Simultaneous Quantification of 3'- and 6'-Sialyllactose in Rat Plasma Using Liquid Chromatography-Tandem Mass Spectrometry and Its Application to a Pharmacokinetic Study.

Sialyllactose (SL), an acidic oligosaccharide, has immune-protective effects against pathogens and helps with the development of the immune system and intestinal microorganisms. To elucidate the pharmacokinetic characterization after oral administration to rats, the simultaneous quantification method for 3'-SL and 6'-SL in rat plasma was validated, using liquid chromatography-tandem mass spectrometry (LC-MS/MS) in an electrospray ionization (ESI) mode. Several types of columns [C18, amide, and hydrophilic interaction liquid chromatography (HILIC) phase] were used to separate the peaks of 3'-SL and 6'-SL, which improved chromatographic selectivity. Ultimately, the HILIC phase column had a good peak shape and quick resolution, with a mobile phase comprising ammonium acetate buffer and acetonitrile obtained by gradient elution. In addition, the simultaneous quantification of 3'-SL and 6'-SL in rat plasma samples were adequately applied to pharmacokinetic study.

Lactose monohydrate flow characterization using shear cell method.

The flow properties of pharmaceutical powders have a great importance in the manufacturing of solid dosage forms. In order to ensure the performance in the production line this parameter must be determined. There are several methods described in European Pharmacopeia that are used to measure these properties. Some of them were used in this study and the results obtained from conventional methods (Conv) and shear cell using the powder flow tester (PFT) showed differences that were more evident in fractions with smaller particle size (F < 63) and for bulk powder (FTotal). The various powder behaviors showed to be related with the size of the particles. An increase of the ffc (Flow Index) was observed with the increase of the particle size. It was also found for the different fractions that the ffc always increases with increasing major principal consolidation stress (σ1). This study shown to be predictive because it also allowed the behavior profiles of other LactMN fractions to be known by interpolation of the median size (Dv50) or σ1 values ranged between the studied intervals. Furthermore, it was also observed that ffc of the FTotal was similar to the F < 63, showing the same behavior under σ1. The occurrence of caking was not observed.

Lactose, Maltose, and Sucrose in Health and Disease.

SCOPE: This review represents a focus on the structure and properties of the common nutritional disaccharides (lactose, maltose, and sucrose) in health and disease. The aim is to provide a comprehensive reference source related to the role of disaccharides in human nutrition. METHODS AND RESULTS: Key reference sources are searched, including Web of Science, PubMed, Science Direct, and Wiley Online, and key reference works are selected to support the factual basis of the text where interpretations and relevance of the works are discussed in the review. There are key nutritional health benefits of receiving dietary energy in the form of sugars, but equally life-threatening issues exist associated with constant/excess consumption. These issues are discussed together with genetic disorders, which impact upon health associated with consumption of the disaccharides (e.g., specific disaccharide intolerance due to deficiency of relevant digestive enzymes). CONCLUSIONS: As the three common dietary disaccharides (lactose, maltose, and sucrose) are consumed on a very regular basis in the human diet, it is critical to understand insofar as possible their role in health and disease. This review provides an insight into the structure and properties of these molecules in health and disease.

Development of M10, myricetin-3-O-ß-d-lactose sodium salt, a derivative of myricetin as a potent agent of anti-chronic colonic inflammation.

Myricetin is a natural dietary flavonoid compound with multiple activities, such as anti-oxidant, anti-inflammatory, anti-carcinogenic and anti-proliferative effects. However, myricetin exhibited substantial limitations, such as poor water-solubility, and low stability in body when it was administrated by oral. To solve these problems, we designed and synthesized a series of derivatives based on the structure of myricetin. M10 was produced by adding a hydrophilic glycosylation group and then forming a sodium salt derivative, which exhibited excellent water-solubility (>100 mg/mL), and better stability in Wistar rat plasma and liver microsomes. In vivo study, M10 exhibited higher efficacy than myricetin and mesalazine in a dextran sulfate sodium (DSS) induced mice model with ulcerative colitis. In addition, M10 also exhibited high safety (LD50 > 5 g/kg) in mice. Based on these results, M10 could be developed as a potential therapeutic agent for treatment of ulcerative colitis.

Highly Soluble Drugs Directly Granulated by Water Dispersions of Insoluble Eudragit® Polymers as a Part of Hypromellose K100M Matrix Systems.

The aim of the present study was to investigate the suitability of insoluble Eudragit® water dispersions (NE, NM, RL, and RS) for direct high-shear granulation of very soluble levetiracetam in order to decrease its burst effect from HPMC K100M matrices. The process characteristics, ss-NMR analysis, in vitro dissolution behavior, drug release mechanism and kinetics, texture profile analysis of the gel layer, and PCA analysis were explored. An application of water dispersions directly on levetiracetam was feasible only in a multistep process. All prepared formulations exhibited a 12-hour sustained release profile characterized by a reduced burst effect in a concentration-dependent manner. No effect on swelling extent of HPMC K100M was observed in the presence of Eudragit®. Contrary, higher rigidity of formed gel layer was observed using combination of HPMC and Eudragit®. Not only the type and concentration of Eudragit®, but also the presence of the surfactant in water dispersions played a key role in the dissolution characteristics. The dissolution profile close to zero-order kinetic was achieved from the sample containing levetiracetam directly granulated by the water dispersion of Eudragit® NE (5% of solid polymer per tablet) with a relatively high amount of surfactant nonoxynol 100 (1.5%). The initial burst release of drug was reduced to 8.04% in 30 min (a 64.2% decrease) while the total amount of the released drug was retained (97.02%).

Spray-Dried PulmoSphere™ Formulations for Inhalation Comprising Crystalline Drug Particles.

Over the past 20 years, solution-based spray dried powders have transformed inhaled product development, enabling aerosol delivery of a wider variety of molecules as dry powders. These include inhaled proteins for systemic action (e.g., Exubera®) and high-dose inhaled antibiotics (e.g., TOBI® Podhaler™). Although engineered particles provide several key advantages over traditional powder processing technologies (e.g., spheronized particles and lactose blends), the physicochemical stability of the amorphous drug present in these formulations brings along its own unique set of constraints. To this end, a number of approaches have been developed to maintain the crystallinity of drugs throughout the spray drying process. One approach is to spray dry suspensions of micronized drug(s) from a liquid feed. In this method, minimization of drug particle dissolution in the liquid feed is critical, as dissolved drug is converted into amorphous domains in the spray-dried drug product. The review explores multiple formulation and engineering strategies for decreasing drug dissolution independent of the physicochemical properties of the drug(s). Strategies to minimize particle dissolution include spray blending of particles of different compositions, formation of respirable agglomerates of micronized drug with small porous carrier particles, and use of common ions. The formulations extend the range of doses that can be delivered with a portable inhaler from about 100 ng to 100 mg. The spray-dried particles exhibit significant advantages in terms of lung targeting and dose consistency relative to conventional lactose blends, while still maintaining the crystallinity of drug(s) in the formulated drug product.

Correlation between the viscoelastic properties of the gel layer of swollen HPMC matrix tablets and their in vitro drug release.

Drug release from hydroxypropyl methylcellulose (HPMC) hydrophilic matrix tablets is controlled by drug diffusion through the gel layer of the matrix-forming polymer upon hydration, matrix erosion or combination of diffusion and erosion mechanisms. In this study, the relationship between viscoelastic properties of the gel layer of swollen intact matrix tablets and drug release was investigated. Two sets of quetiapine fumarate (QF) matrix tablets were prepared using the high viscosity grade HPMC K4M at low (70 mg/tablet) and high (170 mg/tablet) polymer concentrations. Viscoelastic studies using a controlled stress rheometer were performed on swollen matrices following hydration in the dissolution medium for predetermined time intervals. The gel layer of swollen tablets exhibited predominantly elastic behavior. Results from the in vitro release study showed that drug release was strongly influenced by the viscoelastic properties of the gel layer of K4M tablets, which was further corroborated by results from water uptake studies conducted on intact tablets. The results provide evidence that the viscoelastic properties of the gel layer can be exploited to guide the selection of an appropriate matrix-forming polymer, to better understand the rate of drug release from matrix tablets in vitro and to develop hydrophilic controlled-release formulations.

Major human milk oligosaccharides are absorbed into the systemic circulation after oral administration in rats.

Human milk oligosaccharides (HMO) are involved in many biological functions influencing infant health. Although HMO act locally at the intestine, recent evidence has demonstrated that HMO are partially incorporated into the systemic circulation of breast-fed infants. In the last few years, a large amount of research has been conducted using preclinical models to uncover new biological functions of HMO. The aim of this study was to evaluate the absorption and urine excretion of HMO in rats. We administered a single oral dose of the following HMO: 2'-fucosyllactose (2'-FL), 6'-sialyllactose and lacto-N-neotetraose at different concentrations to adult rats. The time course of absorption of HMO into the bloodstream and their appearance in urine was studied. Our results showed that rats, similar to human infants, are able to effectively absorb a portion of HMO from the intestine into plasma and to excrete them in urine. On the basis of this, we also conducted a specific kinetic absorption study with 2'-FL, the most predominant HMO in human milk, in 9-11-d-old rat pups. Our results confirmed that a significant amount of 2'-FL was absorbed into the systemic circulation and subsequently excreted in urine during lactation in rats in a dose-depended manner. We also found basal levels of these HMO in plasma and urine of adult rats as well as rat pups as a natural result of nursing. Our data suggest that the rat may be a useful preclinical model that provides new insights into the metabolism and functions of HMO.

Intestinal Barrier Maturation in Very Low Birthweight Infants: Relationship to Feeding and Antibiotic Exposure.

OBJECTIVE: To test the hypothesis that feeding and antibiotic exposures affect intestinal barrier maturation in preterm infants, we serially measured intestinal permeability (IP) biomarkers in infants <33 weeks gestation (gestational age [GA]) during the first 2 weeks of life. STUDY DESIGN: Eligible infants <33 weeks GA were enrolled within 4 days of birth in a prospective study of IP biomarkers (NCT01756040). Study participants received the nonmetabolized sugars lactulose/rhamnose enterally on study days 1, 8, and 15 and lactulose/rhamnose were measured in urine by high-performance liquid chromatography. Serum zonulin and fecal alpha-1-anti-trypsin, 2 other IP markers, were measured by semiquantitative Western blot and ELISA, respectively. RESULTS: In a cohort of 43 subjects, the lactulose/rhamnose ratio was increased on day 1 and decreased over 2 weeks, but remained higher in infants born at ≤28 weeks of gestation compared with IP in infants born at >28 weeks of gestation. Exclusive breastmilk feeding was associated with more rapid maturation in intestinal barrier function. A cluster analysis of 35 subjects who had urine samples from all time points revealed 3 IP patterns (cluster 1, normal maturation: n = 20 [57%]); cluster 2, decreased IP during the first week and subsequent substantial increase: n = 5 [14%]); and cluster 3, delayed maturation: n = 10 [29%]). There were trends toward more prolonged antibiotic exposure (P = .092) and delayed initiation of feeding ≥4 days (P = .064) in infants with abnormal IP patterns. CONCLUSIONS: Intestinal barrier maturation in preterm infants is GA and postnatal age dependent, and is influenced by feeding with a maturational effect of breastmilk feeding and possibly by antibiotic exposures. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01756040.

Improving Dry Powder Inhaler Performance by Surface Roughening of Lactose Carrier Particles.

PURPOSE: This study investigated the impact of macro-scale carrier surface roughness on the performance of dry powder inhaler (DPI) formulations. METHODS: Fluid-bed processing and roller compaction were explored as processing methods to increase the surface roughness (Ra) of lactose carrier particles. DPI formulations containing either (a) different concentrations of fine lactose at a fixed concentration of micronized drug (isoniazid) or (b) various concentrations of drug in the absence of fine lactose were prepared. The fine particle fraction (FPF) and aerodynamic particle size of micronized drug of all formulations were determined using the Next Generation Impactor. RESULTS: Fluid-bed processing resulted in a modest increase in the Ra from 562 to 907 nm while roller compaction led to significant increases in Ra > 1300 nm. The roller compacted carriers exhibited FPF > 35%, which were twice that of the smoothest carriers. The addition of up to 5%, w/w of fine lactose improved the FPF of smoother carriers by 60-200% whereas only < 30% increase was observed in the rough carriers. Analysis of the FPF in tandem with shifts in the mass median aerodynamic diameter of dispersed drug suggested that the finest drug particles were entrapped on rougher surfaces while larger drug particles were dispersed in the air. CONCLUSIONS: The results showed that the processing of lactose carrier particles by roller compaction was immensely beneficial to improving DPI performance, primarily due to increased surface roughness at the macro-scale.

Clinical evaluation, biochemistry and genetic polymorphism analysis for the diagnosis of lactose intolerance in a population from northeastern Brazil.

OBJECTIVE: This work aimed to evaluate and correlate symptoms, biochemical blood test results and single nucleotide polymorphisms for lactose intolerance diagnosis. METHOD: A cross-sectional study was conducted in Fortaleza, Ceará, Brazil, with a total of 119 patients, 54 of whom were lactose intolerant. Clinical evaluation and biochemical blood tests were conducted after lactose ingestion and blood samples were collected for genotyping evaluation. In particular, the single nucleotide polymorphisms C>T-13910 and G>A-22018 were analyzed by restriction fragment length polymorphism/polymerase chain reaction and validated by DNA sequencing. RESULTS: Lactose-intolerant patients presented with more symptoms of flatulence (81.4%), bloating (68.5%), borborygmus (59.3%) and diarrhea (46.3%) compared with non-lactose-intolerant patients (p<0.05). We observed a significant association between the presence of the alleles T-13910 and A-22018 and the lactose-tolerant phenotype (p<0.05). After evaluation of the biochemical blood test results for lactose, we found that the most effective cutoff for glucose levels obtained for lactose malabsorbers was <15 mg/dL, presenting an area under the receiver operating characteristic curve greater than 80.3%, with satisfactory values for sensitivity and specificity. CONCLUSIONS: These data corroborate the association of these single nucleotide polymorphisms (C>T-13910 and G>A-22018) with lactose tolerance in this population and suggest clinical management for patients with lactose intolerance that considers single nucleotide polymorphism detection and a change in the biochemical blood test cutoff from <25 mg/dL to <15 mg/dL.

Clinical evaluation, biochemistry and genetic polymorphism analysis for the diagnosis of lactose intolerance in a population from northeastern Brazil

OBJECTIVE: This work aimed to evaluate and correlate symptoms, biochemical blood test results and single nucleotide polymorphisms for lactose intolerance diagnosis. METHOD: A cross-sectional study was conducted in Fortaleza, Ceará, Brazil, with a total of 119 patients, 54 of whom were lactose intolerant. Clinical evaluation and biochemical blood tests were conducted after lactose ingestion and blood samples were collected for genotyping evaluation. In particular, the single nucleotide polymorphisms C>T-13910 and G>A-22018 were analyzed by restriction fragment length polymorphism/polymerase chain reaction and validated by DNA sequencing. RESULTS: Lactose-intolerant patients presented with more symptoms of flatulence (81.4%), bloating (68.5%), borborygmus (59.3%) and diarrhea (46.3%) compared with non-lactose-intolerant patients (p<0.05). We observed a significant association between the presence of the alleles T-13910 and A-22018 and the lactose-tolerant phenotype (p<0.05). After evaluation of the biochemical blood test results for lactose, we found that the most effective cutoff for glucose levels obtained for lactose malabsorbers was <15 mg/dL, presenting an area under the receiver operating characteristic curve greater than 80.3%, with satisfactory values for sensitivity and specificity. CONCLUSIONS: These data corroborate the association of these single nucleotide polymorphisms (C>T-13910 and G>A-22018) with lactose tolerance in this population and suggest clinical management for patients with lactose intolerance that considers single nucleotide polymorphism detection and a change in the biochemical blood test cutoff from <25 mg/dL to <15 mg/dL.

Preclinical pharmacokinetic evaluation and metabolites identification of methyl salicylate-2-O-ß-d-lactoside in rats using LC-MS/MS and Q-TOF-MS methods.

Methyl salicylate-2-O-ß-d-lactoside (MSL) is a natural salicylate derivative from the traditional Chinese medicine of Gaultheria yunnanensis (Franch.) Rehder (G. yunnanensis). As a non-steroidal anti-inflammatory drug (NSAID), MSL exerts a significant anti-arthritis effect but hardly has any gastrointestinal toxicity. In this paper, the pharmacokinetics, distribution, excretion and identification of MSL and its metabolites are described following rat oral and intravenous administration. The biological samples were quantified by UPLC-MS/MS and the metabolites in urine and feces were identified by using Q-TOF-MS. These results will support future investigations leading to clinical development of this drug.

Lack of dose dependent kinetics of methyl salicylate-2-O-ß-D-lactoside in rhesus monkeys after oral administration.

ETHNOPHARMACOLOGICAL RELEVANCE: Methyl salicylate-2-O-ß-d-lactoside (MSL) is one of the main active components isolated from Gaultheria yunnanensis, which is a traditional Chinese medicine used to treat arthritis and various aches and pains. Pharmacological researches showed that MSL had various effective activities in both in vivo and in vitro experiments. However, the pharmacokinetics features and oral bioavailability of MSL in primates were not studied up to now. AIM: To study the pharmacokinetics of different doses of MSL in rhesus monkeys and investigate the absolute bioavailability of MSL after oral administration. MATERIALS AND METHODS: Male and female rhesus monkeys were either orally administrated with MSL 200, 400 and 800 mg/kg or received an intravenous dose of 20mg/kg randomly. The levels of MSL and salicylic acid (SA) in plasma were simultaneous measured by a simple, sensitive and reproducible high performance liquid chromatography method. RESULTS: Mean peak plasma concentration values for groups treated with 200, 400 and 800 mg/kg doses ranged from 48.79 to 171.83 µg/mL after single-dose oral administration of MSL, and mean area under the concentration-time curve values ranged from 195.16 to 1107.76 µg/mL h. Poor linearity of the kinetics of SA after oral administration of MSL was observed in the regression analysis of the Cmax-dose plot (r(2)=0.812), CL-dose plot (r(2)=0.225) and AUC(0-t)-dose plot (r(2)=0.938). Absolute bioavailability of MSL was assessed to be 118.89 ± 57.50, 213.54 ± 58.98 and 168.72 ± 76.58%, respectively. CONCLUSIONS: Bioavailability of MSL after oral administration in rhesus monkeys was measured for the first time. Pharmacokinetics parameters did not appear to be dose proportional among the three oral doses of treatments, and MSL showed an apparent absolute bioavailability in excess of 100% in rhesus monkeys based on the present study. In addition, a rapid, sensitive and reliable HPLC method was established and demonstrated for the research of traditional Chinese medicine in this study.

The effect of HPMC particle size on the drug release rate and the percolation threshold in extended-release mini-tablets.

The particle size of HPMC is a critical factor that can influence drug release rate from hydrophilic matrix systems. Percolation theory is a statistical tool which is used to study the disorder of particles in a lattice of a sample. The percolation threshold is the point at which a component is dominant in a cluster resulting in significant changes in drug release rates. Mini-tablets are compact dosage forms of 1.5-4 mm diameter, which have potential benefits in the delivery of drug to some patient groups such as pediatrics. In this study, the effect of HPMC particle size on hydrocortisone release and its associated percolation threshold for mini-tablets and tablets was assessed. For both mini-tablets and tablets, large polymer particles reduced tensile strength, but increased the drug release rate and the percolation threshold. Upon hydration, compacts with 45-125 µm HPMC particles formed a strong gel layer with low porosity, reducing hydrocortisone release rates. In comparison, faster drug release rates were obtained when 125-355 µm HPMC particles were used, due to the greater pore sizes that resulted in the formation of a weaker gel. Using 125-355 µm HPMC particles increased the percolation threshold for tablets and to a greater extent for mini-tablets. This work has demonstrated the importance of HPMC particle size in ER matrices, the effects of which are even more obvious for mini-tablets.

Prevalence of Lactose Malabsorption and Lactose Intolerance in Pediatric Patients with Selected Gastrointestinal Diseases.

BACKGROUND: Lactase is an enzyme involved in the hydrolysis of lactose. Deficiency of the enzyme (hypolactasia) may be determined genetically or arise secondarily to disease of small intestine. Under this condition, lactose enters the colon where it is fermented by intestinal microflora and turns to gases and short-chain fatty acids, causing gastrointestinal symptoms known as lactose intolerance (LI). OBJECTIVES: To investigate the incidence of lactose malabsorption (LM), LI and the coexistence of these two conditions in children with upper gastrointestinal tract diseases (UGTD), malabsorption syndrome, inflammatory bowel disease (IBD) and functional gastrointestinal disorders (FGID). MATERIAL AND METHODS: Hydrogen breath test (HBT) was conducted in 387 pediatric patients in years 2010-2013. Two hundred thirty two children with gastrointestinal tract diseases were selected and assigned to groups - UGTD, malabsorption syndrome, IBD or FGID. For each group the frequency of LM, frequency and severity of LI and the frequency of their co-occurrence were calculated. RESULTS: Lactose malabsorption was observed in 37.08% of patients with gastrointestinal diseases. Positive HBT result was the most common in children with malabsorption syndrome (52.50%) and less common in UGTD (30.85%), especially in ulcer disease (23.53%). Symptoms after lactose ingestion affected 36.64% of the subjects, and were more specific to lactose malabsorbers than to lactose absorbers (72.10% vs. 15.75%). The higher frequency of LI was noted in children with FGID, especially in irritable bowel syndrome (IBS) (65.22%). The lowest incidence of symptoms was obtained in children with UGTD, especially in those with ulcer disease (27.44%). The incidence of LM with LI was noted in 27.16% of all patients and was the highest in IBS (47.83%) and the lowest in ulcer disease (15.78%). CONCLUSIONS: Lactose malabsorption is a common problem in children with gastrointestinal diseases, especially in children with bowel diseases. Lactose intolerance is related to LM, but does not affect all malabsorbers.

Preliminary evaluation of 1'-[(18)F]fluoroethyl-ß-D-lactose ([(18)F]FEL) for detection of pancreatic cancer in nude mouse orthotopic xenografts.

INTRODUCTION: Early detection of pancreatic cancer could save many thousands of lives. Non-invasive diagnostic imaging, including PET with [(18)F]FDG, has inadequate resolution for detection of small (2-3 mm) pancreatic tumours. We demonstrated the efficacy of PET imaging with an (18)F-labelled lactose derivative, [(18)F]FEDL, that targets HIP/PAP, a biomarker that is overexpressed in the peritumoural pancreas. We developed another analogue, 1-[(18)F]fluoroethyl lactose ([(18)F]FEL), which is simpler to synthesise, for the same application. We conducted a preliminary evaluation of the new probe and its efficacy in detecting orthotopic pancreatic carcinoma xenografts in mice. METHODS: Xenografts were developed in nude mice by injecting L3.6 pl/GL(+) pancreatic carcinoma cells into the pancreas of each mouse. Tumour growth was monitored by bioluminescence imaging (BLI); accuracy of BLI tumour size estimates was verified by MRI in two representative mice. When the tumour size reached approximately 2-3mm, the animals were injected with [(18)F]FEL (3.7 MBq) and underwent static PET/CT scans. Blood samples were collected at 2, 5, 10, 20 and 60 min after [(18)F]FEL injection to track blood clearance. Following imaging, animals were sacrificed and their organs and tumours/pancreatic tissue were collected and counted on a gamma counter. Pancreas, including tumour, was frozen, sliced and used for autoradiography and immunohistochemical analysis of HIP/PAP expression. RESULTS: Tumour growth was rapid, as observed by BLI and MRI. Blood clearance of [(18)F]FEL was bi-exponential, with half-lives of approximately 3.5 min and 40 min. Mean accumulation of [(18)F]FEL in the peritumoural pancreatic tissue was 1.29±0.295 %ID/g, and that in the normal pancreas of control animals was 0.090±0.101 %ID/g. [(18)F]FEL was cleared predominantly by the kidneys. Comparative analysis of autoradiographic images and immunostaining results demonstrated a correlation between [(18)F]FEL binding and HIP/PAP expression. CONCLUSION: [(18)F]FEL may be useful for non-invasive imaging of early-stage pancreatic tumours by PET. The results warrant further studies.

Sialyllactose ameliorates myopathic phenotypes in symptomatic GNE myopathy model mice.

Patients with GNE myopathy, a progressive and debilitating disease caused by a genetic defect in sialic acid biosynthesis, rely on supportive care and eventually become wheelchair-bound. To elucidate whether GNE myopathy is treatable at a progressive stage of the disease, we examined the efficacy of sialic acid supplementation on symptomatic old GNE myopathy mice that have ongoing, active muscle degeneration. We examined the therapeutic effect of a less metabolized sialic acid compound (6'-sialyllactose) or free sialic acid (N-acetylneuraminic acid) by oral, continuous administration to 50-week-old GNE myopathy mice for 30 weeks. To evaluate effects on their motor performance in living mice, spontaneous locomotion activity on a running wheel was measured chronologically at 50, 65, 72 and 80 weeks of age. The size, force production, and pathology of isolated gastrocnemius muscle were analysed at the end point. Sialic acid level in skeletal muscle was also measured. Spontaneous locomotion activity was recovered in 6'-sialyllactose-treated mice, while NeuAc-treated mice slowed the disease progression. Treatment with 6'-sialyllactose led to marked restoration of hyposialylation in muscle and consequently to robust improvement in the muscle size, contractile parameters, and pathology as compared to NeuAc. This is due to the fact that 6'-sialyllactose is longer working as it is further metabolized to free sialic acid after initial absorption. 6'-sialyllactose ameliorated muscle atrophy and degeneration in symptomatic GNE myopathy mice. Our results provide evidence that GNE myopathy can be treated even at a progressive stage and 6'-sialyllactose has more remarkable advantage than free sialic acid, providing a conceptual proof for clinical use in patients.

Further constituents of Galianthe thalictroides (Rubiaceae) and inhibition of DNA topoisomerases I and IIα by its cytotoxic ß-carboline alkaloids.

A new cytotoxic ß-carboline alkaloid, 1-methyl-3-(2-hydroxypropan-2-yl)-2-(5-methoxy-9H-ß-carbolin-1-yl)-cyclopentanol (1), was isolated from roots of Galianthe thalictroides, together with the alkaloid 1-(hydroxymethyl)-3-(2-hydroxypropan-2-yl)-2-(5-methoxy-9H-ß-carbolin-1-yl)-cyclopentanol (2), the anthraquinones 1-methyl-alizarin and morindaparvin-A, the coumarin scopoletin, homovanillic alcohol, (-)-epicatechin, and the steroids stigmast-4-en-3-one, 4,22-stigmastadien-3-one, campest-4-en-3-one, stigmast-4-en-3,6-dione, 6-ß-hydroxy-stigmast-4-en-3-one, stigmasterol, campesterol, ß-sitosterol, and ß-sitosterol-3-O-ß-D-glucopyranoside. Among the previously known compounds, homovanillic alcohol is a novel finding in Rubiaceae, while 1-methyl-alizarin, morindaparvin-A, scopoletin, stigmast-4-en-3-one, 4,22-stigmastadien-3-one, campest-4-en-3-one, stigmast-4-en-3,6-dione, and 6-ß-hydroxy-stigmast-4-en-3-one is reported for the first time in the genus Galianthe. The cytotoxic ß-carboline alkaloids 1 and 2 exhibited potent antitopoisomerase I and IIα activities and strong evidence is provided for their action as topoisomerase IIα poisons and redox-independent inhibitors.

Delivery of a lactose derivative of endomorphin 1 to the brain via the olfactory epithelial pathway.

The rapid and direct delivery of a neuroactive endomorphin 1 derivative to the brain via nasal delivery is reported. A synthetic derivative of the native opioid peptide, endomorphin 1 bearing a lactose unit on the N-terminus of the peptide has been previously reported to exhibit antinoceceptive activity similar to morphine after both intravenous and oral administration. This compound has been administered nasally to rats and appeared in the olfactory bulb within 10 min of administration with negligible levels appearing in the circulating blood or in the rest of the brain. These results indicate that the peptide is absorbed into the brain via the olfactory epithelial pathway suggesting nasal delivery may be a viable alternative route of delivery in clinical applications.