Nalbuphine Potentiates Reversal of Fentanyl Overdose by Naloxone.

Developing an effective antidote for fentanyl-induced overdose to achieve timely reversal is an unmet public health need. Previously, we found that naloxone derivative NX90 with mild κ-opioid agonistic properties was three-fold more effective than the parent naloxone in reversing a fentanyl overdose in rats. To investigate whether κ-agonistic properties could indeed augment the robustness of overdose reversal, we evaluated a κ-agonist/µ-antagonist nalbuphine (NB) as well as its combinations with naloxone (NX) in a fentanyl overdose model in rodents. An administration of either NB or NX as single agents at 0.1 mg/kg doses produced a full recovery in 90 ± 9.9 min and 11.4 ± 2.7 min, respectively. A higher dose of NX at 0.2 mg/kg reversed an overdose within 4.8 ± 1.0 min. In contrast to that, the coadministration of NB and NX at 0.1 mg/kg each produced a synergistic effect, with overdose reversal in 3.4 ± 0.2 min. The coadministration of NX and NB at sub-therapeutic doses of 0.05 mg/kg each was also 1.2-fold more effective than NX at 0.2 mg/kg. We further found that co-administration of NB at different doses (0.025, 0.05, 0.1 mg/kg) and ratios (1:4 and 1:1) with NX had differential effects on overdose reversal, cardiorespiratory liabilities, and analgesia.

Modifying naloxone to reverse fentanyl-induced overdose.

Developing an effective antidote for fentanyl-induced overdose (OD) is an unmet medical need that requires both lipophilicity comparable to fentanyl and fast onset of overdose reversal. We synthesized and evaluated a bioreversible derivative of naloxone (NX-90) in silico, in vitro and in vivo to yield a robust reversal of fentanyl-induced OD in rats. All monitored reflexes along with the heart rate (HR) and respiratory rate (RR) were fully restored faster in the NX-90 groups than in naloxone groups on equimolar bases when given intranasally. In NX-90 treated rats RR over the time of observation (RR AUC) was significantly higher at all respective doses with no re-narcotization observed. Apart from the enhanced pharmacodynamics profile, NX-90 was found to have lower circulating levels of naloxone, clean profile in in vitro selectivity panels, as well as Ames and CYP450 counter screens. Finally, we demonstrated a robust release of the parent naloxone in brain matrix, as well as lower peripheral naloxone levels after NX-90 iv administration. With the demonstrated pharmacological profile superior yet congruent to naloxone we nominated NX-90 for preclinical development as an effective intranasal fentanyl antidote.

NB-33, a bioreversible opioid derivative of Nalbuphine, shows enhanced pharmacodynamics.

Developing non-addictive and safer opioids for pain management is unmet medical need. Among a number of bioreversible derivatives of Nalbuphine - an equipotent to morphine opioid without serious side effects - NB-33 was identified in silico and confirmed in vivo as a superior analgesic agent. Apart from enhanced pharmacodynamics profile, NB-33 outperformed the parent compound on equimolar bases in cold ethanol tail-flick and mechanical models of pain in rats. With no ß-arrestin engagement liability, good stability in simulated gastro-intestinal fluid and slow release of Nalbuphine by plasma NB-33 is being developed as an oral and safer alternative of its parent drug.

An Oral Formulation of the Probiotic, Bacillus subtilis HU58, Was Safe and Well Tolerated in a Pilot Study of Patients with Hepatic Encephalopathy.

BACKGROUND: Hepatic encephalopathy often results in high blood ammonia levels because of inefficient ammonia processing by the liver. Lactulose treatment promotes the growth of urease-producing gut bacteria and a reduced colon pH, thus reducing blood ammonia absorption. It is thought that probiotics as an add-on therapy may be beneficial. Patients and Methods. Bacillus subtilis HU58 was tested for safety and tolerability in patients with hepatic encephalopathy taking lactulose in this double-bind, placebo-controlled, 4-week pilot study. Study participants received one dose of B. subtilis HU58 or placebo (orally) for the first five days and two daily doses thereafter. Participants were monitored for safety and blood ammonia levels. RESULTS: Forty patients participated (placebo, 11; probiotic, 29). Baseline characteristics were generally comparable; the mean baseline blood ammonia level was somewhat higher in the probiotic group. Mild or moderate treatment-emergent adverse events (TEAEs) were reported in 27.3% and 17.2% of patients in the placebo and probiotic groups, respectively; no severe TEAEs were reported. One patient (9.1%) taking placebo and two (6.9%) taking the probiotic experienced serious TEAEs (SAEs); none resulted in study discontinuation and all were considered to have no/unlikely relationship to the study product. There were no significant differences in the mean percent change (MPC) of blood ammonia levels between groups, though the probiotic group exhibited a trend toward a milder increase. Stratification of the probiotic group by baseline blood ammonia level (>60 µg/dL and ≤60 µg/dL) resulted in a significantly reduced MPC in the >60 µg/dL subgroup (MPC (SD); ≤60 µg/dL (n = 14), 35.3% (73.3); >60 µg/dL (n = 14), -26.5% (24.4); p = 0.0087). CONCLUSIONS: Daily treatment with oral B. subtilis HU58 was safe and well tolerated over a 4-week period in patients with hepatic encephalopathy, and a significantly reduced MPC of blood ammonia level was observed in patients with a baseline level >60 µg/dL.

Suppressor of sable [Su(s)] and Wdr82 down-regulate RNA from heat-shock-inducible repetitive elements by a mechanism that involves transcription termination.

Although RNA polymerase II (Pol II) productively transcribes very long genes in vivo, transcription through extragenic sequences often terminates in the promoter-proximal region and the nascent RNA is degraded. Mechanisms that induce early termination and RNA degradation are not well understood in multicellular organisms. Here, we present evidence that the suppressor of sable [su(s)] regulatory pathway of Drosophila melanogaster plays a role in this process. We previously showed that Su(s) promotes exosome-mediated degradation of transcripts from endogenous repeated elements at an Hsp70 locus (Hsp70-αß elements). In this report, we identify Wdr82 as a component of this process and show that it works with Su(s) to inhibit Pol II elongation through Hsp70-αß elements. Furthermore, we show that the unstable transcripts produced during this process are polyadenylated at heterogeneous sites that lack canonical polyadenylation signals. We define two distinct regions that mediate this regulation. These results indicate that the Su(s) pathway promotes RNA degradation and transcription termination through a novel mechanism.

Drosophila suppressor of sable protein [Su(s)] promotes degradation of aberrant and transposon-derived RNAs.

RNA-binding proteins act at various stages of gene expression to regulate and fine-tune patterns of mRNA accumulation. One protein in this class is Drosophila Su(s), a nuclear protein that has been previously shown to inhibit the accumulation of mutant transcripts by an unknown mechanism. Here, we have identified several additional RNAs that are downregulated by Su(s). These Su(s) targets include cryptic wild-type transcripts from the developmentally regulated Sgs4 and ng1 genes, noncoding RNAs derived from tandemly repeated alphabeta/alphagamma elements within an Hsp70 locus, and aberrant transcripts induced by Hsp70 promoter transgenes inserted at ectopic sites. We used the alphabeta RNAs to investigate the mechanism of Su(s) function and obtained evidence that these transcripts are degraded by the nuclear exosome and that Su(s) promotes this process. Furthermore, we showed that the RNA binding domains of Su(s) are important for this effect and mapped the sequences involved to a 267-nucleotide region of an alphabeta element. Taken together, these results suggest that Su(s) binds to certain nascent transcripts and stimulates their degradation by the nuclear exosome.

Measuring the glycemic index of foods: interlaboratory study.

BACKGROUND: Many laboratories offer glycemic index (GI) services. OBJECTIVE: We assessed the performance of the method used to measure GI. DESIGN: The GI of cheese-puffs and fruit-leather (centrally provided) was measured in 28 laboratories (n=311 subjects) by using the FAO/WHO method. The laboratories reported the results of their calculations and sent the raw data for recalculation centrally. RESULTS: Values for the incremental area under the curve (AUC) reported by 54% of the laboratories differed from central calculations. Because of this and other differences in data analysis, 19% of reported food GI values differed by >5 units from those calculated centrally. GI values in individual subjects were unrelated to age, sex, ethnicity, body mass index, or AUC but were negatively related to within-individual variation (P=0.033) expressed as the CV of the AUC for repeated reference food tests (refCV). The between-laboratory GI values (mean+/-SD) for cheese-puffs and fruit-leather were 74.3+/-10.5 and 33.2+/-7.2, respectively. The mean laboratory GI was related to refCV (P=0.003) and the type of restrictions on alcohol consumption before the test (P=0.006, r2=0.509 for model). The within-laboratory SD of GI was related to refCV (P<0.001), the glucose analysis method (P=0.010), whether glucose measures were duplicated (P=0.008), and restrictions on dinner the night before (P=0.013, r2=0.810 for model). CONCLUSIONS: The between-laboratory SD of the GI values is approximately 9. Standardized data analysis and low within-subject variation (refCV<30%) are required for accuracy. The results suggest that common misconceptions exist about which factors do and do not need to be controlled to improve precision. Controlled studies and cost-benefit analyses are needed to optimize GI methodology. The trial was registered at clinicaltrials.gov as NCT00260858.

Malignant blue nevus: a case report and molecular analysis.

Malignant blue nevus is a rare melanocytic tumor that is described by some authors as a variant of malignant melanoma, whereas others regard it as a distinct entity. To our knowledge no molecular studies of this tumor have been performed, although the molecular pathogenesis of conventional melanomas has been extensively described. We present a case of malignant blue nevus that developed in a 15-cm congenital blue nevus on the back of a 41-year-old man. Subsequent regional lymph node and lung metastases developed within 1 and 29 months, respectively. We performed a molecular analysis for loss of heterozygosity on microdissected samples from the spectrum of benign to malignant blue nevus, using a panel of eight genes (MTS1, MXI1, CMM1, p53, NF1, L-myc hOGG1, and MCC), many of which are commonly associated with conventional melanomas. No loss of heterozygosity was detected, despite informativeness in seven genes. We suggest that malignant blue nevus may represent a distinct entity with a different molecular pathway to tumorigenesis than that of conventional melanomas.

A predominant IgG4 subclass may be responsible for false-negative direct immunofluorescence in bullous pemphigoid.

BACKGROUND: Bullous pemphigoid (BP) is an immune-mediated blistering disease, usually characterized immunopathologically by the linear deposition of IgG and C3 along the basement membrane zone (BMZ) of skin. However, positive deposition of C3 but negative staining for IgG on direct immunofluorescence (DIF) studies has been noted in some patients. METHODS: Twelve patients known to have BP but with absence of staining for IgG were included in this study. Frozen sections of skin specimens from the 12 patients were subjected to IgG DIF, as well as a sandwich double antibody method of staining for IgG, IgG subclasses, and light chains. Enzyme-linked immunosorbent assay (ELISA) using commercially available human IgG subclasses was used to analyze the subclass restriction of FITC-labeled antihuman IgG conjugates. RESULTS: Of the 12 skin specimens with positive C3 and negative IgG on DIF, nine were positive for IgG with the double antibody sandwich method. In addition, all 12 specimens had positive linear staining for the subclass IgG4 along the BMZ with this method. There was no IgG light chain restriction. Two commercially obtained antihuman IgG conjugates, both commonly used in our laboratory for DIF testing, were analyzed for separate IgG subclass specificity by ELISA. Both conjugates showed high reactivity to IgG1 and IgG3 with less reactivity to IgG2 and IgG4. CONCLUSION: These results suggest that the following factors contribute to false-negative staining for IgG on DIF in some BP patients: (i): subthreshold IgG in skin specimens; (ii) limited reactivity of commercial antihuman IgG conjugates to the IgG4 subclass; and (iii) decreased sensitivity of DIF compared with double antibody methods for the detection of IgG. The use of sandwich double antibody immunofluorescence methods to test for IgG and/or IgG subclasses may be helpful in definitively diagnosing BP in patients with negative IgG and positive C3 staining on DIF.