Discovery of a potent,selective, and orally bioavailable histamine H3 receptor antagonist SAR110068 for the treatment of sleep–wake disorders

Previous studies have shown that compound 1 displayed high affinity towards histamine H₃ receptor (H₃R), (human (h-H₃R), K_i = 8.6 nM, rhesus monkey (rh-H₃R), K_i = 1.2 nM, and rat (r-H₃R), K_i = 16.5 nM), but exhibited high affinity for hERG channel. Herein, we report the discovery of a novel, potent, and highly selective H₃R antagonist/inverse agonist 5a(SS) (SAR110068) with acceptable hERG channel selectivity and desirable pharmacological and pharmacokinetic properties through lead optimization sequence. The significant awakening effects of 5a(SS) on sleep–wake cycles studied by using EEG recording in rats during their light phase support its potential therapeutic utility in human sleep–wake disorders.     

Seasonal effects on the sleep–wake cycle,the rest–activity rhythm and quality of life for Japanese and Thai older people

Background: The sleep-wake cycle and the rest–activity rhythm are known to change with aging, and such changes have been implicated in higher levels of depression as well as an increased incidence of dementia. However, information supporting seasonal changes in the sleep–wake cycle, the rest–activity rhythm and quality of life in older community-dwelling people remains insufficient. The aim of the present study was to prospectively investigate seasonal effects on the sleep–wake cycle, the rest–activity rhythm and quality of life among older people living in areas of Japan or Thailand with different climate classifications.

Method: The survey was conducted from March 2016 to May 2017, and 109 participants were recruited from Japan and Thailand: 47 older people living in Akita prefecture, Japan, and 62 older people living in Chiang Mai or Nakhon Ratchasima, Thailand. According to the Köppen–Geiger classification of Asian climates comprising tropical, desert, steppe, temperate and subarctic climates, Akita prefecture, which is located in northern Japan, is classified as a humid subarctic climate, while the Thai study areas are classified as tropical savanna. To monitor parameters of the sleep–wake cycle during nighttime (e.g. total sleep time, sleep latency, sleep efficiency, awaking time and frequency of sleep interruptions) and to calculate parameters of the rest–activity rhythm over the 24 h profile (e.g., interdaily stability, intradaily variability, relative amplitude, mean of least active 5 h period and mean of most active 10 h period), all the participants from both countries wore an Actiwatch 2 device on their nondominant wrist continuously for 7 days during each local season. The World Health Organization Quality of Life Questionnaire-BREF (WHOQOL-BREF) was also assessed during each local season.

Results: The final sample size was 37 older people living in Akita prefecture, Japan, and 44 older people living in Thailand; these subjects completed the data collections during each local season. The dropout rates were 21% in Japan and 29% in Thailand. The results for the Japanese subjects showed a significantly shorter sleep time with higher levels of activity during the nighttime on summer (p < 0.001) and a fragmented rest–activity rhythm over the 24 h profile on winter (p < 0.001). The older Thai participants exhibited a poor state of night sleeping year-round, and a significant relationship was observed between seasonal variations in motor activity and the social domain of WHOQOL-BREF for each Thai season (|r| = 0.4, p < 0.01).

Conclusion: These findings provide new and important information regarding seasonal effects on the sleep–wake cycle, the rest–activity rhythm and quality of life in older community-dwelling people living in two different Asian climates. Consequently, clinical preventions targeting such seasonal variations might be useful for improving the quality of life of older Japanese and Thai individuals.     

Myotonic dystrophytype 1 – report of non-24-h sleep-wake disorder with excessive daytime sleepiness

ABSTRACT

Myotonic dystrophy (MD) is a neuromuscular disease with myotonia, progressive weakness, and involvement of CNS, heart, and gastrointestinal system. Excessive daytime sleepiness (EDS) in myotonic dystrophy type 1 (MD1) is related to sleep breathing diseases, restless leg syndrome, periodic limb movements during sleep and narcoleptic-like phenotype. However, authors highlight a central dysfunction of sleep regulation.

We describe a 26-year-old, female, MD1 patient with EDS. Sleep diary/actigraphy evidenced two different circadian periods with values of 1442 and 1522 min. Agomelatine, 50 mg at night, was prescribed with improvement of the circadian rhythm and complaints of sleepiness.

The identification of unanticipated causes of EDS, such as circadian rhythm disorders permits an appropriated treatment. As we know, it is the first relate of non-24-h sleep-wake disorder in patient with MD1. Sleep diary and actigraphy could be good options to investigate sleep-wake cycle disorder in patients with MD and EDS.     

Circadian rhythm of activity and sleep‐wakefulness in elderly institutionalized persons

Abstract

The circadian rhythms of blood pressure (BP) and heart rate (HR) were documented in 30 patients for a 24‐hour period before and during the 24 hours that included unilateral surgery for senile cataract or retinal detachment. The patients were premedicated with diazepam. Anaesthesia was induced at a fixed time (09.00) in all patients with thiopentone, and muscle relaxation was with pancuronium. Maintenance was with enflurane in 15 patients and with fentanyl and droperidol in the rest. Though the intraoperative changes in haemodynamic parameters were dissimilar with the two types of maintenance agents, but both types had a similar effect on the circadian rhythms of blood pressure and heart rate. Whereas preoperatively the BP and HR circadian rhythms were nearly in phase, with their peaks in the late morning to early afternoon, the postoperative rhythms underwent a dissociation to a phase shift in the BP 24‐h pattern. The phase effect may be hypothetically attributed to direct pharmacological actions or to masking effects.     

The effects of a split sleep–wake schedule on neurobehavioural performance and predictions of performance under conditions of forced desynchrony

Extended wakefulness, sleep loss, and circadian misalignment are factors associated with an increased accident risk in shiftwork. Splitting shifts into multiple shorter periods per day may mitigate these risks by alleviating prior wake. However, the effect of splitting the sleep–wake schedule on the homeostatic and circadian contributions to neurobehavioural performance and subjective assessments of one’s ability to perform are not known. Twenty-nine male participants lived in a time isolation laboratory for 13?d, assigned to one of two 28-h forced desynchrony (FD) schedules. Depending on the assigned schedule, participants were provided the same total time in bed (TIB) each FD cycle, either consolidated into a single period (9.33?h TIB) or split into two equal halves (2?×?4.67?h TIB). Neurobehavioural performance was regularly assessed with a psychomotor vigilance task (PVT) and subjectively-assessed ability was measured with a prediction of performance on a visual analogue scale. Polysomnography was used to assess sleep, and core body temperature was recorded to assess circadian phase. On average, participants obtained the same amount of sleep in both schedules, but those in the split schedule obtained more slow wave sleep (SWS) on FD days. Mixed-effects ANOVAs indicated no overall difference between the standard and split schedules in neurobehavioural performance or predictions of performance. Main effects of circadian phase and prior wake were present for both schedules, such that performance and subjective ratings of ability were best around the circadian acrophase, worst around the nadir, and declined with increasing prior wake. There was a schedule by circadian phase interaction for all neurobehavioural performance metrics such that performance was better in the split schedule than the standard schedule around the nadir. There was no such interaction for predictions of performance. Performance during the standard schedule was significantly better than the split schedule at 2?h of prior wake, but declined at a steeper rate such that the schedules converged by 4.5–7?h of prior wake. Overall, the results indicate that when the total opportunity for sleep per day is satisfactory, a split sleep–wake schedule is not detrimental to sleep or performance. Indeed, though not reflected in subjective assessments of performance capacity, splitting the schedule may be of some benefit, given its reduction of neurobehavioural impairment at night and its association with increased SWS. Therefore, for some industries that require operations to be sustained around the clock, implementing a split work–rest schedule may be of assistance.     

The effect of consecutive transmeridian flights on alertness,sleep–wake cycles and sleepiness: A case study

ABSTRACT

Travel across time zones disrupts circadian rhythms causing increased daytime sleepiness, impaired alertness and sleep disturbance. However, the effect of repeated consecutive transmeridian travel on sleep–wake cycles and circadian dynamics is unknown. The aim of this study was to investigate changes in alertness, sleep–wake schedule and sleepiness and predict circadian and sleep dynamics of an individual undergoing demanding transmeridian travel. A 47-year-old healthy male flew 16 international flights over 12 consecutive days. He maintained a sleep–wake schedule based on Sydney, Australia time (GMT + 10?h). The participant completed a sleep diary and wore an Actiwatch before, during and after the flights. Subjective alertness, fatigue and sleepiness were rated 4 hourly (08:00–00:00), if awake during the flights. A validated physiologically based mathematical model of arousal dynamics was used to further explore the dynamics and compare sleep time predictions with observational data and to estimate circadian phase changes. The participant completed 191?h and 159 736?km of flying and traversed a total of 144 time-zones. Total sleep time during the flights decreased (357.5?min actigraphy; 292.4?min diary) compared to baseline (430.8?min actigraphy; 472.1?min diary), predominately due to restricted sleep opportunities. The daily range of alertness, sleepiness and fatigue increased compared to baseline, with heightened fatigue towards the end of the flight schedule. The arousal dynamics model predicted sleep/wake states during and post travel with 88% and 95% agreement with sleep diary data. The circadian phase predicted a delay of only 34?min over the 16 transmeridian flights. Despite repeated changes in transmeridian travel direction and flight duration, the participant was able to maintain a stable sleep schedule aligned with the Sydney night. Modelling revealed only minor circadian misalignment during the flying period. This was likely due to the transitory time spent in the overseas airports that did not allow for resynchronisation to the new time zone. The robustness of the arousal model in the real-world was demonstrated for the first time using unique transmeridian travel.     

On the influence of prenatal hypoxia on formation of the orexinergic system and sleep–wake cycle in early ontogenesis of rats

The role of orexin in the organization of the sleep–wake cycle (SWC) is well known. The aim of this study was to examine the timing of the orexinergic system formation in rat postnatal ontogenesis and to assess the role of orexin A in the SWC organization under normal conditions and after prenatal hypoxia undergone on days 14 and 19 of embryogenesis. The SWC was investigated in 30-day-old rats with electrodes implanted into the somatosensory and occipital cortex. Immunoreactivity within the orexigenic structures of the lateral hypothalamus was analyzed. It was shown that in control 14-day-old animals the orexinergic structures were in their formative stage, whereas in 30-day-old rats they were already as formed as in adults. In 14-day-old rats, prenatal hypoxia evoked retarded formation of the orexinergic system. In 30-day-old animals, hypoxia undergone in the prenatal period increased the activity of the orexinergic system, which was higher in animals exposed to hypoxia on day 19 than on day 14 of gestation. In 30-day-old rats, these changes were reflected in the SWC formation in the form of shorter slow-wave sleep, more fitful sleep and increased number of transitions from slow- to fast-wave sleep. The results obtained are discussed in the light of the adaptive-compensatory role of the orexigenic system in postnatal ontogenesis after prenatal damage to the central nervous system.     

Disruptions in sleep–wake cycles in community-dwelling cancer patients receiving palliative care and their correlates

Significant disruptions in sleep–wake cycles have been found in advanced cancer patients in prior research. However, much remains to be known about specific sleep–wake cycle variables that are impaired in patients with a significantly altered performance status. More studies are also needed to explore the extent to which disrupted sleep–wake cycles are related to physical and psychological symptoms, time to death, maladaptive sleep behaviors, quality of life and 24-h light exposure. This study conducted in palliative cancer patients was aimed at characterizing patients’ sleep–wake cycles using various circadian parameters (i.e. amplitude, acrophase, mesor, up-mesor, down-mesor, rhythmicity coefficient). It also aimed to compare rest–activity rhythm variables of participants with a performance status of 2 vs. 3 on the Eastern Cooperative Oncology Group scale (ECOG) and to evaluate the relationships of sleep–wake cycle parameters with several possible correlates. The sample was composed of 55 community-dwelling cancer patients receiving palliative care with an ECOG of 2 or 3. Circadian parameters were assessed using an actigraphic device for seven consecutive 24-h periods. A light recording and a daily pain diary were completed for the same period. A battery of self-report scales was also administered. A dampened circadian rhythm, a low mean activity level, an early mean time of peak activity during the day, a late starting time of activity during the morning and an early time of decline of activity during the evening were observed. In addition, a less rhythmic sleep–wake cycle was associated with a shorter time to death (from the first home visit) and with a lower 24-h light exposure. Sleep–wake cycles are markedly disrupted in palliative cancer patients, especially, near the end of life. Effective non-pharmacological interventions are needed to improve patients’ circadian rhythms, including perhaps bright light therapy.     

Can dim light melatonin onset be predicted by the timing of sleep in patients with possible circadian sleep-wake rhythm disorders?

Dim Light Melatonin Onset (DLMO) is a reliable marker of the endogenous circadian rhythm. To determine if sleep timing can predict DLMO, we investigated the relationship between sleep timing and DLMO in patients in various circadian sleep-wake rhythm disorders (CSWRDs), ages and genders. We found that correlations were only moderate between DLMO and sleep-onset in the complete data-set, but they increased in patients with delayed sleep-wake phase disorder (DSWPD), DSWPD patients with a regular sleep pattern and patients with advanced sleep-wake phase disorder (ASWPD); the levels of correlation were r = 0.542, 0.657, 0.728 and 0.814, respectively. In DSWPD patients with a regular sleep pattern, mid-sleep strongly correlated (r = 0.839) with DLMO. Correlation in other CSRWDs was not significant. DLMO, sleep-onset and age were most discriminated factors between the various CSRWDs. Estimation of DLMO is only possible in patients with ASWPD and in DSWPD patients with a regular sleep pattern.     

Evolutionary aspects of sleep–wake cycle development in vertebrates (Modern state of the I.G. Karmanova’s sleep evolution theory)

The genetic basis of rest–activity circadian alternation in animal behavior is considered in the evolutionary range from bacteria to mammals. We scrutinize various concepts of sleep development in the animal world evolution as well as the I.G. Karmanova’s theory of the sleep–wake cycle evolution in vertebrates, beginning from wakefulness–primary sleep (or protosleep) in fish and amphibians through wakefulness–intermediate sleep in reptiles to wakefulness–slow wave sleep (SWS) and paradoxical sleep (PS) in birds and mammals. Primary sleep is represented by the three major sleep-like immobility states: catalepsy, catatonia and cataplexy. The main behavioral, somatovegetative and neurophysiological characteristics of primary sleep and the ancient activation pattern during primary sleep are described. The issues of which of these sleep manifestations are homologous to SWS, PS, hibernation and stress response are discussed. In conclusion, the general diagram of sleep evolution in vertebrates is presented, and the I.G. Karmanova’s contribution to evolutionary somnology is highlighted.     

Tadpole–odonate larvae interactions: influence of body size and diel rhythm

Several studies have shown that prey and predator body size may affect the outcome of predator–prey interactions. However, few studies have taken in account the changes on predator–prey interactions over 24 h. In a tropical freshwater system I evaluated how predator and prey size, and their diel rhythm in activity influenced the interaction between Physalaemus pustulosus tadpoles and dragonfly larvae. Tadpoles of different size classes were exposed to two size classes of the dragonfly larvae Rhionaeschna spec. Feeding trials were conducted during day and night. Tadpole activity showed a diel rhythm and affected size-selective predation of the smallest dragonfly larvae, but not of the larger ones. Predator and prey size had a significant effect on the prey survivorship and prey size had a significant effect on the preference of the predator. The interaction between both factors was significant, indicating that they did not operate independently. I conclude that the predator–prey interactions between odonate larvae and anuran tadpoles were mainly affected by the size of the prey and the predator, and less by the diel activity pattern of the prey.     

Association of 24 h–systolic blood pressure variability and cardiovascular disease in patients with obstructive sleep apnea

Background

To evaluate association of 24 h–systolic blood pressure (SBP) variability and obstructive sleep apnea (OSA) as defined by the apnea-hypopnea index ≥5/h; and association of 24 h–SBP variability and prevalent cardiovascular disease (CVD) in OSA patients.

Methods

Participants underwent polysomongraphy to evaluate the presence of OSA, and 24 h–ambulatory blood pressure monitoring was applied to evaluate 24 h–SBP variability as indexed by weighted 24 h–standard deviation (SD) of SBP. Between-group differences were evaluated in participants with and without OSA. Participants with OSA were divided into high and low 24 h–SBP variability groups and between-group differences were evaluated.

Results

Mean age of 384 participants was 50 years old and 42.2% had OSA. Mean 24 h–systolic/diastolic BP were 130/78 mmHg, with mean weighted 24 h–SD of systolic/diastolic BP were 12.9/7.3 mmHg. Compared to those without OSA, OSA participants had higher clinic-, 24 h-, daytime- and nighttime-SBP, and weighted 24 h, daytime- and nighttime-SD of SBP. Age, prevalent CVD and OSA, usage of angiotensin converting enzyme inhibitor/angiotensin receptor blocker, calcium channel blocker and diuretic were significantly associated with 24 h–SBP variability. In OSA patients, compared to those with low variability, participants with high variability had higher weighted 24 h, daytime- and nighttime-SD of SBP. After adjusted for covariates including clinic-SBP and 24 h–SBP, per 1-SD increment weighted 24 h–SD of SBP was associated with 21% increased prevalent CVD.

Conclusions

Patients with newly-diagnosed OSA have higher 24 h–SBP variability compared to those without OSA; in OSA patients, increased 24 h–SBP variability is associated with increased prevalence of CVD.

     

Epidemiological study on chronotype among preschool children in Japan: Prevalence,sleep–wake patterns,and associated factors

Our current 24-h society and the weekday–weekend switch of our social clocks may affect young children’s sleep and circadian rhythms. However, such evidence is scarce. We conducted a nationwide epidemiological study of sleep and health in preschool children aged 3–5 years attending kindergarten or childcare centers in Japan, using stratified one-stage cluster sampling. The target population was 2 969 627 individuals (as of 1 April 2013). The Children’s ChronoType Questionnaire was used to measure chronotypes (morning (M)-type, neither (N)-type and evening (E)-type), and weekday and weekend sleep–wake parameters. Randomly sampled population estimates were obtained via respondents with a person-level weight, which accounted for survey responses and poststratification. Standard errors and 95% confidence intervals were adjusted for the complex survey design using jackknife estimation. A linear regression model of the correlation between chronotype and sleep–wake parameters and a multivariate logistic regression model for the links between chronotype and putative associated factors were used for statistical analyses. The estimated prevalence of M-, N- and E-types were 31.6%, 55.9% and 10.0%, respectively. The corresponding numbers of children were 937 910, 1 659 574 and 296 083. The remaining 2.5% was not specified. The proportions of children who woke up by themselves during the weekdays were 55.1%, 43.0% and 1.9% for M-, N- and E-types, respectively. Overall, bedtime, sleep onset time, wake-up time and get-up time during the weekdays were 21:04, 21:26, 6:55 and 6:59, respectively. Nocturnal sleep period, time in bed (TIB) and 24 h TIB (TIB and nap) during the weekdays were 9.49, 9.93 and 10.55 h, respectively. Sleep–wake timings were significantly and linearly delayed from M-, N-, to E-types (p < 0.001). The weekday 24 h TIB (10.47–10.66 h) and weekend nocturnal sleep period (9.58–9.76 h) did not differ significantly among chronotypes. For E-types, socially advanced weekdays rising times (approximately 1 h) caused nocturnal sleep deficit (0.57 h). Children’s socially scheduled times (e.g. start and finish times, mealtimes and daytime nap) and their parents’ diurnal preferences had significant adjusted odds ratios among E-types, while the significant unadjusted odds ratios for morning sunlight and multimedia exposure disappeared. These results suggest the importance of chronobiologically planned sleep discipline at home as well as assessment of socially scheduled times in children.     

Effects of radiation emanating from base transceiver station and mobile phone on sleep,circadian rhythm and cognition in humans – a review

The electromagnetic fields (EMF) are ubiquitous. The base transceiver station (BTS) and mobile phones (MPs) contribute to the generation of EMF around their locations and are regarded as important sources of non-ionizing radiations. The use of mobile phone has increased dramatically in recent years so also the skepticism regarding its effects. In this review, we have made an attempt to scan the key research papers those aimed at elucidating the effects of EMF starting from extreme low frequency (ELF) to radio frequency (RF) through low frequency (LF). We have selected papers that dealt with the effects of radiations emanating from the BTS and MPs on human sleep, circadian rhythm, and cognition. Mostly, we have concentrated on papers published in the last 15 years. We came across conflicting reports. The findings reported in many papers suggest that the exposure to EMF has potentiality to compromise parameters related to sleep quality; in contrast, there are several reports those have given a clean sheet to the EMF exposure. The effects of EMF on circadian rhythms also remain inconclusive. The EMF exposure while did not produce any effect on circadian rhythm of heart rate and blood chemistry, it modulated the rhythms in cortisol and melatonin characterized by a decline in their 24-h circulating levels. The effects of exposure to EMF on cognitive parameters, like performance and memory, are also equivocal. The existing contradictory findings could be attributed to inter-individual variability in tolerance, gender-, and age-dependent differences in response, latitudinal differences in efficacy, variability among employed methodologies and differences in specific absorption rate, frequency of the mobile phone usage, and interaction of EMF with other physiological and environmental factors, among others. The future research should be carried out with added focus on elucidating the modulatory effects of these factors to put an end to the existing controversies on the biological effects of low/RF EMF radiations.     

Duration of activity and period of circadian activity–rest rhythm in a photoperiod-dependent primate,Microcebus murinus

Under free-running conditions, the grey lesser mouse lemur expresses a circadian activity–rest rhythm with a period particularly short (22.50 ± 0.6 h) for a mammal. Light exerts a strong suppressive effect upon activity. After transfer from nycthemeral to free-running conditions the duration of activity was systematically increased. This extension took place in the first cycle and was characterized by both a phase advance in activity onset and an even larger phase delay in activity offset. This phenomenon was more pronounced after long day entrainment. Any shift of the light–dark cycle was followed the next day by a corresponding shift in activity onset. The phase response curve pattern was similar to that already described for nocturnal mammals. Due to the strength of light as a zeitgeber and the plasticity of the response to photic conditions, the mouse lemur appears as a convenient species for chronobiological studies on non-human primates.     

The impact of a delayed sleep–wake schedule on depression is greater in women – A web-based cross-sectional study in Japanese young adults

Sleep-related problems, such as symptoms of insomnia, daytime sleepiness, shorter sleep duration, or a delayed sleep–wake schedule, are known to be risk factors for depression. In general, depression is more prevalent in women than in men, but sleep-related problems do not necessarily show similar gender predominance. Hence, it can be speculated that the impact of sleep-related problems on the development process of depression differs between genders; however, so far, few studies have focused on this issue. The aim of this study was to clarify gender differences in the rates of depression of people with the above sleep-related problems, and to examine gender differences in factors associated with depression in Japanese young adults. A web-based questionnaire survey comprising assessments of demographic variables, sleep-related variables (bed time, wake time, sleep onset latency, frequency of difficulty in initiating sleep and that in maintaining sleep, i.e. symptom components of insomnia, and daytime sleepiness), and the 12-item version of the Center for Epidemiologic Studies Depression Scale was administered to 2502 participants (males:females?=?1144:1358, age range?=?19–25 years). Female predominance in the rate of depression was observed only in subjects with a delayed sleep–wake schedule (χ²₍₁₎?=?15.44, p?<?0.001). In men, daytime sleepiness and difficulty in initiating sleep were significantly associated with depression (odds ratio [OR]?=?2.39, 95% confidence interval [CI]?=?[1.69, 3.39], p?<?0.001; OR?=?3.50, 95% CI?=?[2.29, 5.35], p?<?0.001, respectively), whereas in women, significant associations were found between depression and a delayed sleep–wake schedule (OR?=?1.75, 95% CI?=?[1.28, 2.39], p?<?0.001), daytime sleepiness (OR?=?2.13, 95% CI?=?[1.60, 2.85], p?<?0.001), and difficulty in initiating sleep (OR?=?4.37, 95% CI?=?[3.17, 6.03], p?<?0.001). These results indicate that in younger generations, the impact of a delayed sleep–wake schedule on the development of depression is greater in women; specifically, women are vulnerable to depression when they have an eveningness-type lifestyle, which is possibly attributable to the female-specific intrinsic earlier and shorter circadian rhythm. These results suggest the necessity of gender-based approaches to treating sleep-related problems for alleviating or preventing depressive symptoms in young adults.     

Analysis of lignin–carbohydrate and lignin–lignin linkages after hydrolase treatment of xylan–lignin,glucomannan–lignin and glucan–lignin complexes from spruce wood

Xylan–lignin (XL), glucomannan–lignin (GML) and glucan–lignin (GL) complexes were isolated from spruce wood, hydrolyzed with xylanase or endoglucanase/β-glucosidase, and analyzed by analytical pyrolysis and 2D-NMR. The enzymatic hydrolysis removed most of the polysaccharide moieties in the complexes, and the lignin content and relative abundance of lignin–carbohydrate linkages increased. Analytical pyrolysis confirmed the action of the enzymatic hydrolysis, with strong decreases of levoglucosane and other carbohydrate-derived products. Unexpectedly it also revealed that the hydrolase treatment alters the pattern of lignin breakdown products, resulting in higher amounts of coniferyl alcohol. From the anomeric carbohydrate signals in the 2D-NMR spectra, phenyl glycoside linkages (undetectable in the original complexes) could be identified in the hydrolyzed GML complex. Lower amounts of glucuronosyl and benzyl ether linkages were also observed after the hydrolysis. From the 2D-NMR spectra of the hydrolyzed complexes, it was concluded that the lignin in GML is less condensed than in XL due to its higher content in β-O-4′ ether substructures (62 % of side chains in GML vs 53 % in XL) accompanied by more coniferyl alcohol end units (16 vs 13 %). In contrast, the XL lignin has more pinoresinols (11 vs 6 %) and dibenzodioxocins (9 vs 2 %) than the GML (and both have ~13 % phenylcoumarans and 1 % spirodienones). Direct 2D-NMR analysis of the hydrolyzed GL complex was not possible due to its low solubility. However, after sample acetylation, an even less condensed lignin than in the GML complex was found (with up to 72 % β-O-4′ substructures and only 1 % pinoresinols). The study provides evidence for the existence of structurally different lignins associated to hemicelluloses (xylan and glucomannan) and cellulose in spruce wood and, at the same time, offers information on some of the chemical linkages between the above polymers.     

Molecular mechanism of an adverse drug–drug interaction of allopurinol and furosemide in gout treatment

Gout patients receiving a combination of allopurinol and furosemide require higher allopurinol doses to achieve the target serum urate (SU) of <6 mg/dl (Stamp et al., 2012) [1]. Our study aimed to identify the molecular basis for this observation. We used a fluorimetric assay to determine the impact of furosemide and oxypurinol (the active metabolite of allopurinol) on xanthine oxidase (XO) activity. Immunoblot analysis quantified expression of XO and AMP-kinase (AMPK) in drug-treated human liver (HepG2) and primary kidney (HRCE) cells. In silico analysis identified miR-448 as a potential XO-regulator, whose expression level in HepG2 cells was examined by qPCR.