressed UGT enzymes supplemented with the cofactor UDPGA. Activity correlation analyses with a bank of individual HLMs were performed to identify the main contributing UGT isozymes in hepatic glucuronidation of alpinetin. The effect of BCRP on alpinetin disposition was assessed using HeLa cells overexpressing UGT1A1 (HeLa1A1) cells. Alpinetin underwent extensive glucuronidation in pHLM and pHIM, generating one glucuronide metabolite. Of 12 test UGT enzymes, UGT1A3 was the most active one toward alpinetin with an intrinsic clearance (CLint = Vmax/Km) value of 66.5 μl/min/nmol, followed by UGT1A1 (CLint = 48.6 μl/min/nmol), UGT1A9 (CLint = 21.0 μl/min/nmol), UGT2B15 (CLint = 16.7 μl/min/nmol) and UGT1A10 (CLint = 1.60 μl/min/nmol). Glucuronidation of alpinetin was significantly correlated with glucuronidation of estradiol (an activity marker of UGT1A1), chenodeoxycholic acid (an activity marker of UGT1A3), propofol (an activity marker of UGT1A9) and 5-hydroxyrofecoxib (an activity marker of UGT2B15), confirming the important roles of UGT1A1, UGT1A3, UGT1A9 and UGT2B15 in alpinetin glucuronidation. Inhibition of BCRP by its specific inhibitor Ko143 significantly reduced excretion of alpinetin glucuronide, leading to a significant decrease in cellular glucuronidation of alpinetin. Our data suggest UGTs and BCRP as two important determinants of alpinetin pharmacokinetics.

Hsp90 inhibitor AT-533 blocks HSV-1 nuclear egress and assembly

&NA; Heat shock protein 90 (Hsp90) has been identified as an essential host factor for the infection and replication of several viruses, including HSV‐1. Recent works have clearly shown that Hsp90 plays a role in the early stages of HSV‐1 infection, including nuclear import and DNA replication. However, the role of Hsp90 in the late stages of HSV‐1 infection remains unclear. In this study, we found that Hsp90 was up‐regulated during late viral infection. Treatment with the Hsp90 inhibitor AT‐533 significantly decreased the intracellular and extracellular virus titers, and strongly inhibited nucleocapsid egress from the nucleus. More detailed studies revealed that AT‐533 inhibited the nuclear egress of the viral nucleocapsid by suppressing the expression and translocation of nuclear‐associated proteins pUL31 and pUL34. In addition, we found that AT‐533 hindered the assembly of virus particles possibly though affecting the localization of glycoproteins in the endoplasmic reticulum and Golgi apparatus. These results thus invoke a new role for Hsp90 in the nucleocapsid egress and viral maturation of HSV‐1, and further promote the development of Hsp90 inhibitors as potential anti‐HSV‐1 drugs.