Identification of a Discrete Diglucuronide of GDC-0810 in Human Plasma after Oral Administration.

GDC-0810 is a small molecule therapeutic agent having potential to treat breast cancer. In plasma of the first-in-human study, metabolite M2, accounting for 20.7% of total drug-related materials, was identified as a discrete diglucuronide that was absent in rats. Acyl glucuronide M6 and N-glucuronide M4 were also identified as prominent metabolites in human plasma. Several in vitro studies were conducted in incubations of [14C]GDC-0810, synthetic M6 and M4 with liver microsomes, intestinal microsomes, and hepatocytes of different species as well as recombinant UDP-glucuronosyltransferase (UGT) enzymes to further understand the formation of M2. The results suggested that 1) M2 was more efficiently formed from M6 than from M4, and 2) acyl glucuronidation was mainly catalyzed by UGT1A8/7/1 that is highly expressed in the intestines whereas N-glucuronidation was mainly catalyzed by UGT1A4 that is expressed in the human liver. This complicated mechanism presented challenges in predicting M2 formation using human in vitro systems. The absence of M2 and M4 in rats can be explained by low to no expression of UGT1A4 in rodents. M2 could be the first discrete diglucuronide that was formed from both acyl- and N-glucuronidation on a molecule identified in human plasma. SIGNIFICANCE STATEMENT: A discrete diglucuronidation metabolite of GDC-0810, a breast cancer drug candidate, was characterized as a unique circulating metabolite in humans that was not observed in rats or little formed in human in vitro system.

Iron restriction induces the small-colony variant phenotype in Staphylococcus aureus.

Pathogens such as Staphylococcus aureus must overcome host-induced selective pressures, including limited iron availability. To cope with the harsh conditions of the host environment, S. aureus can adapt its physiology in multiple ways. One of these adaptations is the fermenting small-colony variant (SCV) phenotype, which is known to be inherently tolerant to certain classes of antibiotics and heme toxicity. We hypothesized that SCVs might also behave uniquely in response to iron starvation since one of the major cellular uses of iron is the respiration machinery. In this study, a respiring strain of S. aureus and fermenting SCV strains were treated with different concentrations of the iron chelator, 2,2' dipyridyl (DIP). Our data demonstrate that a major impact of iron starvation in S. aureus is the repression of respiration and the induction of the SCV phenotype. We demonstrate that the SCV phenotype transiently induced by iron starvation mimics the aminoglycoside recalcitrance exhibited by genetic SCVs. Furthermore, prolonged growth in iron starvation promotes increased emergence of stable aminoglycoside-resistant SCVs relative to the naturally occurring subpopulation of SCVs within an S. aureus community. These findings may have relevance to physiological and evolutionary processes occurring within bacterial populations infecting iron-limited host environments.

Effect of Photobiomodulation Therapy on Reducing Acute Pain and Inflammation Following Surgical Removal of Impacted Mandibular Third Molars: A Randomized, Split-Mouth Clinical Trial.

Objective: This study aimed to compare the effects of photobiomodulation therapy (PBMT) on analgesic and inflammatory reduction with that of ibuprofen following surgical removal of impacted mandibular third molars (IMTMs). Methods: A randomized, split-mouth clinical trial was performed on patients undergoing bilateral IMTM removal. PBMT [gallium aluminum arsenide (GaAlAs) laser] with specific parameters (wavelength of 810 nm, power of 0.5% ± 20% W, and energy density of 4 J/cm2) was applied randomly on one side of the mouth immediately after surgery and 1 and 2 days after surgery. The pain level was self-rated with a Likert scale at 2, 4, 6, 24, and 48 h postoperatively. Swelling and trismus were measured on the first and second day after surgery. Saliva was collected for measuring pre- and postoperative salivary immunoglobulin A (sIgA) concentrations with the sandwich ELISA test. Results: The study sample included 25 patients (average age of 22.88 years) with 50 bilateral symmetrical IMTMs. Pain level was highest at 2 h after surgery in both groups and gradually decreased over time (p < 0.01). Swelling and trismus at 48 h were higher than at 24 h (p < 0.01). Within the first 48 h postoperatively, pain level, swelling, and trismus were significantly lower in the PBMT group (p < 0.05). Postoperative sIgA was also significantly lower in the PBMT group (p < 0.05). Conclusions: In short-term and specific conditions of this study, it was found that PBMT helped promote postoperative pain relief and anti-inflammation after surgical removal of IMTMs. The results suggested that there may be a link between a decrease in salivary sIgA levels and decrease in inflammatory processes after PBMT. Trial Registration No. NCT04280809 at ClinicalTrials.gov.

Risk Score Using Demographic and Clinical Risk Factors Predicts Gastric Intestinal Metaplasia Risk in a U.S. Population.

BACKGROUND/AIMS: Screening for gastric intestinal metaplasia (GIM) may lead to early gastric cancer detection. We developed and validated a pre-endoscopy risk prediction model for detection of GIM based on patient-level risk factors in a U.S. METHODS: We used data from 423 GIM cases and 1796 controls from a cross-sectional study among primary care and endoscopy clinic patients at the Houston VA. We developed the model using backwards stepwise regression and assessed discrimination using area under the receiver operating characteristic (AUROC). The model was internally validated using cross-validation and bootstrapping. The final expanded model was compared to a model including H. pylori infection alone and a baseline model including remaining terms without H. pylori. RESULTS: Male sex, older age, non-white race/ethnicity, smoking status, and H. pylori were associated with GIM risk. The expanded model including these terms had AUROC 0.73 (95%CI 0.71-0.76) for predicting GIM and AUROC 0.82 (95%CI 0.79-0.86) for extensive GIM. This model discriminated better than a model including only H. pylori (AUROC 0.66; 95%CI 0.63-0.68) and the baseline model (AUROC 0.67; 95%CI 0.64-0.70). The expanded model performed similarly among primary care (AUROC 0.75) and endoscopy (AUROC 0.73) patients. The expanded model showed sufficient internal validity (cross-validation AUROC 0.72) with little evidence of over-fitting. CONCLUSIONS: We develop and validate a non-invasive risk model for GIM detection in a U.S. population that included terms for sex, age, race/ethnicity, smoking status, and H. pylori infection. Validated risk models would identify individuals with GIM who should be referred for endoscopic screening.

Dietary Factors and Gastric Intestinal Metaplasia Risk Among US Veterans.

BACKGROUND: Studies on diet and gastric intestinal metaplasia (GIM) risk are lacking in US populations. AIM: To determine the associations of dietary factors and risk of GIM among a US population with typical American diet. METHODS: We analyzed data from a cross-sectional study of veterans attending primary care and endoscopy clinics at the Houston VA Medical Center. Patients completed a 110-item Block Food Frequency Questionnaire then underwent upper endoscopy with gastric mapping biopsies. We compared cases defined by GIM on ≥ 1 non-cardia gastric biopsy to controls without GIM. Associations of dietary factors and GIM were estimated using logistic regression models as odds ratios (OR) and 95% confidence intervals (CI). RESULTS: Among 423 GIM cases and 1796 controls, cases were older (62.1 vs. 59.9 years) and more likely to be male (97.2% vs. 90.8%) and non-White (58.6% vs. 39.0%). GIM cases had lower fat intake (percent kcal from fat tertile 1: 43.6% vs. 33.4%) and higher carbohydrate intake (percent kcal from carbohydrate T3: 41.8% vs. 33.3%) than controls. Adjusting for age, gender, race, smoking, and Helicobacter pylori, percent kcal from carbohydrates (T3 vs. T1: OR 1.35, 95% CI 1.08-1.67), fruit intake (T3 vs. T1: OR 1.28, 95% CI 1.02-1.61), and fiber intake (T3 vs. T1: OR 1.37, 95% CI 1.04-1.80) were associated with GIM. In subgroup analyses, these associations were primarily seen in non-White patients. CONCLUSIONS: Few dietary factors, including high carbohydrate intake, are associated with increased risk of GIM in US populations, independent of H. pylori or smoking.

Evaluating the in vitro inhibition of UGT1A1, OATP1B1, OATP1B3, MRP2, and BSEP in predicting drug-induced hyperbilirubinemia.

Hyperbilirubinemia may arise due to inadequate clearance of bilirubin from the body. Bilirubin elimination is a multifaceted process consisting of uptake of bilirubin into the hepatocytes facilitated by OATP1B1 and OATP1B3. Once in the hepatocytes, it is extensively glucuronidated by UGT1A1. Eventually, the glucuronide metabolite is excreted into the bile via MRP2. UGT1A1 inhibition has been previously shown to be linked with hyperbilirubinemia. However, because drug transporters also contribute to bilirubin elimination, the purpose of this work was to investigate the in vitro inhibition of OATP1B1, OATP1B3, MRP2, and BSEP of select test drugs known to elicit hyperbilirubinemia. Test drugs investigated in this study were atazanavir and indinavir, which are associated with hyperbilirubinemia and elevations in serum transaminase; ritonavir and nelfinavir, which are not associated with hyperbilirubinemia; and bromfenac, troglitazone, and trovafloxacin, which are associated with severe idiosyncratic hepatotoxicity exhibiting elevations in serum bilirubin and transaminase. Due to limited solubility and poor ionization of bilirubin and its glucuronide, the formation of estradiol 3-glucuronide was used as a surrogate to assess UGT1A1 activity, while the transport of pitavastatin, CDCF, and taurocholate were used as surrogate probe substrates to monitor the function of OATP1B1/OATP1B3, MRP2, and BSEP, respectively. It was assumed that any inhibition of the surrogate probe substrates by test drugs is indicative of the potential impact of test drugs to modulate the function of proteins involved in bilirubin disposition. In vitro inhibition was determined by calculating IC50. Moreover, Cmax and Cmax,free were integrated with IC50 values to calculate R and Rfree, respectively, which represents the ratio of probe drug glucuronidation/transport in the absence and presence of test drugs. Analysis of the data showed that Rfree demonstrated the best correlation to hyperbilirubinemia. Specifically, Rfree was above the 1.1 target threshold against UGT1A1, OATP1B1, and BSEP for atazanavir and indinavir. In contrast, Rfree was below this threshold for ritonavir and nelfinavir as well as for bromfenac, troglitazone, and trovafloxacin. For all test drugs examined, only minor inhibition against OATP1B3 and MRP2 were observed. These data suggest that the proposed surrogate probe substrates to evaluate the in vitro inhibition of UGT1A1, OATP1B1, and BSEP may be suitable to assess bilirubin disposition. For protease inhibitors, inclusion of OATP1B1 and BSEP inhibition may improve the predictability of hyperbilirubinemia.

Expression, purification, and characterization of Pseudomonas aeruginosa SecA.

A secA gene from Pseudomonas aeruginosa PAO1 was amplified and expressed in Escherichia coli BL21.19 (secA13) under conditions where E. coli SecA was depleted. The binding of P. aeruginosa SecA (PaSecA) to the SP-Sepharose column was facilitated by ammonium sulfate fractionation but was not necessary for E. coli SecA (EcSecA) as the later bound more efficiently. PaSecA and EcSecA were purified by the single chromatographic step to greater than 98% purity and had a recovery of more than 20 and 40%, respectively, from the soluble fraction. This simple step purification obtained a higher homogeneity than previously reported. Cross-reactivity by immunoblotting showed that the purified PaSecA contained little EcSecA if any. The purified PaSecA is a dimer in solution, as judged by size exclusion chromatography, and is slightly larger than its counterpart EcSecA with an estimated molecular weight of 240 kDa. Further studies by the sedimentation velocity method indicate that PaSecA tends to remain as a monomer in solution. The purified PaSecA possessed ATPase activity; the intrinsic and liposome-stimulated ATPase specific activities of PaSecA were approximately 50% of EcSecA.