In Vitro Evaluation of the Adsorption Efficacy of Biochar Materials on Aflatoxin B1, Ochratoxin A, and Zearalenone.

Mycotoxin sequestration materials are important tools to reduce mycotoxin illness and enable proper handling of mycotoxin-contaminated commodities. Three food-grade bentonite clays and four generally recognized as safe (GRAS) charcoal/biochar carbon materials that are marketed as feed additives and supplements were evaluated for their ability to sequester the mycotoxins aflatoxin B1, ochratoxin A, and zearalenone. The surface area of the clays varied between 32.1 to 51.4 mg2/g, and the surface area of the carbon-based materials varied from 1.7 to 1735 mg2/g. In vitro, gastric fluid studies indicated that certain pine biochar and activated coconut charcoal could sequester high amounts (85+%) of the mycotoxins at 1 ppm levels or below. However, some biochar materials with lower surface area properties lacked binding capacity. The coconut shell charcoal and pine biochar utilize agricultural waste products in a manner that significantly reduces carbon emissions and provides valuable materials to minimize exposure to toxins found in food and feed.

Biophysical characterization of α-glucan nanoparticles encapsulating feruloylated soy glycerides (FSG).

Water insoluble α-glucans that were enzymatically synthesized using glucansucrase that was cloned from Leuconostoc mesenteroides NRRL B-1118 were previously shown to form nanoparticles via high pressure homogenization. These α-glucan nanoparticles were previously shown capable of encapsulating a small hydrophobic molecule. This work demonstrates that the same α-glucan can be formed into nanoparticles that encapsulate feruloylated soy glycerides from modified soybean oil, a product of interest to the cosmetic and skin care industries because of the UV absorbance and antioxidant properties of the feruloyl moiety. It is demonstrated that the feruloylated soy glyceride/α-glucan nanoparticles have distinct size, zeta potential and thermal profiles from that of nanoparticles made from α-glucan alone or feruloylated soy glyceride alone. Thermal analysis also demonstrates the release of feruloylated soy glycerides from the α-glucan nanoparticles.

Preclinical studies in support of phase I/II clinical trials to treat GUCY2D-associated Leber congenital amaurosis.

Mutations in GUCY2D are associated with severe early-onset retinal dystrophy, Leber congenital amaurosis type 1 (LCA1), a leading cause of blindness in children. Despite a high degree of visual disturbance stemming from photoreceptor dysfunction, patients with LCA1 largely retain normal photoreceptor structure, suggesting that they are good candidates for gene replacement therapy. The purpose of this study was to conduct the preclinical and IND-enabling experiments required to support clinical application of AAV5-hGRK1-GUCY2D in patients harboring biallelic recessive mutations in GUCY2D. Preclinical studies were conducted in mice to evaluate the effect of vector manufacturing platforms and transgene species on the therapeutic response. Dose-ranging studies were conducted in cynomolgus monkeys to establish the minimum dose required for efficient photoreceptor transduction. Good laboratory practice (GLP) studies evaluated systemic biodistribution in rats and toxicology in non-human primates (NHPs). These results expanded our knowledge of dose response for an AAV5-vectored transgene under control of the human rhodopsin kinase (hGRK1) promoter in NHPs with respect to photoreceptor transduction and safety and, in combination with the rat biodistribution and mouse efficacy studies, informed the design of a first-in-human clinical study in patients with LCA1.

AAV2-hAADC (Eladocagene Exuparvovec) Biodistribution and Expression: Superiority of Intraputaminal versus Intracerebroventricular and Intrathecal (Lumbar) Routes of Administration.

Aromatic L-amino acid decarboxylase deficiency is a genetic disorder of enzyme loss with decreased neurotransmitter synthesis, and it is characterized by symptoms of impaired motor development and cognitive function, hypotonia, dystonia, and oculogyric crises. Though symptomatic severity varies, the majority of patients experience severe motor impairments, including an inability to sit, stand, or walk. One approved therapy for Aromatic L-amino acid decarboxylase deficiency involves intraputaminal delivery of an adeno-associated virus packaging the human Aromatic L-amino acid decarboxylase enzyme (hAADC) cDNA. The objective of this study in monkeys was to determine the acceptability of ICV/IT as minimally invasive dosing options by evaluating hAADC biodistribution and expression following intraputaminal, intracerebroventricular (ICV), or intrathecal (IT, lumbar) administration. Results show that all routes produced comparable CSF transgene levels and were well-tolerated. The intraputaminal route yielded the highest levels of transgene-derived mRNA expression in the putamen, caudate, and globus pallidus, while expression levels in the spinal cord and dorsal root ganglia (DRG, a target of special toxicological concern) were undetectable. In contrast, the highest transgene levels in ICV/IT groups were observed in the spinal cord and DRG, but levels were too low to result in expression in the putamen, caudate, and globus pallidus. Unlike ICV/IT, the intraputaminal route produced no transgene in blood, suggesting a lower likelihood of off-target toxicities. Additionally, intraputaminal dosing resulted in the lowest anti-AAV2 antibody (anti-drug antibody) levels. Together, these data demonstrate the superiority of intraputaminal administration over ICV/IT routes in achieving AAV2-hAADC transgene DNA distribution and mRNA expression in target therapeutic areas while minimizing risk of toxicity.

Impact of a Prenatal Vitamin D Supplementation Program on Vitamin D Deficiency, Rickets and Early Childhood Caries in an Alaska Native Population.

Background: Early childhood rickets increased in Alaska Native children after decreases in vitamin D-rich subsistence diet in childbearing-aged women. We evaluated the impact of routine prenatal vitamin D supplementation initiated in Alaska's Yukon Kuskokwim Delta in Fall 2016. Methods: We queried electronic health records of prenatal women with 25(OH) vitamin D testing during the period 2015−2019. We evaluated 25(OH)D concentrations, vitamin D3 supplement refills, and decayed, missing, and filled teeth (dmft) scores and rickets in offspring. Results: Mean 25(OH)D concentrations increased 36.5% from pre- to post-supplementation; the percentage with deficient 25(OH)D decreased by 66.4%. Women with ≥ 60 vitamin D3 refill days had higher late pregnancy 25(OH)D concentrations than those with no refill days (p < 0.0001). Women with late pregnancy insufficient 25(OH)D concentrations had offspring with higher dmft scores than those with sufficient 25(OH)D (RR 1.3, p < 0.0001). Three children were diagnosed with nutritional rickets during the period 2001−2021, and none after 2017. Conclusions: These findings suggest that prenatal vitamin D supplementation can improve childhood outcomes in high-risk populations with high rates of rickets.

Characterization and properties of starch-dicarboxylic acid inclusion complexes prepared by excess steam jet cooking.

A series of dicarboxylic-amylose inclusion complexes (AIC) were prepared by excess steam jet-cooking high amylose corn starch with linear C10, C12, C14, and C16 dicarboxylic acids to examine the influence of two polar head groups on complex formation. The C12, C14, and C16 dicarboxylic acid AIC were prepared in 48-63 % yields and contained 8.9-11.8 % diacid while the C10 AIC gave 30 % and contained 2.6 % diacid. These AIC had V6 helical amylose structures by XRD and complexation was further confirmed by DSC, FTIR, and TGA. SEM of the C12-C16 AIC revealed micron-sized toroidal spherulites while the C10 AIC was predominantly amorphous. DSC showed two AIC related transitions. This work provides a better understanding of the formation and physicochemical properties of these diacid AIC. Preparation by excess steam jet cooking demonstrates practical and commercial utility to prepare AIC as off-the-shelf materials for food and nonfood applications.

Raman spectral analysis for rapid determination of zearalenone and alpha-zearalanol.

Mycotoxins, including zearalenone, are important natural products produced by fungi that occasionally contaminate agricultural commodities and pose serious health risks to consumers of food and feed. Zearalenone and its metabolite, α-zearalanol, are of significant concern due to their estrogenic and anabolic steroid activity. Several governments have regulatory standards and advisory guidelines for zearalenone and α-zearalanol. Raman and ultraviolet spectroscopy were employed with density functional theory methods to evaluate spectroscopic properties to distinguish between zearalenone and α-zearalanol systematically. Raman bands were assigned based on vibrational frequency calculations. A portable Raman spectroscopy instrument (785 nm laser) distinguished between zearalenone and α-zearalanol in a label-free manner. Many vibrational bands of zearalenone and α-zearalanol are similar, including high-intensity peaks at 1315 cm-1 and 1650 cm-1. However, the intensities in the Raman spectra at 1465 cm-1, 1495 cm-1, and 1620 cm-1 enabled the identification of zearalenone. The Raman peak at 1450 cm-1 is associated with α-zearalanol. These vibrational bands serve as spectral indicators to differentiate between the structurally similar zearalenone and α-zearalanol.

Gene therapy in the putamen for curing AADC deficiency and Parkinson's disease.

This commentary provides an overview of the putamen as an established target site for gene therapy in treating aromatic l-amino acid decarboxylase (AADC) deficiency and Parkinson's disease, two debilitating neurological disorders that involve motor dysfunction caused by dopamine deficiencies. The neuroanatomy and the function of the putamen in motor control provide good rationales for targeting this brain structure. Additionally, the efficacy and safety of intraputaminal gene therapy demonstrate that restoration of dopamine synthesis in the putamen by using low doses of adeno-associated viral vector serotype 2 to deliver the hAADC gene is well tolerated. This restoration leads to sustained improvements in motor and nonmotor symptoms of AADC deficiency and improved uptake and conversion of exogenous l-DOPA into dopamine in Parkinson's patients.

Predictive Quantitative Structure-Activity Relationship Modeling of the Antifungal and Antibiotic Properties of Triazolothiadiazine Compounds.

Predictive models were developed using two-dimensional quantitative structure activity relationship (QSAR) methods coupled with B3LYP/6-311+G** density functional theory modeling that describe the antimicrobial properties of twenty-four triazolothiadiazine compounds against Aspergillus niger, Aspergillus flavus and Penicillium sp., as well as the bacteria Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa. B3LYP/6-311+G** density functional theory calculations indicated the triazolothiadiazine derivatives possess only modest variation between the frontier orbital properties. Genetic function approximation (GFA) analysis identified the topological and density functional theory derived descriptors for antimicrobial models using a population of 200 models with one to three descriptors that were crossed for 10,000 generations. Two or three descriptor models provided validated predictive models for antifungal and antibiotic properties with R 2 values between 0.725 and 0.768 and no outliers. The best models to describe antimicrobial activities include descriptors related to connectivity, electronegativity, polarizability, and van der Waals properties. The reported method provided robust two-dimensional QSAR models with topological and density functional theory descriptors that explain a variety of antifungal and antibiotic activities for structurally related heterocyclic compounds.

Nonclinical Studies that Support Viral Vector-Delivered Gene Therapies: An EFPIA Gene Therapy Working Group Perspective.

Nonclinical development strategies for gene therapies are unique from other modalities. The European Federation of Pharmaceutical Industries and Associates (EFPIA) Gene Therapy Working Group surveyed EFPIA member and nonmember pharmaceutical and biotechnology companies about their current practices for designing and implementing nonclinical toxicology studies to support the development of viral vector-delivered in vivo gene therapies. Compiled responses from 17 companies indicated that these studies had some variability in species selection, study-design elements, biodistribution, immunogenicity or genomic insertion assessments, safety pharmacology, and regulatory interactions. Although there was some consistency in general practice, there were examples of extreme case-by-case differences. The responses and variability are discussed herein. Key development challenges were also identified. Results from this survey emphasize the importance for harmonization of regulatory guidelines for the development of gene-therapy products, while still allowing for case-by-case flexibility in nonclinical toxicology studies. However, the appropriate timing for a harmonized guidance, particularly with a platform that continues to rapidly evolve, remains in question.

Development and Physical Characterization of α-Glucan Nanoparticles.

α-Glucans that were enzymatically synthesized from sucrose using glucansucrase cloned from Leuconostoc mesenteroides NRRL B-1118 were found to have a glass transition temperature of approximately 80 °C. Using high-pressure homogenization (~70 MPa), the α-glucans were converted into nanoparticles of ~120 nm in diameter with a surface potential of ~-3 mV. Fluorescence measurements using 1,6-diphenyl-1,3,5-hexatriene (DPH) indicate that the α-glucan nanoparticles have a hydrophobic core that remains intact from 10 to 85 °C. α-Glucan nanoparticles were found to be stable for over 220 days and able to form at three pH levels. Accelerated exposure measurements demonstrated that the α-glucan nanoparticles can endure exposure to elevated temperatures up to 60 °C for 6 h intervals.

Enzymatic Synthesis and Flash Chromatography Separation of 1,3-Diferuloyl-sn-Glycerol and 1-Feruloyl-sn-Glycerol.

Ethyl ferulate was transesterified with Enova Oil (a soy-based vegetable oil containing 80-85% diacylglycerol) using Novozym 435 at 60 °C. The resultant feruloylated vegetable oil reaction product produced a precipitate (96.4 g, 4.02 wt%) after 7 d of standing at room temperature. Preliminary characterization of the precipitate identified the natural phenylpropenoids 1,3-diferuloyl-sn-glycerol (F2G) and 1-feruloyl-sn-glycerol (FG) as the major components. A flash chromatography method was developed and optimized (e.g., mass of sample load, flow rate, binary solvent gradient slope, and separation run length) using a binary gradient of hexane and acetone mobile phase and silica gel stationary phase to separate and isolate F2G and FG. The optimized parameters afforded F2G (1.188 ± 0.052 g, 39.6 ± 1.7%) and FG (0.313 ± 0.038 g, 10.4 ± 1.3%) from 3.0 g of the transesterification precipitate, n = 10 trials. Overall, all flash chromatography separations combined, F2G (39.1 g, 40.6%) and FG (9.4 g, 9.8%) were isolated in a combined yield of 48.5 g (51.4%), relative to the 96.4 g of transesterification precipitate collected. The optimized flash chromatography method was a necessary improvement over previously reported preparative HPLC and column chromatography methods used to purify milligram to low gram quantities of F2G and FG to be able to process ~100 g of material in a timely, efficient manner.

Rapid Raman spectroscopic determination of 1-feruloyl-sn-glycerol and 1,3-diferuloyl-sn-glycerol.

Ferulic acid and its derivatives are important natural products found throughout the plant kingdom and are of special interest due to their health benefits. 1-Feruloyl-sn-glycerol (FG) and 1,3-diferuloyl-sn-glycerol (F2G) are two common bioproducts of ferulic acid that co-occur in nature and during the biocatalytic production of feruloylated lipids. In this paper, we report a comprehensive characterization of FG and F2G using Raman and UV spectroscopies and theoretical density functional theory calculations at the B3LYP/6-311+G** level. UV spectroscopy produced spectra for FG and F2G with similar peak shape, but difference intensities. The vibrational frequency calculations aided in the assignment of the Raman bands. The Raman analysis demonstrates that Raman spectroscopy is a rapid label free method to clearly distinguish between FG and F2G.

Charged phospholipid effects on AAPH oxidation assay as determined using liposomes.

The capacity of molecules to inhibit oxidation is widely tested using liposomes as host matrices of the antioxidant molecule of interest. Spectroscopic assays are readily used for this purpose, specifically assays using 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH). In this work the effect that charged lipids have on an AAPH antioxidation assay using 4,4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid (C11-BODIPY® 581/591) as the reporter molecule was investigated. We measured the diameter, zeta potential and spectroscopic rate of decay and area-under-the-curve (AUC) associated with liposomes containing C11-BODIPY® 581/591 at varying molar percentages (0-10 mol%) of charged (cationic or anionic) lipids and compared the results. We showed that although increasing amounts of cationic or anionic lipids did change the diameter of the liposomes, size had little to no effect on the area-under-the-curve or decay rate of fluorescence. Increased (more positive) or decreased (more negative) zeta potentials did, on the other hand, affect the spectroscopic decay rates and area-under-the-curve. The results demonstrate the importance of considering the presence of charged lipids in the AAPH antioxidation assay.

Determination of pH Effects on Phosphatidyl-Hydroxytyrosol and Phosphatidyl-Tyrosol Bilayer Behavior.

A robust method was developed to investigate the liposomal behavior of novel enzymatically-synthesized hydroxytyrosol and tyrosol phospholipids. Bilayer characteristic obtained by this method, including bilayer formation stability and adsorption properties, were explored using dynamic light scattering, zeta-potential measurements, and quartz crystal microbalance with dissipation monitoring (QCMD), respectively. Liposome diameters were found to typically increase from pH 5.5 to pH 10. Zeta potentials values, on the other hand, were found to be well below -25 mV at all pH conditions explored, with the lowest values (and thus, the best liposome stability) at pH 5.5 or pH 10. Quartz crystal microbalance with dissipation monitoring measurements demonstrated that 100% 1,2-dioloeoylphosphatidyl-hydroxytyrosol (DOPHT) liposomes adsorbed intact onto silica in buffer conditions at pH 5.5 and with no calcium, or at pH 7.5 with calcium (no adsorption was detected at pH 10). 1,2-Dioleoylphosphatidyl-tyrosol (DOPT) liposomes were shown to adsorb intact under buffer conditions only at pH 5.5 with and without calcium. 1,2-Dioleoylphosphatidyl-2-phenolethanol (DOPPE), in comparison, readily adsorbed intact at pH 7.5 without calcium and just slightly at pH 5.5 with calcium present, but formed a supported bilayer over hours at pH 5.5 in the absence of calcium ions.

Abuse liability assessment for biologic drugs - All molecules are not created equal.

The development of novel drug candidates involves the thorough evaluation of potential efficacy and safety. To facilitate the safety assessment in light of global increases in prescription drug misuse/abuse, health authorities have developed guidance documents which provide a framework for evaluating the abuse liability of candidate therapeutics. The guidances do not distinguish between small molecules and biologics/biotherapeutics; however, there are key differences between these classes of therapeutics which are important drivers of concern for abuse. An analysis of these properties, including ability to distribute to the central nervous system, pharmacokinetic properties (e.g., half-life and metabolism), potential for off-target binding, and the physiochemical characteristics of biologic drug products suggests that the potential for abuse of a biologic is limited. Many marketed antibodies and recombinant proteins have been associated with adverse effects such as headache and dizziness. However, biologics have not historically engendered the rapid-onset psychoactive effects typically present for drugs of abuse, thus further underscoring their low risk for abuse potential. The factors to be taken into consideration before conducting nonclinical abuse liability studies with biologics are described herein; importantly, the aggregate assessment of these factors leads to the conclusion that abuse liability studies are unlikely to be necessary for this class of therapeutics.

Phosphatidyl-hydroxytyrosol and phosphatidyl-tyrosol bilayer properties.

Hydroxytyrosol and tyrosol phospholipids were enzymatically synthesized and investigated for their bilayer properties. Dynamic light scattering demonstrated that hand extrusion at 100nm consistently resulted in liposomes of nearly 85nm diameter for both phosphatidyl-hydroxytyrosol (DOPHT) and phosphatidyl-tyrosol (DOPT). Transmission electron microscopy showed DOPT and DOPHT liposomes extruded at 100-nm to be spherical and non-distinctive from one another. Zeta potential measurements resulted in surface charges<-25mV, demonstrating both DOPT and DOPHT form highly stable liposomes. Quartz crystal microbalance with dissipation monitoring measurements demonstrated that liposomal adsorption was dependent on a combination of DOPT (or DOPHT) mole-percent and calcium ions concentration. Fluorescence anisotropy measurements indicated that melting temperatures of DOPT and DOPHT were below 4°C, suggesting that adsorption behavior and liposome formation was limited by electrostatic interactions and not gel-state formation.

Experimental and theoretical study of the influence of water on hydrolyzed product formation during the feruloylation of vegetable oil.

BACKGROUND: Feruloylated vegetable oil is a valuable green bioproduct that has several cosmeceutical applications associated with its inherent anti-oxidant and ultraviolet-absorption properties. Hydrolyzed vegetable oil by-products can influence product quality and consistency. RESULTS: The formation of by-products by residual water in the enzymatic synthesis of feruloylated vegetable oil was investigated using chemical theory and experimental studies by monitoring the reaction over a 22-day period. The hydrolysis of vegetable oil is thermodynamically favored over the hydrolysis of the ethyl ferulate starting material. These results suggest that hydrolyzed vegetable oil products will be experimentally observed in greater concentrations compared to hydrolyzed ethyl ferulate products. CONCLUSION: Quantum chemical studies identified several reaction mechanisms that explain the formation of side products by water, suggesting that residual water influences product quality. Efforts to reduce residual water can improve product consistency and reduce purification costs. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

Proarrhythmic mechanisms of the common anti-diarrheal medication loperamide: revelations from the opioid abuse epidemic.

Loperamide is a µ-opioid receptor agonist commonly used to treat diarrhea and often available as an over-the-counter medication. Recently, numerous reports of QRS widening accompanied by dramatic QT interval prolongation, torsades de pointe arrhythmia, and death have been reported in opioid abusers consuming large amounts of the drug to produce euphoria or prevent opiate withdrawal. The present study was undertaken to determine the mechanisms of this cardiotoxicity. Using whole-cell patch clamp electrophysiology, we tested loperamide on the cloned human cardiac sodium channel (Nav1.5) and the two main repolarizing cardiac K(+) channels cloned from the human heart: KvLQT1/minK and the human ether-a-go-go-related gene (hERG) channel. Loperamide inhibited Nav1.5 with IC50 values of 297 and 239 nM at holding potentials of -90 and -70 mV, respectively. Loperamide was weakly active on KvLQT1/minK producing 17 and 65 % inhibition at concentrations of 1 and 10 µM, respectively. Conversely, loperamide was found to be a very high affinity inhibitor of the hERG channel with an IC50 value of 89 nM at room temperature and 33 nM when measured at physiological temperature. The QRS and QT interval prolongation and the attending arrhythmias, produced by loperamide, derive from high affinity inhibition of Nav1.5 and especially hERG. Since the drug has been widely available and safely used as directed for many years, we believe that the potent inhibition loperamide possesses for cardiac ion channels has only been uncovered because of the excessive misuse of the drug as a consequence of the recent opioid abuse epidemic.

Formation of inclusion complexes between high amylose starch and octadecyl ferulate via steam jet cooking.

Amylose-ligand inclusion complexes represent an interesting approach to deliver bioactive molecules. However, ferulic acid has been shown not to form single helical inclusion complexes with amylose from high amylose maize starch. To overcome this problem a lipophilic ferulic acid ester, octadecyl ferulate, was prepared and complexed with amylose via excess steam jet cooking. Jet-cooking octadecyl ferulate and high amylose starch gave an amylose-octadecyl ferulate inclusion complex in 51.0% isolated yield. X-ray diffraction (XRD) and differential scanning calorimetry (DSC) confirmed that a 61 V-type inclusion complex was formed. Amylose and extraction assays showed the complex to be enriched in amylose (91.9±4.3%) and contain 70.6±5.6mgg(-1) octadecyl ferulate, although, minor hydrolysis (∼4%) of the octadecyl ferulate was observed under the excess steam jet-cooking conditions utilized. This study demonstrates that steam jet cooking is a rapid and scalable process in which to prepare amylose-octadecyl ferulate inclusion complexes.