Analysis of N1‐acetyl‐N2‐formyl‐5‐methoxykynuramine/N1‐acetyl‐5‐methoxy‐kynuramine formation from melatonin in mice

Abstract: The interactions of melatonin, a potent endogenous antioxidant, with reactive oxygen species generate several products that include N¹‐acetyl‐N²‐formyl‐5‐methoxykynuramine (AFMK) and N¹‐acetyl‐5‐methoxy‐kynuramine (AMK). The physiological or pathological significance of AFMK/AMK formation during the process of melatonin metabolism in mammals has not been clarified. Using a metabolomic approach in the current study, the AFMK/AMK pathway was thoroughly investigated both in mice and humans. Unexpectedly, AFMK and AMK were not identified in the urine of humans nor in the urine, feces or tissues (including liver, brain, and eyes) in mice under the current experimental conditions. Metabolomic analysis did identify novel metabolites of AMK, i.e. hydroxy‐AMK and glucuronide‐conjugated hydroxy‐AMK. These two newly identified metabolites were, however, not found in the urine of humans. In addition, oxidative stress induced by acetaminophen in the mouse model did not boost AFMK/AMK formation. These data suggest that AFMK/AMK formation is not a significant pathway of melatonin disposition in mice, even under conditions of oxidative stress.     

Rebaudioside A directly stimulates insulin secretion from pancreatic beta cells: a glucose-dependent action via inhibition of ATP-sensitive K-channels

Recently, we showed that rebaudioside A potently stimulates the insulin secretion from isolated mouse islets in a dose-, glucose- and Ca(2+)-dependent manner. Little is known about the mechanisms underlying the insulinotropic action of rebaudioside A. The aim of this study was to define the signalling system by which, rebaudioside A acts. Isolated mouse islets were used in the cAMP[(125)I] scintillation proximity assay to measure total cAMP level, and in a luminometric method to measure intracellular ATP and ADP concentrations. Conventional and permeabilized whole-cell configuration of the patch-clamp technique was used to verify the effect of rebaudioside A on ATP-sensitive K(+)-channels from dispersed single beta cells from isolated mouse islets. Insulin was measured by radioimmunoassay from insulinoma MIN6 cells. In the presence of 16.7 mM glucose, the addition of the maximally effective concentration of rebaudioside A (10(-9) M) increased the ATP/ADP ratio significantly, while it did not change the intracellular cAMP level. Rebaudioside A (10(-9) M) and stevioside (10(-6) M) reduced the ATP-sensitive potassium channel (K(ATP)) conductance in a glucose-dependent manner. Moreover, rebaudioside A stimulated the insulin secretion from MIN6 cells in a dose- and glucose-dependent manner. In conclusion, the insulinotropic effect of rebaudioside A is mediated via inhibition of ATP-sensitive K(+)-channels and requires the presence of high glucose. The inhibition of ATP-sensitive K(+)-channels is probably induced by changes in the ATP/ADP ratio. The results indicate that rebaudioside A may offer a distinct therapeutic advantage over sulphonylureas because of less risk of causing hypoglycaemia.     

An international collaborative study to establish the WHO 1st international standard for alpha-1-antitrypsin

Background and Objectives The aim was to establish the 1st International Standard (IS) for alpha‐1‐antitrypsin (AAT) to standardise potency assignment of therapeutic products, calibrated in moles and mg active AAT in line with product labelling practice. Assigning total protein and antigen values to the IS was also investigated. Materials and Methods The active concentration of four candidate AAT preparations was determined in an international collaborative study by inhibition of trypsin (calibrated by active‐site titration). Total protein and antigen content were determined for each candidate using local methods and in‐house standards, and a common AAT preparation. The total protein content of the IS was also determined by amino acid analysis. Potency determination of recombinant and transgenic materials against the IS was investigated in a follow‐up study. Results Data analysis for potency determination indicated no statistical difference between any of the candidates, or between the results for recombinant and plasma‐derived products. Total protein content of the IS determined by amino acid analysis was consistent with the potency value. The variability in the total protein and antigen results for the other candidates was reduced when the data were recalculated relative to the IS. Conclusions Candidate C (05/162) was established by the WHO Expert Committee on Biological Standardization (ECBS) in 2006 as the WHO 1st IS for AAT with a potency of 243 nmoles (12·4 mg) active inhibitor per ampoule. In 2008, ECBS approved the IS for potency determination of recombinant material and assigned a total protein and antigen value of 12·4 mg.     

The melatonin metabolite N1‐acetyl‐5‐methoxykynuramine facilitates long‐term object memory in young and aging mice

Melatonin (MEL) has been reported to enhance cognitive processes, making it a potential treatment for cognitive decline. However, the role of MEL’s metabolites, N1‐acetyl‐N2‐formyl‐5‐methoxykynuramine (AFMK) and N1‐acetyl‐5‐methoxykynuramine (AMK), in these effects are unknown. The current study directly investigated the acute effects of systemic MEL, AFMK, and AMK on novel object recognition. We also analyzed MEL, AFMK, and AMK levels in hippocampus and temporal lobe containing the perirhinal cortex following systemic MEL and AMK treatment. AMK administered post‐training had a more potent effect on object memory than MEL and AFMK. AMK was also able to rescue age‐associated declines in memory impairments when object memory was tested up to 4 days following training. Results from administering AMK at varying times around the training trial and the metabolism time course in brain tissue suggest that AMK’s memory‐enhancing effects reflect memory consolidation. Furthermore, inhibiting the MEL‐to‐AMK metabolic pathway disrupted object memory at 24 hours post‐training, suggesting that endogenous AMK might play an important role in long‐term memory formation. This is the first study to report that AMK facilitates long‐term object memory performance in mice, and that MEL crosses the blood‐brain barrier and is immediately converted to AMK in brain tissue. Overall, these results support AMK as a potential therapeutic agent to improve or prevent memory decline.     

On the free radical scavenging activities of melatonin's metabolites,AFMK and AMK

The reactions of N¹‐acetyl‐N²‐formyl‐5‐methoxykynuramine (AFMK) and N¹‐acetyl‐5‐methoxykynuramine (AMK) with ^?OH, ^?OOH, and ?OOCCl₃ radicals have been studied using the density functional theory. Three mechanisms of reaction have been considered: radical adduct formation (RAF), hydrogen transfer (HT), and single electron transfer (SET). Their relative importance for the free radical scavenging activity of AFMK and AMK has been assessed. It was found that AFMK and AMK react with ?OH at diffusion‐limited rates, regardless of the polarity of the environment, which supports their excellent ?OH radical scavenging activity. Both compounds were found to be also very efficient for scavenging ?OOCCl₃, but rather ineffective for scavenging ?OOH. Regarding their relative activity, it was found that AFMK systematically is a poorer scavenger than AMK and melatonin. In aqueous solution, AMK was found to react faster than melatonin with all the studied free radicals, while in nonpolar environments, the relative efficiency of AMK and melatonin as free radical scavengers depends on the radical with which they are reacting. Under such conditions, melatonin is predicted to be a better ?OOH and ?OOCCl₃ scavenger than AMK, while AMK is predicted to be slightly better than melatonin for scavenging ?OH. Accordingly it seems that melatonin and its metabolite AMK constitute an efficient team of scavengers able of deactivating a wide variety of reactive oxygen species, under different conditions. Thus, the presented results support the continuous protection exerted by melatonin, through the free radical scavenging cascade.     

Metabolism of melatonin by cytochrome P450s in rat liver mitochondria and microsomes

Abstract: In the present study we provide direct evidence for the involvement of rat microsomal cytochrome P450s in melatonin O‐demethylation and hydroxylation at two different positions: 2 and 6, as well as generation of N¹‐acetyl‐N²‐formyl‐5‐methoxy‐kynuramine (AFMK) and two unknown products. Moreover, we found that mitochondrial cytochrome P450s also converts melatonin into AFMK, N‐acetylserotonin, 2‐hydroxymelatonin, 6‐hydroxymelatonin and the same two unknown products. Eadie–Hofstee plots for 6‐hydroxylation and O‐demethylation reactions were curvilinear for all tested fractions, suggestive of involvement of at least two components, one with a high affinity and low capacity, and another with a low affinity and high capacity. Mitochondrial cytochrome P450s exhibited higher affinity (suggesting lower K_m value) and higher V_max for melatonin 6‐hydroxylation and O‐demethylation for both high‐affinity and low‐affinity components as compared with microsomal enzymes. The intrinsic clearance for melatonin hydroxylation by high‐ and low‐affinity components displayed the highest values in all tested fractions, indicating that both mitochondrial and microsomal cytochrome P450s metabolize melatonin principally by 6‐hydroxylation, with O‐demethylation representing a minor metabolic pathway.     

Pre-engraftment graft-versus-host disease after allogeneic hematopoietic cell transplantation for acute leukemia

Objectives: Acute graft‐versus‐host disease (GVHD) usually occurs with neutrophil engraftment following allogeneic hematopoietic cell transplantation (HCT), but it can also occur before engraftment. We intended to analyze the effects of timing of acute GVHD on leukemia relapse and mortality. Methods: The outcomes of pre‐ and postengraftment GVHD were investigated in 384 patients who underwent allogeneic HCT for acute leukemia. Results: Acute GVHD occurred in 100 patients, pre‐engraftment in 22 and postengraftment in 78. Compared with postengraftment GVHD, pre‐engraftment GVHD was more severe, as assessed by overall grade, with more frequent and more severe skin involvement and higher incidences of non‐infectious fever, diarrhea, hepatic dysfunction, renal insufficiency, and weight gain. Compared with patients without acute GVHD, those with postengraftment GVHD had lower cumulative incidence of relapse [CIR; hazard ratio (HR), 0.470; P = 0.006] and higher cumulative incidence of non‐relapse mortality (CINRM; HR, 2.568; P < 0.001), while those with pre‐engraftment GVHD had similar CIR (HR, 0.815; P = 0.059) and higher CINRM (HR, 2.872; P = 0.036). Overall survival of patients with pre‐engraftment GVHD was lower than that of those without acute GVHD (HR, 1.976; P = 0.017), which was similar to that of those with postengraftment GVHD (HR, 0.969; P = 0.878). Separate analyses of the effects of timing of acute GVHD on post‐transplant outcomes in patients with acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) showed similar trends. Conclusion: Pre‐engraftment GVHD might be a ‘cytokine storm’ type syndrome rather than ‘real’ GVHD, indicating the need for separate analyses of pre‐ and postengraftment GVHD in future trials.     

Successful liver transplantation despite donor‐transmitted ESBL‐producing Klebsiella pneumoniae infection: Case report and review of the literature

Donor‐derived bacterial infection is a recognized complication of solid organ transplantation. Patients admitted to the intensive care unit are increasingly exposed to infection with multidrug‐resistant microorganisms. However, no specific recommendations are available about their suitability as donors. We report a case of donor‐transmitted extended‐spectrum beta‐lactamase‐producing Klebsiella pneumoniae infection in a liver recipient, and review the related literature.     

Controversies on the prognostic value of interim FDG‐PET in advanced‐stage Hodgkin lymphoma

Hodgkin lymphoma, even in advanced‐stage, is a highly curable malignancy, but treatment is associated with short‐term toxicity and long‐term side effects. Early predictive markers are required to identify those patients who do not require the full‐length standard therapy (and thus qualify for therapy de‐escalation) and those patients who will not be cured by standard therapy (and thus qualify for therapy escalation). Multiple trials have assessed the value of ¹⁸F‐fluoro‐2‐deoxy‐d ‐glucose positron emission tomography (FDG‐PET) after a few cycles of chemotherapy (also known as ‘interim FDG‐PET’) in predicting outcome in advanced‐stage Hodgkin lymphoma. Furthermore, multiple interim FDG‐PET‐adapted trials, in which patients with positive interim FDG‐PET scans are assigned to escalated therapies, and patients with negative interim FDG‐PET scans are assigned to de‐escalated therapies, have recently been published or are currently ongoing, with generally heterogeneous results. The present article reports the currently available evidence (and controversies) on the prognostic value of interim FDG‐PET in advanced‐stage Hodgkin lymphoma in patients with positive and negative interim FDG‐PET findings following continuation of standard chemotherapy or escalated/de‐escalated therapy.     

Initial clinical experience of selective coronary angiography using one prolonged injection and a 180 degrees rotational trajectory.

OBJECTIVE: Evaluate the safety of prolonged coronary injections during a rotational acquisition covering 180 degrees. BACKGROUND: Rotational angiography has been adapted to coronary angiography and shown to reduce radiation and contrast exposure. Three-dimensional (3D) reconstructions and other advanced applications require imaging over a 180 degrees -arc with a single but longer injection of larger contrast volumes. METHODS: Thirty patients referred for angiography were enrolled. Blood pressure (BP), heart rate (HR), symptoms, and ectopy were recorded before-and-after injections. RESULTS: Pre and post-injection HRs for the LCA/RCA were not statistically different (LCA-pre-injection 63+/-13 bpm vs. LCA-post-injection 62+/-11 bpm, P=0.54 and RCA-pre-injection 65+/-12 bpm vs. RCA-post-injection 65+/-10, P=0.88). Central aortic pressure values were not statistically different for the RCA injections (RCA-systolic-pre-injection 118+/-14 mm Hg vs. RCA-systolic-post-injection 112+/-25 mm Hg, P=0.15, and RCA diastolic-pre-injection 69+/-9 mm Hg vs. RCA-diastolic-post-injection 60+/-10 mm Hg, P=0.88) but were statistically significant for the LCA injections (LCA systolic-pre-injection 122+/-19 mm Hg vs. LCA-systolic-post-injection 116+/-17 mm Hg, P=0.0004, and LCA-diastolic-pre-injection 69+/-10 mm Hg vs. LCA-diastolic-post-injection 65+/-9 mm Hg, P=0.0007). There were no symptoms or electrical events documented during or immediately post-injection. CONCLUSION: This study demonstrates the feasibility and safety of longer coronary injections. There were no significant HR changes, clinically insignificant pressure changes, and no adverse reactions. Additional studies will be needed to assure its safety in a larger and clinically more varied patient population.     

Serelaxin in acute heart failure: Most recent update on clinical and preclinical evidence

Heart failure continues to be a widely prevalent disease across the world, affecting millions of Americans annually. Acute heart failure (AHF) has a substantial effect on rising healthcare costs and is one of the major causes of morbidity and mortality. The search for new drugs for symptom relief and to improve long‐term outcomes in heart failure has led to development of serelaxin, a recombinant human relaxin‐2 hormone. Relaxin was discovered in pregnancy, but eventually found to have a number of other physiological actions, not only in pregnancy, but also in nonpregnant women and men. The actions of serelaxin are primarily via nitric oxide, leading to the observed vasodilatory effects, and increase in renal plasma flow. It has also been found to increase expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase (MMP)‐2 and MMP‐9. The antifibrotic and antiinflammatory effects of the drug also play a role in heart failure. In Phase II studies, serelaxin has shown reduction in pulmonary arterial pressure, pulmonary capillary wedge pressure, and NT‐proBNP. The recently published results of the RELAX‐AHF, a phase III clinical trial on serelaxin, has opened new avenues into our understanding of its effects in heart failure. The trial showed improvement in short‐term dyspnea scores and 180‐day mortality, but, interestingly, failed to show any improvement of the secondary endpoints of death or readmission at 60 days. Ongoing Phase III trials like RELAX‐AHF‐2 and RELAX‐AHF‐ASIA would explain these data better and improve understanding of the use of serelaxin in clinical practice. This article summarizes the most updated published preclinical and clinical study data on serelaxin, including pharmacokinetic, pharmacodynamic, safety studies in hepatic, renal impaired patients, Phase II and Phase III trials.