We know that the gut microbiota is important for human health and well-being, regulating host metabolism, shaping the immune system and preventing pathogen colonization.
Restoring balance of diverse microbiota through fecal microbiota transplantation (FMT) has emerged as a potential therapeutic strategy and promising tool for studying the causal relationship of microbiota in disease pathogenesis.
However, FMT poses logistical challenges and potential safety risks, such as the transfer of pathogenic organisms, the potential metastasis of undesirable phenotypes (e.g., obesity), or an increased risk of developing disease later in life.
Therefore, a more controlled and personalized blend of cultured beneficial microorganisms may be a better option.
Most of these beneficial microorganisms will be endogenous symbionts of the host with no long-term history of safe and beneficial use, and are therefore often referred to as next-generation probiotics (NGPs) or live biotherapeutics (LBPs).
The Lactobacillus plantarum strain, with its probiotic and functional properties and its health-promoting effects, stands out for regulating the composition of the intestinal flora well.
An FMT study found that co-production of butyric acid bacteria Anaerobutyricum spp. (formerly known as Eubacterium hallii) was associated with improved insulin sensitivity in subjects with metabolic syndrome. Therefore, further research and development of this potentially beneficial microorganism was set out, with a focus on Anaerobutyricum soehngenii L2-7, among others, because it has the best characteristics.
After completing preclinical trials with Anaerobutyricum soehngenii in a mouse model, the strain was produced under controlled conditions and several clinical studies were conducted to evaluate its safety and efficacy in humans.
This article will take Lactobacillus plantarum as an example to introduce its probiotic characteristics; Taking A. soehingeii as an example, the development for clinical use is introduced, which provides practical guidance for the development and testing of the next generation of probiotics.
01 Definition of next-generation probiotics and live biotherapeutics
Traditional probiotics are defined as "live microorganisms that, when given in sufficient amounts, provide health benefits to the host." These microorganisms have a long history of use and are considered safe.
Note: Generally Recognized as Safe (GRAS) status in the United States and Qualified Safety Presumption (QPS) status in the European Union.
The use of probiotics may represent a therapeutic strategy to modulate the gut microbiota and improve human disease.
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In contrast, next-generation probiotics (NGPs) are microorganisms that have no long-term history of safe beneficial use, and as with traditional probiotics, next-generation probiotics are beneficial to host health when administered in adequate amounts.
In 2012, the U.S. Food and Drug Administration introduced the term Live Biotherapeutic Product (LBP), defined as "a biological product," which:
(1) Contains living organisms, such as bacteria;
(2) suitable for the prevention, treatment or cure of human disease or condition;
(3) Not a vaccine.
LBP is defined in the European Pharmacopoeia (Ph.Eur.) as "a medicinal product for human use containing live microorganisms (bacteria or yeast)". However, because LBP includes formulations of the final product in addition to microorganisms and is defined as a pharmaceutical product, the term should not be systematically used in place of NGP.
NGP is a broader term to include microorganisms present in LBP and microorganisms currently under study that have not yet been formulated in the final product. In addition, NGPs can be used both as food supplements such as traditional probiotics and as pharmaceutical products that prevent, treat or cure diseases. Finally, GM microbes can also be considered NGPs, although most likely marketed as LBPs.
The following diagram illustrates the various definitions.
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02 Lactobacillus plantarum as a probiotic property
Lactobacillus plantarum is one of the most important members of Lactobacillus and is often used as a probiotic due to its excellent probiotic properties (good GI tolerance, adhesion, oxidation, and antimicrobial).
✔ Fights gastrointestinal disorders
An essential characteristic of considering microbes as probiotics is the ability to survive the harsh conditions of the human gastrointestinal tract.
Lactobacillus plantarum MA2 and B23 strains show good tolerability and can survive low pH (2.5-3). Lactobacillus plantarum KU15149 is gastric and bile salt resistant.
✔ Adhesion to intestinal mucosa and / or extracellular matrix components
Mucosa adhering to epithelial cells or components adhering to the extracellular matrix of the intestine are ideal characteristics of probiotic microorganisms, as they will favor colonization and persistence of probiotics in the host.
Two Lactobacillus plantarum strains, DKL3 and JGR2, showed adhesion of 82.8% and 79.6%, respectively.
The intestinal epithelial adhesion rate of Lactobacillus plantarum strains KACC11451 and Wikim0112 is approximately 60–62%.
✔ Antioxidant activity
Some probiotics have been shown to have antioxidant activity that reduces damage caused by oxidative reactions.
- Lactobacillus plantarum DMDL 9010 has excellent antioxidant capacity
- Antioxidant compounds are present in intact cells of Lactobacillus plantarum N-1
- Lactobacillus plantarum Wikim0112 and KACC11451 exhibit over 70% activity similar to superoxide dismutase (SOD)
- Lactobacillus plantarum KU15149 is a highly abundant antioxidant
- Lactobacillus plantarum MA2 has a high antioxidant capacity
✔ Bacteriocin production
Bacteriocins can exert various benefits in food and host, as they extend shelf life and prevent unwanted colonization, respectively. Many strains of Lactobacillus plantarum have been shown to produce bacteriocins that give this microorganism probiotic properties.
Lactobacillus plantarum produces bacteriocins commonly known as plantaritin.
KLDS1.0391, ZJ5, TN635, B23, and AA135 strains are producers of bacteriocin Plantaricin MG, Plantaricin ZJ5, bacteriocin ST28MS and ST26MS, bacteriocin BacTN635, bacteriocin Lac-B23, and Plantaricin AA135, respectively, which have antimicrobial effects against several gram-negative bacteria.
✔ Antimicrobial activity
Probiotics are characterized by inhibition of the growth, development and colonization of pathogenic microorganisms.
During the fermentation metabolism of Lactobacillus plantarum, it produces a variety of antimicrobial compounds (in addition to bacteriocins), which may include organic acids such as lactic acid, citric acid, isobutyric acid and acetic acid, ethanol, diacetyl, and H2O2. Lactobacillus plantarum can also produce exopolysaccharides with natural antifungal activity.
- Cell-free supernatants of Lactobacillus plantarum WiKim0112 and KACC11451 showed the ability to inhibit six foodborne pathogens, i.e., Escherichia coli
- Lactobacillus plantarum DMDL 9010 has potent antimicrobial ingredients, including a variety of organic acids against 4 different pathogens
- Antibacterial ability against pathogenic and opportunistic bacteria (gram-negative and gram-positive) and antifungal ability against 8 fungi were determined in Lactobacillus plantarum SJ14
✔ Native intestinal regulation
The ecological balance of the different species that make up the gut microbiome is essential to prevent communicable and non-communicable diseases and to stop disturbances in the balance of the microbiota. Probiotics have the ability to adjust the composition of the intestinal flora and correct abnormal responses of the immune system, resulting in different beneficial effects on the host.
Lactobacillus plantarum ZJ316 plays a regulatory role in the microbiota in vitro intestinal models, increasing the growth of Veillonella, which can improve the immunity of the human respiratory and digestive systems. At the same time, the presence of Blautia is reduced.
Note: Blautia is associated with intestinal inflammation in obese children.
In addition, the ZJ316 strain reduced the Enterobacteriaceae, including commensal organisms and primary and opportunistic pathogens.
Note: These pathogens can easily multiply in the inflamed gut, leading to an imbalance in the microbiota.
Effects of different strains of Lactobacillus plantarum on intestinal flora
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More information about Lactobacillus plantarum can be found at:
Objective understanding of Lactobacillus plantarum (L. plantarum) and its health benefits
03 Discovery and isolation of Anaerobutyricum soehngenii
As the global obesity epidemic worsens, the incidence of metabolic syndrome increases dramatically, and it is more prone to cardiovascular disease and type 2 diabetes. Dynamic changes in the gut microbiota are associated with the emergence of metabolic syndrome.
Further study of the causal role of the gut microbiota in metabolic syndrome ↓↓↓
Researchers previously infused male subjects with metabolic syndrome with fecal microbiota from lean healthy donors. After 6 weeks of infusion of donor microbiota, peripheral insulin sensitivity increased with the level of butyric acid-producing bacteria compared to the autologous FMT group.
Among these butyric acid-producing bacteria, anaerobic butyric acid producing bacteria are more abundant in the small intestine, suggesting a potential role in regulating insulin sensitivity through butyric acid production.
Since subjects with insulin-resistant metabolic syndrome are characterized by reduced levels of short-chain fatty acid-producing bacteria, oral butyrate improves diet-induced insulin resistance and dyslipidemia in obese mice.
The researchers therefore concluded that A. soehingenii could be a promising next-generation probiotic that could improve insulin resistance.
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In 1996, the A. soehngenii strain L2-7, previously named E. hallii, isolated from infant feces, is a strictly anaerobic, gram-positive, catalase-negative Lachnospiracae bacteria. A. soehngenii is part of the core microbiota of the human gastrointestinal tract. Unlike other known butyrate-producing species, such as Roseburia and Faecalibacterium spp., A. soehingenii has the ability to utilize D- and L-lactate in the presence of acetate. In addition, the genome contains sodium bile acid co-transporter and cholinergic hydrolase genes, suggesting that A. soehnii can influence host bile acid metabolism.
Learn the main points and directions
The development of next-generation probiotics typically employs two strategies.
1
The first approach is to correlate the presence of a particular strain with a healthy phenotype and explore whether that strain has a causal effect on the disease phenotype.
To date, a number of NGP candidates have been identified using sequencing techniques to select strains that are depleted in abundance in diseased subjects or strains associated with successful FMT treatment.
The second strategy is to take a probiotic strain with good characteristics and genetically modify that strain, for example through the production and delivery of bioactive molecules, thereby confering health benefits.
The latter approach will result in genetically modified organisms (GMOs) being subject to specific regulations around the world, such as in the European Union.
2
Regardless of the strategy used to identify or generate NGPs, candidate strains need to be adequately characterized in vitro before any health benefits can be studied in vivo.
The figure below summarizes the most important characteristics that must be evaluated in addition to the genotyping and phenotype of the strain.
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In addition, the origin of the strain and subsequent manipulation or genetic modification must be documented. If any antimicrobial resistance genes or virulence genes are present, the likelihood of transmission of the human microbiota to other microorganisms should be assessed and measures taken to mitigate this risk.
When next-generation probiotics are used in patients with immunosuppressive epithelial barrier damage, the risk of bacterial translocation should be determined. Thorough strain characterization assessment is critical for potential safety concerns regarding the use of NGP in healthy or diseased populations.
04 Anaerobutyricum soehngenii preclinical development
After in vitro testing on A. soehngenii, the researchers turned to animal models to assess the strain's safety and efficacy against insulin sensitivity.
First, a batch of preclinical A. soehngenii was produced under anaerobic conditions.
Briefly, cultures are grown under anaerobic conditions to the end of the exponential stage, concentrated by anaerobic centrifugation, washed with phosphate-buffered saline (PBS), and finally diluted with 10% glycerol to 100 μl of 106, 108, and 1010 colony forming units (CFU) concentrations.
Purity is assessed by 16S rRNA sequencing and microscopic evaluation of cell morphology.
Viability is assessed by the most likely number (MPN) analysis and confirmed by microscopic analysis. Samples were stored directly at −80°C and used within 6 months of production, during which survivability was stable.
In addition, some of these samples were tested for stability over a 2-year period to support product development for clinical trials.
Next, the researchers conducted a dose-finding study in male diabetic (db/db) mice to test the safety and efficacy of oral administration of A. soehingeii for insulin sensitivity and lipid metabolism.
Mice were treated daily with A. soehingeii or placebo (10% glycerol) for 4 weeks, during which no adverse events (normal vital signs) were observed. A significant improvement in insulin sensitivity was observed during the insulin resistance test, which was strongest at the 108CFU dose. This is accompanied by a decrease in liver fat and a decrease in the expression of the Fasn and Acc1 genes, both of which are involved in adipogenesis.
To confirm these findings and further dissect the therapeutic mechanism of A. soehingeii, Prof. Bäckhed's lab conducted a second study of db/db mice.
Mice are treated with 108 CFU of A. soehingeii or heat-inactivated A. soehingeii for 4 weeks. When the body weight remained constant, an increase in resting energy expenditure was observed after active A. soehingeii treatment. In addition, active A. Soehingeii increased fecal butyric acid levels and altered bile acid metabolism compared to heat-inactivated A. soehingeii.
These two mouse studies showed that treatment with A. soehingeii is safe and has beneficial effects on metabolism, which may be mediated by butyrate production and changes in bile acid metabolism. This data is used to obtain ethical approval for our clinical studies conducted in humans.
Recently, a toxicological safety assessment of A. soehingeii CH106, a tetracycline-sensitive derivative from the A. soehingeii strain L2-7T, was performed on a toxicological safety evaluation, indicating that it is safe to ingest at recommended doses.
A. soehingeii was evaluated for genotoxicity and subchronic toxicity in accordance with the requirements of the European Food Safety Authority (EFSA) and the US Food and Drug Administration (FDA) for safety assessments of new non-absorbable food ingredients. Neither bacterial reverse mutation nor in vitro mammalian cell micronucleus tests showed genotoxic effects.
In addition, no adverse events associated with A. soehingeii feeding were found in 90-day subchronic toxicity in rats, and no adverse events exceeding the recommended daily intake for humans were not found even at the highest dose (5×1011 CFU/kg body weight/day).
These findings support the safety of oral A. soehingeii as a food supplement.
Learn the main points and directions
During preclinical development, sufficient pharmacological and toxicological information should be provided to support the proposed clinical trial.
Safety and toxicity studies of NGP are challenging↓↓
Since the product does not usually reach the systemic circulation, but its metabolites or its activity may directly or indirectly affect the physiological functions of the body, efficacy and toxicity are not necessarily dose-dependent.
Other factors, such as human physiology and microbiota composition, may affect safety and efficacy.
Since most NGPs co-evolve with a human host (the all-living concept), it is difficult to translate the results of animal studies into the human environment.
Therefore, it is strongly recommended to combine in vitro, ex vitro, and in vivo models to establish a global safety profile adapted to the risk of the intended population.
Safety and toxicity studies are typically conducted in accordance with the Good Laboratory Practice (GLP) principles of the Organisation for Economic Co-operation and Development (OECD). However, this can be difficult due to the need for innovative methods and models (e.g., artificial models of the human gastrointestinal tract) that may be neither verifiable nor validated at the GLP level.
For food ingredients and dietary supplements, EFSA recommends a grading approach to toxicology studies.
This grading method assesses the toxicokinetics, genotoxicity, subchronic and chronic toxicity, carcinogenicity, and teratogenicity of NGP, balancing data requirements and risks. This method is also used for the toxicological safety assessment of A. soehingeii CH106. If NGP is intended to be used as a pharmaceutical product in a sick population, it must demonstrate safety in the target population.
The preceding figure summarizes the most important issues that must be addressed, such as the effect of therapeutic dose and duration on toxicity, and the potential for teratogenicity, carcinogenicity, and genotoxicity.
05 Production of A. soehngenii suitable for clinical trials
Before A. soehngenii can be given orally to humans, a product suitable for clinical trials must be manufactured.
At the time of approval by an independent ethics committee (2014), A. soehngenii was considered a probiotic and must comply with the Dutch "Warenwet", which complies with the EU dietary supplement regulation. This means that production must be carried out according to Hazard Analysis and Critical Control Points (HACCP) standards. Clinical intervention studies can be conducted according to HACCP criteria.
Growth medium
First, the growth medium was further optimized for the mass production of food-grade products. The composition is based on previous experience:
(1) Conversion of laboratory chemicals into food-grade sources
(2) Use only animal-free components (no heme or meat peptone)
(3) Complexity reduction (removal/reduction of trace minerals, vitamins, carbon sources and organic acids)
(4) Biomass production has been further improved. Raw materials are sourced from audited and reliable suppliers to ensure high quality. Prior to fermentation, growth medium is prepared and sterilized in a large fermenter system, which is completely anaerobic by nitrogen (N2) rinsing.
Fermentation
Fermentation takes place in four sequential steps, as shown in the figure below:
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First, inoculate a small amount of food-grade medium with carefully prepared A. soehnii frozen seed stock. The same strain was used in animal studies, therefore, the strain had good properties, was viable, pure, and did not have any bacterial or viral contaminants. After 24 h of fermentation at 37 °C, inoculate with 1 L of medium using culture and ferment again for 18 h.
Then, use this secondary seed culture to inoculate 30 L of medium in a small fermenter. The fermentation tank ferments for 17 hours as a test run for large-scale fermentation.
Finally, inoculate 290 L medium in a large fermenter with inoculum from a 10 L small fermenter. Control the temperature, pH, and oxygen levels of small and large fermenters and determine the fermentation time (14 to 18 h) using the optical density (OD) of the culture. After 16 hours of fermentation in a large fermenter, A. soehngenii grows to an OD of about 10.
Concentrate and wash
Use of hollow fiber membranes (Koch membrane systems; HF3043-25-43-PM500； HF3043-16-106-PM500) and PBS diafiltration, concentrate and wash cells. The fermentation broth is cooled to 10°C, pumped through an anaerobic membrane unit, and concentrated to 40–50 L in 3 hours.
Diafiltration is performed in the second stage to reduce the level of medium components and fermentation products. The wash buffer is sterilized, degassed, and added directly to the returned cell stream into the fermenter using ultra-high heat. After 6 h, cells are concentrated approximately 20-fold to 15 liters, 99.8% of the medium compound is discarded as waste, and only 2.9% of the medium component remains in the final concentrate.
Finally, 9 L of product can be harvested from the system into a 10 L sterile N2 rinse container.
Preparation of the final product
Four different batches were produced for clinical studies, including 600 tubes and a placebo lot, in which PBS contained 10 mL of A. soehngenii, PBS + 10% glycerol, and only 10% glycerol in PBS at concentrations of 106, 108, and 1010 CFU/mL.
For each batch, prepare 7 L bottles with glycerol and PBS for further dilution, autoclave, cool and rinse with N2. From the 9 L harvested concentrate, add the necessary volume to these bottles to obtain the correct concentration. With continuous stirring and N2 rinsing, place the bottle on ice.
The 10 mL tube is first filled with N2 and then the 10 mL product is filled with a dosing tube pump. Close the tube immediately, label it, and place it in the -30 °C freezer within 10 minutes of filling. All filling takes place inside a disinfection laminar flow cabinet.
quality control
During the manufacturing process, temperature, pH and oxygen levels are continuously monitored. In addition, cell count and OD were determined at each step of the process, as well as the presence of any contaminants. Since anaerobic bacteria are difficult to count quantitatively on agar plates, MPN analysis is performed under anaerobic conditions to obtain the number of viable cells and cell morphology is assessed with a microscope. All of the above quality controls are carried out for packaged vials that meet human consumption standards.
Anaerobutyricum soehingeii intermediate and final product specification
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Subsequently, the stability of the vials produced is tested every 6 months. After production, these vials have a "shelf life" of 6 months, which is required by Dutch law for food. If the viability and purity criteria are met, there is an opportunity to extend the expiration date of the vial.
The table below shows the potency and purity of vials with the highest dose of A. soehngenii over a 3-year time period.
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Learn the main points and directions
▸ Industrial-scale production is a technical challenge
Producing strains on an industrial scale has different strain and media requirements than lab-scale culture. Therefore, when a strain qualifies for potential NGP, steps should be taken to see if the strain can be cultured on an industrial scale.
One of the technical challenges is the stringent conditions required to grow NGPs, such as the need for specific nutrients, lack of oxygen, stable temperature, and suitable pH. In addition, longer holding times, absolute pressure pumping, downstream purification processes, and storage can negatively affect the viability of bacterial cells.
▸ Addition of the strain to the product requires an effective strategy to deliver it to the site of action
Next, the strain must be added to the product, such as capsules, powders or liquid suspensions. Since most NGPs are strictly anaerobic or facultative anaerobics, exposure to oxygen should be minimized. For this purpose, oxygen permeability in the vessel should be reduced, and antioxidants can be added to reduce the redox potential.
After ingesting the product, NGP must survive in the harsh environment of the gastrointestinal tract. Enteric-coated capsules and microcapsules are an effective strategy to protect bacteria and deliver them to their site of action.
▸ Deliver a sufficient amount of dose before expiry date
Ultimately, manufacturing needs to produce a robust, stable product that will allow effective doses to be delivered in sufficient quantities of NGP before the expiration date.
▸ Quality control and quality assurance programs need to be in place
For pharmaceutical products or LBP, it needs to be manufactured in accordance with Good Manufacturing Practices (GMP). For food and dietary supplements, production in HACCP certified factories is standard. In any case, quality control and quality assurance programs need to be in place to ensure consistent quality of ingredients and final products, as well as a reliable production process.
The manufacturing process of the strain should be clearly documented from the raw materials used, cell banking systems, cell growth and harvesting, purification and downstream processing to in-process testing.
▸ Thoroughly describe the manufacture of the final product
Similarly, the manufacture of the final product must be thoroughly described, including production records and recipes, filling, labeling, and packaging instructions. For strain and product manufacturing, the risk of cross-contamination with other products produced in the same room or in the same contact equipment must be assessed.
▸ The strain and product specifications must be described
Includes a description of the sampling procedure and verification test methods. These specifications should describe identity, potency, purity, contamination, appearance and, if applicable, additional tests for percentage of viable cells, particulate matter, pyrogen, pH, and residual moisture.
▸ Stability data must be generated
Demonstrate that the product is stable in terms of potency and contamination for the planned life of use.
For frozen products, the effects of multiple freeze-thaw cycles should be assessed, while for lyophilized products, the shelf life after reconstitution should be discussed.
▸ The environmental impact of the product needs to be assessed
Especially when the strain is genetically modified, pathogenic, ecologically more suitable than wild type, or difficult to eradicate.
06 Clinical trial of Anaerobutyricum soehingeii
Safety/dose discovery trials
To validate mouse data in the human environment, the researchers established a single-blind, phase I/II dose-escalation trial to determine the safety and efficacy of Anaerobutyricum soehingeii in obese, insulin-resistant subjects.
In the study, 27 obese Caucasian men with metabolic syndrome were enrolled and assigned to receive Soehngenii at doses of 107, 109 or 1011 cells/day for 28 days.
When subjects blind test their respective therapeutic doses, the first 9 subjects must successfully complete the study protocol with the lowest dose before the dose is increased to a higher concentration.
Participants store cryoval at −20°C at home, thaw one 10 mL bottle per day, mix it with 100 mL of milk, and take it orally. Milk is added to increase the pH in the stomach, thus protecting living cells during gastrointestinal passage. The primary outcome was safety, in addition, the effect on insulin sensitivity and lipolysis was evaluated after 4 weeks of treatment.
Treatment with A. soehngenii up to 1011 cells/day was well tolerated without any serious adverse events.
When all treatments are combined, fecal abundance of A. soehngenii is associated with improved peripheral insulin sensitivity, accompanied by beneficial changes in the distribution of bile acids.
Unexpectedly, no increase in fecal butyrate levels was observed, which can be explained by the volatility of short-chain fatty acids and the detection limits of assays, which makes butyrate difficult to measure.
The increase in the abundance of A. soehngenii is short-lived, most disappearing 2 weeks after cessation. The viability of the administered strain is negatively affected by gastric acid and oxygen.
However, as indicated by the highest replication signal in the feces of subjects receiving the highest dose, A. soehngenii was partially able to survive in the gastrointestinal tract. Better protection of strains from acidic and oxygenated environments through encapsulation and/or freeze-drying can further improve viability (and therapeutic effectiveness).
Different management methods and behaviors
To further elucidate the mode of action of A. soehngenii in humans, a randomized placebo-controlled crossover trial was conducted in which the strain was administered directly in the duodenum, thereby avoiding gastric acid and reducing oxygen exposure.
Since the small intestine plays a central role in glucose sensitization, regulation of insulin sensitivity/secretion, and glucose homeostasis, it is hypothesized that direct duodenal infusion of A. soehngenii can further improve therapeutic efficacy.
Similarly, obese subjects with metabolic syndrome (N = 12) were enrolled and randomized to receive a single nasalduodenal infusion with the highest dose of A. soehngenii (1011 cells) or placebo (10% glycerol in PBS). After 6 hours, a duodenal biopsy and mixed diet test are performed.
In addition, subjects monitored blood glucose for 24 hours and collected some stool samples. After a 4-week flush period, subjects switched to another treatment group, which in the first trial was determined to be long enough to reduce stress.
Again, this study shows that A. soehngenii is safe and well tolerated for administration. Treatment with this strain increases the postprandial drift of the insulin-stimulating hormone glucagon-like peptide 1 (GLP-1), accompanied by a decrease in glucose variability.
Given that A. soehngenii has the ability to produce butyrate, and that butyrate levels in feces tend to be higher after A. soehngenii treatment, the increased GLP-1 secretion may be a result of butyrate's activation of G protein-coupled receptor 43 (GPR43) on intestinal L cells.
Since A. soehngenii expresses sodium bile acid co-transporter and bile acid hydrolase, and plasma levels of secondary bile acids are elevated, increased GLP-1 expression may also be a result of TGR5 activation by secondary bile acids.
Note: TGR5 is a member of the G protein-coupled receptor superfamily, TGR5 is not only a bile acid receptor, but also a receptor for a variety of selective synthetic agonists, a derivative that regulates different signaling pathways. Involved in energy homeostasis, bile acid balance and glucose metabolism.
In addition, treatment with A. soehingeii resulted in a decrease in the expression of the duodenal nuclear farnesin X receptor (FXR) and its target gene OSTa, which may also be responsible for the increased availability of GLP-1.
Finally, the improvement in glucose variability can be explained by the insulin sensitization of GLP-1 and butyrate.
In addition, A. soehingeii altered duodenal transcription in 73 genes, most notably by inducing the expression of REG1B and REG1A, which encode the production of islet-derived protein 1A/B.
Note: Reg1A and Reg1B are strongly expressed in Paneth cells at the bottom of the intestinal crypt, secreted in the lumen, and may act locally by inducing progenitor cell or L cell proliferation.
In addition, induction of REG1B was found to be associated with increased GLP-1 secretion and decreased glucose variability 24 h after administration of A. soehngenii. Treatment with a single dose of A. soehingeii does not affect the composition or diversity of the microbiota, as seen in previous studies.
In addition, the abundance of fecal A. soehngenii did not change over time, excluding microbiota-mediated carryover effects at the time of crossover.
Learn the main points and directions
The main purpose of the first clinical study is to determine safety and determine the appropriate dosage range and regimen based on the tolerability of the product. This includes determining the minimum effective dose or optimal effective dose range and, if possible, the maximum safe dose.
In addition to drug delivery, the focus should be on obtaining safety data to identify common product-related adverse events. These early-stage clinical studies are typically conducted in healthy volunteers, but it may be more appropriate to include patients, such as when NGP should correct the biological dysregulation. Risk mitigation measures to ensure the safety of study participants, such as continuous enrollment, dose escalation, and monitoring by independent data monitoring committees, should be considered.
In addition, it is advantageous to monitor translocation, inflammation, and infection, as well as to determine the persistence of NGP and its role after final administration.
It is important to consider other confounding factors that affect the function or composition of the microbiota, such as age, diet, lifestyle, and environmental factors. In this regard, studies employing placebo-controlled crossover designs are useful because they limit the influence of this extrinsic and intrinsic confounding factor, allowing for smaller sample sizes. Needless to say, blinds are very important and the flushing period should be carefully considered.
Increasingly, baseline microbiota composition is also being incorporated into screening criteria, such as looking for the presence or cluster of specific bacterial populations in specific gut types. This will lead to more comparable study groups and optimize the effect of the intervention when specific flora are involved in the mechanism of action.
07 Regulatory framework: Next generation probiotics
According to the FAO and WHO definitions of probiotics, probiotics can be divided into dietary supplements and medicines, but there are huge regulatory differences between the two. Similarly, products containing next-generation probiotics can enter the market as food, dietary supplements, or pharmaceuticals, depending on the intended use.
In the European Union, food is regulated by the European Food Safety Authority and medicines by the European Medicines Agency, while in the United States, the Food and Drug Administration is responsible for both types of products. The product is considered a medical product or medical device when the intended use is related to the prevention, mitigation or treatment of disease.
Oral intake products associated with enhanced physiological function or reduced disease risk factors may be classified as functional foods or food supplements. In addition, topical products with pure cosmetic functions can be rated as cosmetic. To ensure compliance, prior to preclinical research and manufacturing, the use of indentation and subsequent regulatory classification must be decided.
Functional foods or dietary supplements
In the European Union, "food" is defined as "any substance or product, whether processed, partially processed or unprocessed, intended or reasonably expected to be ingested by humans". Each category is managed accordingly in accordance with the general requirements and regulations for labeling, display, and advertising.
When NGPs are used as food or dietary supplements, they are likely to be considered a novel food. However, if NGP is genetically modified, it will be regulated as a genetically modified food. In order for NGP to enter the market as a novel food product, it needs to be authorized and listed in the European Union.
One of the most important conditions is that NGP does not pose a risk to human health, which must be supported by scientific evidence. This includes a comprehensive risk assessment to assess potential risks to human health in conjunction with biological and toxicological studies of anticipated human exposure. In addition, the application should contain a detailed description of the NGP, manufacturing process, product composition, analytical methods used, labeling, and conditions of intended use.
In addition to safety, the product must not facilitate the spread of antimicrobial resistance in the food chain or in the environment, requiring phenotypic and genotypic assessment of antimicrobial resistance.
Even claims of "containing probiotics/prebiotics" are considered health claims in the EU. In order to accept health claims, NGPs need to be properly described and proven by high-quality studies of their beneficial effects and causation.
Live biotherapeutic products
Since 2012 and 2019, the FDA and EDQM have clarified the quality requirements for LBP, which is described as a pharmaceutical product containing live microorganisms intended for human use. Other than these quality requirements, there are currently no specific LBP regulations.
However, because LBPs contain live microorganisms, they are considered biomedical products and therefore must comply with legislative and regulatory frameworks. Without a specific LBP subcategory, developers will have to rely on regulatory concepts for biomedical products in other subcategories.
One of the concepts is a thorough risk-benefit analysis based on quality, safety, and efficacy data obtained from preclinical and clinical studies.
Other relevant guidelines for preclinical and clinical study design include:
- International Council for Harmonization of Technical Requirements for Medicinal Products for Human Use (ICH) Guidelines on General Considerations in Clinical Trials (ICHE8);
- Council for Medicinal Products for Human Use (CHMP) strategic guidance on identifying and mitigating the risks of first human and early clinical trials of investigational drugs;
- CHMP guidelines for human cell-based medicines.
To date, no LBPs have entered the EU market, partly due to the lack of a clear regulatory framework. In the absence of clear guidelines, engage with competent authorities early to discuss uncertainty and the importance of risk reduction.
08 Conclusion
As more is known about our gut microbiome, more and more potential next-generation probiotics will be discovered and developed. This article uses A. soehingeii as an example to introduce the experience of developing it as a next-generation probiotic.
Importantly, these new strains have good characteristics, high quality, and safety. A thorough safety assessment of NGPs is important (albeit complex), especially since efficacy and toxicity are not necessarily dose-dependent.
Due to the relative immaturity of this field, there is currently no specific LBP regulation, so communicating with regulators in the early stages of development can help reduce risks and clarify any uncertainties. This requires a clear view of the route of the market (food or drug) at the early stages of development.
After FMT intervention, A. soehingeii was identified as a potentially beneficial microorganism and showed promising results in preclinical in vitro and in vivo studies, as well as in human studies. It has shown good results in improving insulin sensitivity, increasing GLP-1 secretion, and reducing glucose variability.
These effects may be mediated by the production of butyric acid and secondary bile acids. By better protecting the strain from acidic and oxygenated environments, for example through freeze-drying and encapsulation, viability can be potentially improved and thus therapeutic efficacy can be enhanced.
Main references:
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