Trehalose in Machado-Joseph Disease: Safety, Tolerability, and Efficacy.

Machado-Joseph disease (MJD) is relatively prevalent among the Yemenite Jewish subpopulation living in Israel. Currently, there is no treatment able to modify the disease progression. Trehalose is a disaccharide with protein-stabilizing and autophagy-enhancing properties. In animal models of MJD, trehalose showed reduction of cerebellar lesion size and improved motor function. This study was designed to be a proof-of-concept, phase 2 study lasting 6 to 12 months, to determine the safety, tolerability, and efficacy of weekly IV administration of 15 g or 30 g 10% trehalose solution in 14 MJD patients. Primary endpoints were safety and tolerability, which were assessed by various clinical and laboratory tests. Secondary endpoints were changes in the Scale for Assessment and Rating of Ataxia (SARA) score, Neurological Examination Score for Spinocerebellar Ataxia (NESSCA), time to do 9-hole peg test (9HPT), time to do 8-meter walk (8MW), and quality of life assessed by the World Health Organization Quality-of-Life Questionnaire-BREF (WHOQoL-BREF). Trehalose was well tolerated, and no serious drug-related adverse events were noted. The average SARA score, NESSCA, and time to do 9HPT and 8MW and the WHOQoL-BREF for all patients remained stable at 6 months. Six patients received treatment for as long as 12 months and continued to remain stable on all the above tests. IV trehalose seems to be safe in humans and probably effective to stabilize neurological impairment in MJD.

Foodborne transmission of Clostridioides difficile: a review.

PURPOSE OF REVIEW: The epidemiology of Clostridioides difficile infection (CDI) is changing, with increasing rates of community-acquired infections. In light of recent advances in understanding C. difficile transmission networks with whole-genome sequencing, new routes of spread outside the hospital need to be considered. This review examines the evidence behind food as a driver of C. difficile dissemination. RECENT FINDINGS: Recently published studies adding to the existing body of literature supporting C. difficile as a foodborne pathogen are discussed. Specifically, new evidence on the presence of C. difficile in root vegetables is reviewed. Whole genome sequencing studies delineating local and global transmission networks, in which the food chain may play a large role, are presented. Additional research implicating trehalose in the food industry and C. difficile is examined. SUMMARY: Genomic studies show that a new approach to studying C. difficile transmission is needed. Further research on C. difficile epidemiology should shift from a primarily nosocomial setting to include the community and environment at large, and attention given to implications of the food chain in the spread of this pathogen.

Excipients: not so inert? When the excipient plays the role of an active substance, as exemplified by systemic lupus.

It is well recognised that the historical timeline required for developing a drug, beginning with target identification and validation, is long and often tedious. It requires a large set of competences in various areas of molecular and cellular biology, chemistry, pharmacology, imaging, and model animal experimentation. Once the active molecule appears to be ready for human testing in controlled clinical trials, then the question arises of how to formulate it to render it stable, adequately packaged, according to the chosen route of administration, and bioavailable to reach its target in the affected organs. Historically, excipients have been considered inert and devoid of medicinal effect or influence. In fact, excipients are seldom neutral and some of them have been found to play a significant role, for example by initiating or participating in chemical and physical interactions with the active substance, leading in certain cases to compromise its therapeutic activity. It is difficult today to appreciate the number of potential drugs that have been discarded as a result of limited efficacy due to inappropriate excipients. This matter is presented here, with the peptide P140 (Lupuzor™) as example. Two formulations of P140, differing in the excipients used (mannitol or trehalose), have been evaluated in patients affected by systemic lupus erythematosus in two successive phase IIb clinical trials. P140 was shown to reduce excessive autophagy activity discovered in some lupus immune cell subsets. One of the two excipients, namely trehalose, has been claimed to exert an intrinsic stimulating activity on autophagy process, which was found therefore to counteract the beneficial peptide effects.

A randomized, controlled study of the efficacy and safety of a new eyedrop formulation for moderate to severe dry eye syndrome.

PURPOSE: This study compared the efficacy and safety of hyaluronic acid (HA)-trehalose, a new eyedrop containing trehalose (a natural bioprotectant) and HA, to an established formulation containing only HA. METHODS: This was a phase III, randomized, active-controlled, investigator-masked, multicenter study in France and Tunisia. In all, 105 adult patients (≥18 years) with moderate to severe dry eye disease (DED) received either HA-trehalose (n = 52) or HA (n = 53) 3-6 times per day for 84 days. The primary efficacy variable was the Oxford grading score at day 35. A questionnaire on dry eye and symptoms, Schirmer test, tear break-up time, conjunctival hyperemia, and global performance were assessed as secondary efficacy criteria at baseline, day 35, and day 84. Safety assessments were standard. RESULTS: Noninferiority of HA-trehalose to HA for keratoconjunctivitis sicca assessed by Oxford grading score was demonstrated at day 35. For the secondary efficacy parameters, reductions in dry eye questionnaire classes of none or mild at day 84, dry eye symptoms of stinging, itching, and blurred vision at day 35, and investigator (days 35 and 84) and patient assessments (day 35) of global performance were significantly better for HA-trehalose. There were no clinically meaningful differences between groups for the other secondary criteria. Both treatments were well-tolerated, and there were fewer ocular symptoms upon instillation and fewer adverse events for HA-trehalose than for HA. CONCLUSIONS: Hyaluronic acid-trehalose is effective and safe, with better patient satisfaction, than existing HA-only eyedrops particularly from the first month of treatment, and offers a therapeutic advancement in the treatment of moderate to severe DED.

Engineered Trehalose Permeable to Mammalian Cells.

Trehalose is a naturally occurring disaccharide which is associated with extraordinary stress-tolerance capacity in certain species of unicellular and multicellular organisms. In mammalian cells, presence of intra- and extracellular trehalose has been shown to confer improved tolerance against freezing and desiccation. Since mammalian cells do not synthesize nor import trehalose, the development of novel methods for efficient intracellular delivery of trehalose has been an ongoing investigation. Herein, we studied the membrane permeability of engineered lipophilic derivatives of trehalose. Trehalose conjugated with 6 acetyl groups (trehalose hexaacetate or 6-O-Ac-Tre) demonstrated superior permeability in rat hepatocytes compared with regular trehalose, trehalose diacetate (2-O-Ac-Tre) and trehalose tetraacetate (4-O-Ac-Tre). Once in the cell, intracellular esterases hydrolyzed the 6-O-Ac-Tre molecules, releasing free trehalose into the cytoplasm. The total concentration of intracellular trehalose (plus acetylated variants) reached as high as 10 fold the extracellular concentration of 6-O-Ac-Tre, attaining concentrations suitable for applications in biopreservation. To describe this accumulation phenomenon, a diffusion-reaction model was proposed and the permeability and reaction kinetics of 6-O-Ac-Tre were determined by fitting to experimental data. Further studies suggested that the impact of the loading and the presence of intracellular trehalose on cellular viability and function were negligible. Engineering of trehalose chemical structure rather than manipulating the cell, is an innocuous, cell-friendly method for trehalose delivery, with demonstrated potential for trehalose loading in different types of cells and cell lines, and can facilitate the wide-spread application of trehalose as an intracellular protective agent in biopreservation studies.

Mediated trehalose un-loading for reduced erythrocyte osmotic fragility and phosphatidylserine translocation.

Recently, high concentrations of intracellular trehalose (>200mM) were employed to enhance the cryoprotection and desiccation protection of human erythrocytes. However, significant challenges must be overcome if this advancement is to be translated into clinical practice. It is here demonstrated that 247 ± 5 mM intracellular trehalose caused the lysis of 60 ± 2 percent of erythrocytes upon resuspension in PBS of physiological osmolality (300 mOsm) and caused surviving cells to swell up to 140 ± 2 percent of isotonic cell volume. Trehalose loaded cells also exhibited 24 ± 1 percent incidence of phosphatidylserine translocation upon resuspension in 300 mOsm PBS, likely due to loading induced cell swelling. Un-loading of trehalose from erythrocytes using the membrane-permeabilizing biopolymer PP-50 was investigated as a technique to mitigate these damaging effects. After erythrocyte un-loading from 247 ± 5 mM to 39 ± 2 mM intracellular trehalose, cell lysis at 300 mOsm PBS was reduced from 60 ± 2 percent to 17 ± 3 percent. Un-loading also reduced cellular incidence of PS translocation in resuspended cells from 24 ± 1 percent to 13 ± 1 percent.

Quantitative microinjection of trehalose into mouse oocytes and zygotes, and its effect on development.

Sugars such as trehalose are effectively used by various organisms as protective agents to undergo anhydrobiosis and cryobiosis. The objective of this study was first to establish a method for quantitative delivery of trehalose as a model sugar into oocytes, and then to evaluate its effect on development of mouse zygotes. To this end, a quantitative microinjection technique was developed using volumetric response of microdroplets suspended in dimethylpolysilaxene. To verify accuracy of this technique, both microdroplets and oocytes were microinjected with fluorophore-labeled dextran. Thereafter, injection volumes were calculated from fluorescence intensity, and volumetric responses of both microdroplets and oocytes. Comparison of calculated injection volumes revealed that this technique reflects microinjection into oocytes with pL-accuracy. The next series of experiments focused on toxicity of injection buffers (i.e., 10mM Tris and 15mM Hepes) and trehalose. Microinjection of Hepes and Tris buffer in the presence of 0.1M trehalose resulted in blastocyst rates of 86 and 72%, respectively, without a significant difference when compared to controls (86%). In subsequent experiments, Hepes was used as the injection buffer, and embryonic development of zygotes was studied as a function of intracellular trehalose concentrations. Microinjection of trehalose up to 0.15M resulted in development to blastocyst stage similar to controls (85 and 87%, respectively) while the blastocyst rate was significantly decreased (43%) in the presence of 0.20M intracellular trehalose. When transferred to foster mothers, trehalose-injected zygotes (0.1M) implanted and developed to day 16 fetuses similar to controls, healthy pups were born. The findings of this study suggest that trehalose at effective intracellular concentrations does not impair development of mouse zygotes.

Trehalose: a review of properties, history of use and human tolerance, and results of multiple safety studies.

This paper contains a review of the history, natural occurrence, human consumption, metabolism, manufacture, and the results of eight standardized animal safety studies using trehalose. Trehalose (alpha,alpha-trehalose) is a naturally occurring sugar containing two D-glucose units in an alpha,alpha-1,1 linkage. Trehalose functions in many organisms as an energy source or a protectant against the effects of freezing or dehydration. It also possesses physical and/or chemical properties that are different than other sugars, which may make trehalose an attractive ingredient in food, health and beauty and pharmaceutical products. Data are presented supporting safe human consumption of trehalose in doses up to 50 g, and the physiologic ability of humans to digest it. No consistent treatment-related, dose-dependent adverse effects were observed in any of the eight safety studies performed at doses up to 10% of the diets. On the basis of these toxicity studies, human studies in which doses of trehalose were administered to various populations, and consumption of trehalose in commercial products in Japan, it is concluded that trehalose is safe for use as an ingredient in consumer products when used in accordance with current Good Manufacturing Practices.

Transitory laxative threshold of trehalose and lactulose in healthy women.

The transitory laxative threshold of a partially digestible disaccharide, trehalose, and an undigestible disaccharide, lactulose, was estimated by the dose-response relation between the test substance and the prevalence of diarrhea in 20 healthy female subjects. The subjects ingested several indicated amounts of trehalose or lactulose once daily 2 to 3 h after a meal. The intake of the test substance was stopped at the dose level that caused diarrhea or when the dose reached the maximal level. A record of physical conditions, gastrointestinal symptoms, and fecal conditions was made by all subjects before and after each ingestion of the test substance. Half the subjects experienced no diarrhea even with the ingestion of the maximal dose level (60 g) of trehalose in this study, and the ingestion of up to 40 g of lactulose caused diarrhea in 75% of all subjects. Abdominal symptoms such as flatus, distension, and borborygmus appeared at high prevalence with lactulose and trehalose ingestion, and the effect of lactulose was significantly stronger than that of trehalose at the same dose level (p < 0.05). The quantity of trehalose and lactulose that induced diarrhea differed greatly from person to person. The transitory laxative threshold was estimated as 0.65 g/kg body weight for trehalose and 0.26 g/kg body weight for lactulose by using the regression equation between the dose levels of the test substances and the cumulative incidence of diarrhea. These results suggest that it would be quite acceptable to administer trehalose up to 33 g and lactulose up to 13 g in a person weighing 50 kg.