Neonatal subchronic toxicity and in vitro genotoxicity studies of the human-identical milk oligosaccharide 3-fucosyllactose.

Human milk oligosaccharides, such as 3-fucosyllactose (3-FL), are bioactive components of breast milk associated with benefits for infant growth and development. Structurally identical compounds (human-identical milk oligosaccharides-HiMOs) can be produced using microbial fermentation, allowing their use in infant formula to increase its similarity with human milk. Toxicological studies are required to demonstrate safety of HiMOs and that of any impurities potentially carried over from the manufacturing process. Biotechnologically produced 3-FL was tested for potential genotoxicity (bacterial reverse mutation test and in vitro mammalian micronucleus test) and subchronic toxicity (90-day study with neonatal rats). In the 90-day study, 3-FL was administered by gavage to rats once daily from Day 7 of age, at doses up to 4000 mg/kg body weight (bw)/day (the maximum feasible dose), followed by a 4-week recovery period. Reference controls received 4000 mg/kg bw/day of oligofructose, an ingredient permitted for use in infant formula. Results for the genotoxicity studies were negative. In the 90-day study, there were no adverse effects of 3-FL on any of the parameters measured; thus, the no-observed-adverse-effect level (NOAEL) was 4000 mg/kg bw/day (the highest dose tested). These results support the safety of biotechnologically produced 3-FL for use in infant formula and other foods.

Genotoxicity and neonatal subchronic toxicity assessment of a novel mixture of the human-identical milk oligosaccharides lacto-N-fucopentaose I and 2'-fucosyllactose.

Human milk oligosaccharides (HMOs) are a complex group of bioactive molecules largely observed in human breast milk but also occurring in limited amounts in other mammalian milks. Advances in biotechnology have enabled production of human-identical milk oligosaccharides (HiMOs), structurally identical molecules to HMOs found naturally in human milk, intended for addition to infant formula to more closely replicate breast milk. Biosynthesis of a novel mixture of two major HMOs, lacto-N-fucopentaose I and 2'-fucosyllactose (LNFP-I/2'-FL), recently became possible. To support the safety of LNFP-I/2'-FL for use in infant formula and other foods, it was subject to a safety assessment comprising a bacterial reverse mutation test, an in vitro mammalian cell micronucleus test, and a 90-day oral gavage study in neonatal rats. In the 90-day study (the first HiMO study to include the new endocrine-sensitive endpoints described in the 2018 version of OECD Test Guideline 408), LNFP-I/2'-FL was administered by oral gavage to neonatal rats once daily (from Day 7 of age) for 90 consecutive days, at doses up to 5000 mg/kg bw/day, followed by a 4-week recovery period. Concurrent reference controls received 5000 mg/kg bw/day of the approved infant formula ingredient oligofructose. LNFP-I/2'-FL was nongenotoxic in vitro. The highest dose tested (5000 mg/kg bw/day) was established as the no-observed-adverse-effect level in the 90-day study, as there were no test article-related adverse effects on clinical observations, body weight, food consumption, clinical pathology, and organ weights nor any noteworthy macroscopic or microscopic findings. This supports the safety of LNFP-I/2'-FL for its intended uses in food.

Toxicological safety evaluation of the human-identical milk oligosaccharide 6'-sialyllactose sodium salt.

Human milk oligosaccharides (HMOs) are abundant in breastmilk, but their presence in infant formula is negligible. Sialylated HMOs, such as 6'-sialyllactose, constitute a significant portion of the HMO fraction of human milk and are linked to important biological functions. To produce infant formula that is more comparable with human milk, biosynthesized sialyllactoses known as human-identical milk oligosaccharides (structurally identical counterparts to their respective naturally occurring HMOs in breastmilk) are proposed for use in infant formula and other functional foods for the general population. To support the safety of 6'-sialyllactose sodium salt (6'-SL), a 90-day oral (gavage) toxicity study and in vitro genotoxicity tests were conducted. The 90-day study is the first to be conducted with 6'-SL using neonatal rats (day 7 of age at the start of dosing), thus addressing safety of 6'-SL for consumption by the most sensitive age group (infants). In the 90-day study, neonatal rats received 6'-SL at doses up to 5000 mg/kg body weight (BW)/day and reference controls received 5000 mg/kg BW/day of fructooligosaccharide (an ingredient approved for use in infant formula) for comparison with the high-dose 6'-SL group, followed by a 4-week recovery period. There was no evidence of genotoxicity in vitro. No test item-related adverse effects were observed on any parameter in the 90-day study, thus the high dose (5000 mg/kg BW/day) was established as the no-observed-adverse-effect level. These results confirm that 6'-SL is safe for use in formula milk for infants and in other functional foods for the general population.

Toxicological safety assessment of the human-identical milk oligosaccharide 3'-sialyllactose sodium salt.

Human breastmilk is a mixture of nutrients, hormones and bioactive molecules that are vital for infant growth and development. Infant formula (IF) lacks many of these compounds, most notably human milk oligosaccharides (HMOs), which are abundant in breastmilk but scarce in IF. Sialyllactoses, such as 3'-sialyllactose, constitute a large portion of the HMO fraction. To produce IF that matches breastmilk more closely, biosynthesized human-identical milk oligosaccharides (structurally identical to HMOs) such as 3'-sialyllactose sodium salt (3'-SL) are proposed for use in IF and foods for the general population. The safety assessment of 3'-SL comprised in vitro genotoxicity tests and a 90-day oral (gavage) toxicity study. This is the first 90-day study conducted with 3'-SL using neonatal rats (7 days old at the start of dosing-equivalent age to newborn human infants in terms of central nervous system and reproductive development), demonstrating the safety of 3'-SL for consumption by infants, the most sensitive age group. The neonatal rats received 3'-SL at doses up to 5,000 mg/kg body weight (BW)/day and reference controls received 5,000 mg/kg BW/day of fructooligosaccharide (an ingredient approved for use in IF) for comparison with the high-dose 3'-SL group, followed by a 4-week recovery period. There was no evidence of genotoxicity in vitro. In the absence of any test item-related adverse effects in the 90-day study, the high dose (5,000 mg/kg BW/day) was established as the no-observed-adverse-effect level. This confirms the safety of 3'-SL for use in IF for infants, as well as in functional foods for the general population.

Preclinical safety evaluation of the human-identical milk oligosaccharide lacto-N-tetraose.

Lacto-N-tetraose (LNT) is one of the most abundant oligosaccharides that are endogenously present in human breast milk. To simulate the composition of human breast milk more closely, commercial infant formula can be supplemented with human-identical milk oligosaccharides, which are manufactured structurally identical versions of their naturally occurring counterparts. As part of the safety evaluation of LNT, in vitro genotoxicity tests and a subchronic oral gavage toxicity study (in neonatal Sprague-Dawley rats) were conducted. In the subchronic study, LNT was administered at dose levels of 0, 1,000, 2500 or 4000 mg/kg body weight (bw)/day, once daily for at least 90 days, followed by a 4-week treatment-free period. An identically comprised reference control group received fructooligosaccharides powder (a non-digestible oligosaccharide used in infant formula) at 4000 mg/kg bw/day, to allow for direct comparison against the high-dose LNT group. LNT was non-genotoxic in the in vitro tests. There were no compound-related adverse effects in the 90-day study; therefore, 4000 mg/kg bw/day (the highest feasible dose) was established as the no-observed-adverse-effect-level. These results support the safe use of LNT in infant formula and as a food ingredient, at levels not exceeding those found naturally in human breast milk.

The nutritional role of free sialic acid, a human milk monosaccharide, and its application as a functional food ingredient.

N-Acetyl-d-neuraminic acid (NANA), more commonly known by its trivial name sialic acid, is an endogenous human and ubiquitous nutritional monosaccharide. As a bound sugar at the terminal positions of glycans NANA is known to play important roles in many biological events. The data that exist on the occurrence of the free monosaccharide in breast milk and nutrition, however, are less commonly discussed. In most foods of animal origin, sialic acid occurs as a mixture of NANA and N-glycolyl-d-neuraminic acid (NGNA), a hydroxylated derivative of NANA that is not found in humans. The dietary intake of NGNA has been identified as a risk factor for long-term adverse health effects. Therefore, we present summaries on the biochemistry, metabolism, bioavailability, and the data on NANA and NGNA levels that occur in diverse foods. Finally, we discuss the emerging data demonstrating that free NANA is linked to positive nutritional effects including pronounced antioxidative properties. These data and the extremely high safety profile of NANA justify dietary enrichment at levels that correspond to the dietary intake of NANA in infants through breast milk.

Safety evaluation of the human-identical milk monosaccharide, l-fucose.

l-Fucose is a natural monosaccharide present in mammals where it is found predominantly as an O-glycosidically linked component of glycoproteins, glycolipids, and oligosaccharides. It is also present in its free form in human breast milk (human milk monosaccharide). l-Fucose plays important roles in the development of the immune and nervous systems and is involved in cognitive function and memory formation. The human-identical milk monosaccharide l-fucose is therefore proposed for use in infant formulas to better simulate the free saccharides present in human breast milk. As part of the safety evaluation of l-fucose, a subchronic dietary toxicity study preceded by an in utero phase was conducted in Sprague-Dawley rats. l-Fucose was without maternal toxicity or compound-related adverse effects on female reproduction and general growth and development of offspring at a maternal dietary level up to 1%, equivalent to a dose of 1655 mg/kg body weight (bw)/day. During the subchronic phase, no compound-related adverse effects were observed in first generation rats at dietary levels of up to 1% (highest level tested), corresponding to doses of 516 and 665 mg/kg bw/day in males and females, respectively. l-Fucose was non-genotoxic in a series of in vitro genotoxicity/mutagenicity tests. These results support the safe use of l-fucose in infant formula and as a food ingredient at levels equivalent to those present in human breast milk.

Safety evaluation of the human-identical milk monosaccharide sialic acid (N-acetyl-d-neuraminic acid) in Sprague-Dawley rats.

N-Acetyl-d-neuraminic acid (Neu5Ac) is the predominant form of sialic acid (Sia) in humans, while other mammals express Sia as a mixture with N-glycolyl-d-neuraminic acid (Neu5Gc). Neu5Ac occurs in highest levels in the brain and in breast milk, and is therefore, coined a human-specific milk monosaccharide, and is thought to play an important nutritional role in the developing infant. Synthesized human-identical milk monosaccharide (HiMM) Neu5Ac is proposed for use in infant formulas to better simulate the free saccharides present in human breast milk. As part of the safety evaluation of HiMM Neu5Ac, a subchronic dietary toxicity study preceded by an in utero phase was conducted in Sprague-Dawley rats. Neu5Ac was without maternal toxicity or compound-related adverse effects on female reproduction and on the general growth and development of offspring at a maternal dietary level of up to 2%, equivalent to a dose of 1895mg/kg body weight (bw)/day. During the subchronic phase, no compound-related adverse effects were observed in first generation rats at dietary levels of up to 2% (highest level tested), corresponding to doses of 974 and 1246mg/kgbw/day in males and females, respectively. Neu5Ac also was non-genotoxic in a series of in vitro genotoxicity/mutagenicity tests. These results support the safe use of Neu5Ac both in infant formula and as a food ingredient at levels equivalent to those found naturally in human breast milk.

Safety evaluation of a mixture of the human-identical milk oligosaccharides 2'-fucosyllactose and difucosyllactose.

Human milk oligosaccharides (HMOs) are endogenous indigestible carbohydrates representing the largest compositional difference between human breastmilk and infant formula (IF). Two major HMOs in human breastmilk are 2'-fucosyllactose (2'-FL) and difucosyllactose (DFL); commercial IF can be supplemented with manufactured structurally identical versions of HMOs [known as human-identical milk oligosaccharides (HiMOs)] to better replicate the composition of human milk. As 2'-FL and DFL are always found together in human milk, a mixture of these HiMOs (2'-FL/DFL) has been proposed for use in IF and as a food ingredient. Safety assessment of 2'-FL/DFL included conduct of in vitro genotoxicity tests and a subchronic oral toxicity study. In the subchronic study, 2'-FL/DFL (8:1 ratio) was administered to neonatal rats at doses up to 5000 mg/kg body weight (bw)/day, once daily for 90 days, followed by a 4-week recovery period. A concurrent reference control group received 5000 mg/kg bw/day of an oligosaccharide already used in IF (fructooligosaccharide), for direct comparison with the high-dose 2'-FL/DFL group. No evidence of genotoxicity was observed. In the absence of compound-related adverse effects in the 90-day study, 5000 mg/kg bw/day was established as the no-observed-adverse-effect-level. These results support the use of 2'-FL/DFL in IF and as a food ingredient.

Fragment-based synthesis and SAR of modified FKBP ligands: influence of different linking on binding affinity.

The viability of the fragment-based approach for lead discovery depends on reliable fragment-screening methods combined with straightforward fragment-linking- or fragment-growing-chemistry. In the present study we sought a flexible synthetic approach that would allow efficient synthesis of a variety of linkers that can subsequently be tested for biological activity. We applied this approach to fragments known to bind to FKBP12 (FK506 binding protein), a peptidyl-prolyl isomerase involved in immunosuppression and neural functioning. In our set of linked FKBP ligands, ester and thioester linkages resulted in high-affinity ligands, whereas an amide linkage decreased affinity remarkably; oxime and triazole linkages were not tolerated by the target protein's binding pocket, rendering these ligands ineffective. By investigating corresponding derivatized non-linked fragments and docking studies of linked fragments, we were able to evaluate the effect of the linker region on ligand binding affinity.

A new strategy for the synthesis of dinucleotides loaded with glycosylated amino acids--investigations on in vitro non-natural amino acid mutagenesis for glycoprotein synthesis.

The in vitro non-natural amino acid mutagenesis method provides the opportunity to introduce non-natural amino acids site-specifically into proteins. To this end, a chemically synthesised aminoacylated dinucleotide is enzymatically ligated to a truncated suppressor transfer RNA. The loaded suppressor tRNA is then used in translation reactions to read an internal stop codon. Here we report an advanced and general strategy for the synthesis of the aminoacyl dinucleotide. The protecting group pattern developed for the dinucleotide facilitates highly efficient aminoacylation, followed by one-step global deprotection. The strategy was applied to the synthesis of dinucleotides loaded with 2-acetamido-2-deoxy-glycosylated amino acids, including N- and O-beta-glycosides and O- and C-alpha-glycosides of amino acids, thus enabling the extension of in vitro non-natural amino acid mutagenesis towards the synthesis of natural glycoproteins of high biological interest. We demonstrate the incorporation of the glycosylamino acids--although with low suppression efficiency--into the human interleukin granulocyte-colony stimulating factor (hG-CSF), as verified by the ELISA technique.

In vitro non-natural amino acid mutagenesis using a suppressor tRNA generated by the cis-acting hepatitis delta virus ribozyme.

In vitro non-natural amino acid mutagenesis requires aminoacyl-charged suppressor transfer RNAs which read an internal stop codon. For the synthesis of aminoacyl-tRNAs loaded with non-natural amino acids, T4 RNA ligase is used to ligate a chemically synthesised aminoacyl-dinucleotide to a truncated 74mer tRNA(-CA) lacking the two 3' end nucleotides. The 74mer tRNA(-CA) in turn is generated by run-off transcription from a linearised plasmid encoding the tRNA sequence under control of the T7 promoter. Transcripts with heterogeneous ends are commonly obtained, which interfere with subsequent reactions such as ligation or translation. Here we report an improved procedure for the generation and chromatographic purification of large amounts of homogeneous 3' end tRNA(-CA) by hepatitis delta virus ribozyme cis-cleavage and the first application of this tRNA to in vitro non-natural amino acid mutagenesis. Stop codon suppression is increased compared to conventionally synthesised suppressor tRNA; 2.5 microg of mutated protein was synthesised in a 50 microl batch reaction.