Probiotics: From Bench to Market

Web Sites, Books, and Articles

International Scientific Association for Probiotics and Prebiotics
This association is a nonprofit collaboration of scientists dedicated to advancing the science of probiotics and prebiotics.

Group Danone
Group Danone is a health and nutrition company that produces dairy products, bottled waters, and baby and medical nutrition products.

WGO Practice Guideline — Probiotics and Prebiotics
The WGO Practice Guideline is a set of clinical practice guidelines for the use of probiotics and prebiotics from the World Gastroenterology Organisation.

Human Microbiome Project
The Human Microbiome Project is a research program initiated by the NIH Roadmap intended to generate resources leading to a comprehensive characterization of the human microbiota and its role in human health and disease.

An Introduction to Probiotics
The National Center for Complementary and Alternative Medicine has developed an informational page on probiotics.

Dietary supplements: Industry Information and Regulations
This page provides FDA guidance on dietary supplement labeling, new dietary ingredients, and other areas of interest to probiotic manufacturers.

FDA Guidance for Industry: Evidence-Based Review System for the Scientific Evaluation of Health Claims
This page provides recent FDA guidance on the use of health claims in food product labeling.

Duan F, March JC. 2010. Engineered bacterial communication prevents Vibrio cholerae virulence in an infant mouse model. Proc. Natl. Acad. Sci. USA 107: 11260-11264.

Gartlehner G, Jonas DE, Morgan LC, et al. 2007. Drug Class Review on Constipation Drugs: Final Report [Internet]. Oregon Health & Science University, Oregon.

Articles

Mary Ellen Sanders

Sanders ME, Merenstein DJ. 2010. Use of probiotic yogurts in health and disease. In Floch MH, Kim AS, eds., Probiotics: A Clinical Guide. Slack Inc., Thorofare, NJ.

Douglas LC, Sanders ME. 2008. Probiotics and prebiotics in dietetics practice. J. Am. Diet Assoc. 108: 510-521.

Lenoir-Wijnkoop I, Sanders ME, Van Loo J, et al. 2007. Probiotic and prebiotic influence beyond the intestinal tract. Nutr. Rev. 65: 469-489.

Sanders ME. 2009. How do we know when something called "probiotic" is really a probiotic? A guideline for consumers and healthcare professionals. Functional Food Rev. 1: 3-12. (PDF, 205 KB) Full Text

Sanders ME. 2008. Clinical use of probiotics: what physicians need to know. Am. Fam. Physician. 78: 1026. Full Text

Sanders ME. 2008. Probiotics: definition, sources, selection, and uses. Clin. Infect. Dis. 46 Suppl 2: S58-S61; discussion S144-S151.

Sanders ME, Akkermans LMA, Haller D, et al. 2010. Safety assessment of probiotics for human use. Gut Microbes 1:164-185. (PDF, 1.69 MB) Full Text

Sanders ME, Gibson GR, Gill H, Guarner F. 2007. Probiotics in food: their potential to impact human health. Council for Agricultural Science and Technology (CAST), Issue Paper 36, CAST, Ames, Iowa.

Sanders ME, Marco M. 2010. Food formats for effective delivery of probiotics. Ann. Rev. Food Sci. Technol. 1: 65–85.

Shane AL, Cabana MD, Vidry S, et al. 2010. Guide to designing, conducting, publishing and communicating results of clinical studies involving probiotic applications in human participants. Gut Microbes 1: 1-10. (PDF, 905 KB) Full Text

Yehuda Ringel

Ringel-Kulka T,Ringel Y. 2007. Probiotics in irritable bowel syndrome: has the time arrived? Gastroenterology 132: 813-816; discussion 816.

Ringel Y,Carroll IM. 2009. Alterations in the intestinal microbiota and functional bowel symptoms. Gastrointest. Endosc. Clin. N. Am. 19: 141-150, vii.

Emeran Mayer

Jarcho JM, Chang L, Berman M, et al. 2008. Neural and psychological predictors of treatment response in irritable bowel syndrome patients with a 5-HT3 receptor antagonist: a pilot study. Aliment. Pharmacol. Ther. 28: 344-352.

Kilpatrick LA, Ornitz E, Ibrahimovic H, et al. 2010. Sex-related differences in prepulse inhibition of startle in irritable bowel syndrome (IBS). Biol. Psychol. 84: 272-278.

Labus JS, Naliboff BD, Berman SM, et al. 2009. Brain networks underlying perceptual habituation to repeated aversive visceral stimuli in patients with irritable bowel syndrome. Neuroimage. 47: 952-960.

Mayer EA, Aziz Q, Coen S, et al. 2009. Brain imaging approaches to the study of functional GI disorders: a Rome working team report. Neurogastroenterol. Motil. 21: 579-596.

Rhee SH, Pothoulakis C, Mayer EA. 2009. Principles and clinical implications of the brain-gut-enteric microbiota axis. Nat. Rev. Gastroenterol. Hepatol. 6: 306-314.

Saito YA, Mitra N,Mayer EA. 2010. Genetic approaches to functional gastrointestinal disorders. Gastroenterology 138: 1276-1285.

Seminowicz DA, Labus JS, Bueller JA, et al. 2010. Regional gray matter density changes in brains of patients with irritable bowel syndrome. Gastroenterology 139: 48-57 e2.

Tillisch K, Wang Z, Kilpatrick L, et al. 2008. Studying the brain-gut axis with pharmacological imaging. In Goetzl EJ, ed. Neural Signaling: Opportunities for Novel Diagnostic Approaches and Therapies. Annals of the New York Academy of Sciences Vol. 1144.

Videlock EJ, Adeyemo M, Licudine A, et al. 2009. Childhood trauma is associated with hypothalamic-pituitary-adrenal axis responsiveness in irritable bowel syndrome. Gastroenterology 137: 1954-1962.

Mansour Mohamadzadeh

Curiel TJ, Morris C, Brumlik M, et al. 2004. Peptides identified through phage display direct immunogenic antigen to dendritic cells. J. Immunol. 172: 7425-7431. Full Text

Mohamadzadeh M. 2010. Induction of protective immunity against microbial challenge by targeting antigens expressed by probiotic bacteria to mucosal dendritic cells. Curr. HIV Res. 8: 323-329.

Mohamadzadeh M, Duong T, Hoover T, et al. 2008. Targeting mucosal dendritic cells with microbial antigens from probiotic lactic acid bacteria. Expert Rev. Vaccines 7: 163-174.

Mohamadzadeh M, Duong T, Sandwick SJ, et al. 2009. Dendritic cell targeting of Bacillus anthracis protective antigen expressed by Lactobacillus acidophilus protects mice from lethal challenge. Proc. Natl. Acad. Sci. USA 106: 4331-4336. Full Text

Mohamadzadeh M, Klaenhammer TR. 2008. Specific Lactobacillus species differentially activate Toll-like receptors and downstream signals in dendritic cells. Expert Rev. Vaccines 7: 1155-1164.

Mohamadzadeh M, Olson S, Kalina WV, et al. 2005. Lactobacilli activate human dendritic cells that skew T cells toward T helper 1 polarization. Proc. Natl. Acad. Sci. USA 102: 2880-2885. Full Text

Tournier JN,Mohamadzadeh M. 2010. Key roles of dendritic cells in lung infection and improving anthrax vaccines. Trends Mol. Med. 16: 303-312.

Justin Sonnenburg

Backhed F, Ley RE, Sonnenburg JL, et al. 2005. Host-bacterial mutualism in the human intestine. Science 307: 1915-1920.

Lecuit M, Sonnenburg JL, Cossart P, et al. 2007. Functional genomic studies of the intestinal response to a foodborne enteropathogen in a humanized gnotobiotic mouse model. J. Biol. Chem. 282: 15065-15072. Full Text

Marco ML, Peters TH, Bongers RS, et al. 2009. Lifestyle of Lactobacillus plantarum in the mouse caecum. Environ. Microbiol. 11: 2747-2757.

Sonnenburg ED, Sonnenburg JL, Manchester JK, et al. 2006. A hybrid two-component system protein of a prominent human gut symbiont couples glycan sensing in vivo to carbohydrate metabolism. Proc. Natl. Acad. Sci. USA 103: 8834-8839. Full Text

Sonnenburg ED, Zheng H, Joglekar P, et al. 2010. Specificity of polysaccharide use in intestinal bacteroides species determines diet-induced microbiota alterations. Cell 141: 1241-1252.

Sonnenburg JL, Angenent LT, Gordon JI. 2004. Getting a grip on things: how do communities of bacterial symbionts become established in our intestine? Nat. Immunol. 5: 569-573.

Sonnenburg JL, Chen CT, Gordon JI. 2006. Genomic and metabolic studies of the impact of probiotics on a model gut symbiont and host. PLoS Biol. 4: e413. Full Text

Sonnenburg JL, Xu J, Leip DD, et al. 2005. Glycan foraging in vivo by an intestine-adapted bacterial symbiont. Science 307: 1955-1959.

Colin Hill

Corr SC, Hill C, Gahan CG. 2009. Understanding the mechanisms by which probiotics inhibit gastrointestinal pathogens. Adv. Food Nutr. Res. 56: 1-15.

Cotter PD, Draper LA, Lawton EM, et al. 2008. Listeriolysin S, a novel peptide haemolysin associated with a subset of lineage I Listeria monocytogenes. PLoS Pathog. 4: e1000144. Full Text

O'Shea EF, O'Connor PM, Cotter PD, et al. 2010. Synthesis of trypsin-resistant variants of the Listeria-active bacteriocin salivaricin P. Appl. Environ. Microbiol. Jun 25. [Epub ahead of print]

Liu L, O'Conner P, Cotter PD, et al. 2008. Controlling Listeria monocytogenes in Cottage cheese through heterologous production of enterocin A by Lactococcus lactis. J. Appl. Microbiol. 104: 1059-1066.

O'Shea EF, Gardiner GE, O'Connor PM, et al. 2009. Characterization of enterocin- and salivaricin-producing lactic acid bacteria from the mammalian gastrointestinal tract. FEMS Microbiol Lett. 291: 24-34.

Piper C, Draper LA, Cotter PD, et al. 2009. A comparison of the activities of lacticin 3147 and nisin against drug-resistant Staphylococcus aureus and Enterococcus species. J. Antimicrob. Chemother. 64: 546-551.

Rea MC, Sit CS, Clayton E, et al. 2010. Thuricin CD, a posttranslationally modified bacteriocin with a narrow spectrum of activity against Clostridium difficile. Proc. Natl. Acad. Sci. USA 107: 9352-9357.

Walsh MC, Gardiner GE, Hart OM, et al. 2008. Predominance of a bacteriocin-producing Lactobacillus salivarius component of a five-strain probiotic in the porcine ileum and effects on host immune phenotype. FEMS Microbiol. Ecol. 64: 317-327.

Glenn Gibson

Bialonska D, Ramnani P, Kasimsetty SG, et al. 2010. The influence of pomegranate by-product and punicalagins on selected groups of human intestinal microbiota. Int. J. Food Microbiol. 140: 175-182.

Costabile A, Kolida S, Klinder A, et al. 2010. A double-blind, placebo-controlled, cross-over study to establish the bifidogenic effect of a very-long-chain inulin extracted from globe artichoke (Cynara scolymus) in healthy human subjects. Br. J. Nutr. 1-11.

Goulas T, Goulas A, Tzortzis G, et al. 2009. Comparative analysis of four β-galactosidases from Bifidobacterium bifidum NCIMB41171: purification and biochemical characterisation. Appl. Microbiol. Biotechnol. 82: 1079-1088.

Goulas T, Goulas A, Tzortzis G, et al. 2009. Expression of four β-galactosidases from Bifidobacterium bifidum NCIMB41171 and their contribution on the hydrolysis and synthesis of galactooligosaccharides. Appl. Microbiol. Biotechnol. 84: 899-907.

Ogue-Bon E, Khoo C, McCartney AL, et al. 2010. In vitro effects of synbiotic fermentation on the canine faecal microbiota. FEMS Microbiol. Ecol.

Ramnani P, Gaudier E, Bingham M, et al. 2010. Prebiotic effect of fruit and vegetable shots containing Jerusalem artichoke inulin: a human intervention study. Br. J. Nutr. 1-8.

Reid G, Gibson G, Sanders ME, et al. 2008. Probiotic prophylaxis in predicted severe acute pancreatitis. Lancet 372: 112-113; author reply 114.

Saulnier DM, Kolida S, Gibson GR. 2009. Microbiology of the human intestinal tract and approaches for its dietary modulation. Curr. Pharm. Des. 15: 1403-1414.

Saulnier DM, Spinler JK, Gibson GR, et al. 2009. Mechanisms of probiosis and prebiosis: considerations for enhanced functional foods. Curr. Opin. Biotechnol. 20: 135-141. Full Text

Silk DB, Davis A, Vulevic J, et al. 2009. Clinical trial: the effects of a trans-galactooligosaccharide prebiotic on faecal microbiota and symptoms in irritable bowel syndrome. Aliment. Pharmacol. Ther. 29: 508-518.

Dan Merenstein

Merenstein D, Murphy M, Fokar A, et al. 2010. Use of a fermented dairy probiotic drink containing Lactobacillus casei (DN-114 001) to decrease the rate of illness in kids: the DRINK study. A patient-oriented, double-blind, cluster-randomized, placebo-controlled, clinical trial. Eur. J. Clin. Nutr. 64: 669-677.

Merenstein DJ, Foster J, D'Amico F. 2009. A randomized clinical trial measuring the influence of kefir on antibiotic-associated diarrhea: the measuring the influence of Kefir (MILK) Study. Arch. Pediatr. Adolesc. Med. 163: 750-754.

Merenstein DJ, Smith KH, Scriven M, et al. 2010. The study to investigate the potential benefits of probiotics in yogurt, a patient-oriented, double-blind, cluster-randomised, placebo-controlled, clinical trial. Eur. J. Clin. Nutr. 64: 685-691.

Patricia Hibberd

Bousvaros A, Guandalini S, Baldassano RN, et al. 2005. A randomized, double-blind trial of Lactobacillus GG versus placebo in addition to standard maintenance therapy for children with Crohn's disease. Inflamm. Bowel Dis. 11: 833-839.

Doron SI, Hibberd PL, Gorbach SL. 2008. Probiotics for prevention of antibiotic-associated diarrhea. J. Clin. Gastroenterol. 42 Suppl 2: S58-S63.

Floch MH, Walker WA, Guandalini S, et al. 2008. Recommendations for probiotic use — 2008. J. Clin. Gastroenterol. 42 Suppl 2: S104-S108.

Hibberd PL, Davidson L. 2008. Probiotic foods and drugs: impact of US regulatory status on design of clinical trials. Clin. Infect. Dis. 46 Suppl 2: S137-S140; discussion S144-S151.

Sponsors

Grant Support

This event was supported by an educational grant from The Dannon Company, Inc.

Funding for this conference was made possible [in part] by 1 R13 AI 088836 - 01 from the National Institute of Allergy and Infectious Diseases. The views expressed in written conference materials or publications and by speakers and moderators do not necessarily reflect the official policies of the Department of Health and Human Services; nor does mention of trade names, commercial practices, or organizations imply endorsement by the U.S. Government.

One of the consequences of the genetic revolution in biology has been the increasing realization that much of the genetic material found in and on the surface of the human body does not belong to us. It belongs instead to the huge numbers of microorganisms that call our bodies their homes; organisms that have been estimated to outnumber human cells by a factor of ten to one. The National Institutes of Health has established a core research program, the Human Microbiome Project, to develop the resources needed to identify the thousands of species involved, and to investigate their roles in human health and disease.

Nowhere is the importance of these microorganisms more apparent than in the complex ecosystem of the human gut, where an important component, known as the gut microbiota, flourishes on the food we ingest, often providing digestive capabilities that we ourselves lack. Putting two and two together, biologists and medical researchers have come to suspect that this microbiota is strongly influenced by the foods we eat, including foods that contain live microorganisms (eg. live and active cultures) and probiotics. Many of these foods—for example, fermented dairy products such as yogurt—have long been associated with maintaining & promoting human health. The most widely used definition for probiotics, developed jointly by the World Health Organization and the Food and Agriculture Organization, is that they are "live microorganisms, which when administered in adequate amounts confer a health benefit on the host." Certain food ingredients, non-digestible by people but a food source for our native beneficial bacteria, known as prebiotics, have also been shown to have beneficial effects on health by promoting changes in the composition of the pre-existing gut microbiota.

Many of these products have been consumed for centuries in cultures all over the world, and manufacturers would like to promote them for their specific benefits. Likewise, physicians and other health care professionals who have witnessed the benefits of these products in their patients would like to see them more widely adopted, particularly in Western medicine where they are largely considered alternative, unproven therapies. Until recently, these efforts have been stymied by a lack of well conducted clinical trials needed to prove benefits. In June 2010, representatives from these groups, as well as researchers who are interested in the basic mechanisms by which these products exert their effects, regulatory experts, and others, met to examine the current state of the science and to identify what still needs to be done to fully legitimize these products in the eyes of consumers, health care professionals, and the U.S. Food and Drug Administration (FDA).

Keynote speaker Mary Ellen Sanders of Dairy and Food Culture Technologies kicked off the meeting with a succinct description of probiotics and their actions, including a number of myths and misconceptions. This was followed by several clinicians who provided cogent summaries of the current state of the science, including Yehuda Ringel, of the University of North Carolina at Chapel Hill, who described research linking probiotics to improvements in functional bowel disorders; Emeran Mayer of the University of California at Los Angeles, who discussed our emerging understanding of the connection between the brain, the gut, and the gut microbiota and probiotics; and Mansour Mohamadzadeh of Northwestern University, who described his efforts to develop probiotic-based vaccines that harness the immunologic capabilities of the gut.

One factor that holds back the acceptance of pre- and probiotics as therapeutic agents is a lack of understanding of their molecular mechanisms, a deficit that is further hampered by lack of knowledge about the gut microbiota as a whole. Three researchers presented basic science and clinical research intended to fill these needs, including the development of in vivo and in vitro models. Justin Sonnenburg of Stanford University described his efforts to develop a simplified model of the gut microbiota in germ-free mice, allowing the investigation of fundamental properties and effects of host diet and genetics. Colin Hill of University College Cork, Ireland, is investigating specific mechanisms used by probiotic bacterial strains to kill pathogenic bacteria, perhaps underlying the anti-infective properties of these agents. And Glenn Gibson of the University of Reading, UK, described the development and use of a fermentor-based model of the human gut to investigate pre- and probiotic effects and their mechanisms. This session was followed by a data blitz that provided snapshots of many types of probiotic research currently going on around the world.

Any substance that is intended to be used to prevent, diagnose, mitigate, treat, or cure a disease is regulated as a drug in the U.S., regardless of the composition or appearance of the product. A product that appears to be a food will be considered to be a drug in the eyes of the FDA if it is intended to prevent, diagnose, mitigate, treat or cure a disease. The final session of the day brought together regulatory experts with physician-researchers who have embarked on the process of understanding the fine line between probiotic foods and drugs. Cary Frye of the International Dairy Foods Association described the standards of evidence that must be met to substantiate product labeling claims for pre- and probiotics when they are intended to improve a structure or function of the body or reduce risk of disease. Cara Fiore of the Office of Vaccines Research and Review at the FDA discussed the conditions under which a probiotic may be labeled as a drug intended to treat a specific medical condition. The physician-researchers, Dan Merenstein of Georgetown University and Patricia Hibberd of Tufts University, described their groundbreaking experiences with conducting studies of probiotics under the "Investigational New Drug Application" regulatory framework and provided insights that might ease others through the process and perhaps stimulate changes in FDA regulations to facilitate this type of research.

One of the meeting organizers, Howard Young of the National Cancer Institute, noted at the beginning of the meeting that, "with this field, we really have the ability to benefit the human condition, but it needs to be done right." Although there is still much to be done, it was apparent from the wide range of research presented at the meeting that the field of probiotics is off to a great start on the task of providing the scientific and clinical evidence that will allow these useful products to be more widely disseminated and utilized for the alleviation of human health conditions.

Speaker:
Mary Ellen Sanders, Dairy and Food Culture Technologies

Highlights

• The ideal composition of gut microbiota, or even whether there is one, is still unknown.
• Many parameters that could impact the effectiveness of probiotics added to foods or other products have not yet been investigated comprehensively.
• Probiotics are largely designed to benefit gastrointestinal conditions that for the most part do not have validated biomarkers.

Mary Ellen Sanders of Dairy and Food Culture Technologies opened the meeting with a discussion of some misconceptions surrounding probiotics. While probiotics have been used for centuries and have great potential to improve human health, the science surrounding their use is still quite new. It is important for investigators to examine previously held assumptions if they are to provide a solid, evidence-based footing on which to build future research.

One commonly held belief among lay people and health professionals alike is that probiotics help to “balance” the gut microbiota. This idea is difficult to substantiate because the normal gut microbiota is not well defined, and can differ significantly among normal individuals. The ideal composition, or even whether there is one, is still unknown. But simply increasing levels of “probiotic” genera such as Lactobacillus or Bifidobacterium cannot be considered clear evidence of facilitating “balance”. In contrast, however, there is evidence to show that probiotics can reduce the degree of disturbance of the gut microbiota or facilitate a quicker return to normal in individuals taking antibiotics and that this reduction is associated with fewer diarrheal symptoms (Engelbrektson et al., 2009, Kubota et al., 2009). These types of studies are useful for establishing the impact of probiotics on balance in the microbial ecosystem.

Another potential misconception is the idea that probiotics are the same as “live, active cultures.” Live, active cultures are used primarily for their fermentative capabilities; they may or may not have been tested and shown to confer a benefit. Until they are, they cannot be presumed to be probiotics.

Many parameters that could impact the effectiveness of probiotics added to foods or other products have not yet been investigated comprehensively. These parameters include the characteristics of the product containing the probiotics, such as moisture content, oxygen, temperature, or pH. There are multiple production parameters that could be researched and optimized, including how the microbial cultures are grown, harvested, and processed for use. There are also numerous parameters related to the host organism (whether humans or animals) whose effects are as yet unknown, such as the composition of the pre-existing host microbiota; the effects of bile, acid, and digestive enzymes; and the ability of probiotic bacteria to survive at the intended site of action.

The scarcity of research data in many of these areas has created some confusion about the uses of probiotics that are already on the market. While surveys have shown that both consumers and health care professionals are aware of probiotics, many still do not have a clear idea of their intended benefits. Evidence-based practice guidelines for the use of probiotics have been released by professional organizations, including the World Gastroenterology Organisation, but studies have shown that physicians may be using probiotics for conditions where there is currently no evidence of benefit, such as Crohn's disease.

Manufacturers of probiotics foods must meet the FDA- and FTC-specified standards of scientific proof before their products can be marketed with a claim for a benefit. Sanders described some of the difficulties that are likely to be involved in conducting clinical trials in this area. One particularly important barrier is posed by the fact that probiotics are largely designed to benefit gastrointestinal conditions that for the most part do not have validated biomarkers. Researchers designing clinical trials must depend on clinical endpoints, often prolonging the research process. This may change, however, if the Human Microbiome Project is able to identify a core human gut microbiota, as well as changes in the microbiota that can be correlated with human health or disease conditions. Whether such markers are developed or not, clinical trials will be needed to convincingly establish a scientific basis for health benefits.

Speakers:
Yehuda Ringel, University of North Carolina at Chapel Hill
Emeran Mayer, University of California at Los Angeles
Mansour Mohamadzadeh, Northwestern University

Highlights

• Current clinical data suggests that functional bowel disorders involve changes in the gut microbiota, and that specific probiotics are useful in treating these conditions and managing related symptoms.
• The gut microbiota is both influenced by and influences ongoing communication between the gut and the brain in both animal models and humans.
• Probiotic strains can be used to deliver vaccine antigens, leading to substantial immune responses in animal models and protection from certain pathogens.

The effect of probiotics on intestinal function and symptoms

In the first session of the meeting, researchers described the current state of probiotics research, including investigations into mechanisms, basic information about the human gut microbiota, clinical studies that suggest the potential benefits of probiotics, and the development of model systems to promote more efficient research in this area.

Yehuda Ringel of the University of North Carolina at Chapel Hill provided a comprehensive look at the current state of the science regarding probiotics and their effectiveness in treating human gastrointestinal (GI) disorders. Specific probiotics have been suggested to have beneficial effects on disorders including acute infectious diarrhea, antibiotic-associated diarrhea, and functional bowel disorders such as irritable bowel syndrome. Ringel reviewed multiple types of studies that suggest a potential role for certain probiotics in maintaining intestinal health, focusing primarily on available research on functional bowel disorders (Carroll and Ringel, 2009).

A number of observations suggest that changes in the composition of the gut microbiota may be involved in the development of functional bowel disorders. Microbiological studies have shown that patients with such disorders have gut microbiota that is altered in both quantity and composition compared to healthy individuals. Epidemiological studies suggest that disturbance of the gut microbiota is associated with the development of these conditions, since 10% to 30% of patients who contract acute GI infections develop chronic irritable bowel-like symptoms, and 22% to 40% of individuals treated with antibiotics have irritable bowel-like symptoms 4 months after treatment.

Studies in germ-free animals that lack gut microbiota, usually rats or mice raised in a sterile environment, have also provided data on the relationship between gut microbiota and bowel function. Germ-free animals show multiple abnormalities in intestinal function, including delayed gastric emptying, delayed intestinal transit, and enlarged cecal size, symptoms that are relevant to irritable bowel syndrome and other functional bowel disorders. The introduction of gut microbiota from mice raised under typical conditions can reverse these abnormalities (Husebye et al., 2001). Taken altogether, the microbiological, epidemiological, and physiological data now available support the idea that altered gut microbiota is an important factor in the pathophysiology of functional bowel disorders, and lead to the hypothesis that probiotics could be used to prevent or manage these conditions.

These lines of evidence have provided the rationale for clinical research in this area. Studies have shown that probiotics can improve measures of GI function, relieve symptoms, and confer overall improvements in health and well-being in some patients (McFarland et al, 2008). However, more research is needed to assess their direct effects on intestinal physiology and to identify the mechanisms by which these effects are achieved. In addition, studies to date have used a wide range of different probiotic preparations, containing different mixtures of microorganisms, and it is still unknown to what extent these results can be generalized.

Impact of probiotics on the central nervous system?

Most people are well aware of interactions between the gut and the nervous system. Neurophysiological states, such as stress, pain, and depression, can have effects on GI function, e.g. "nervous stomach," diarrhea, and changes in appetite. The reverse is also true: the physiological state of the gut can have effects on mood, the perception of pain, and behavior. Emeran Mayer of the University of California at Los Angeles reviewed considerable evidence suggesting that gut microbiota may play an integral role in this ongoing conversation between gut and brain.

Stress has multiple effects on the gut, including increased gastric acid production and increased GI motility, which would affect the gut environment and thus the growth and physiology of bacteria living there. Stress has also been shown to causes changes in the composition of the gut microbiota in studies of infant rats and monkeys subjected to maternal separation (Bailey and Coe, 2004). Certain probiotics have been shown to reverse these effects in animals (Gareau et al., 2007). Stress is also known to lead to changes in neurotransmitter levels, which could affect not only the gut itself but also the bacteria living there. For example, the neurotransmitter norepinephrine has been shown to promote virulence in highly pathogenic strains of Escherichia coli and in another intestinal pathogen, Campylobacter jejuni (Waldor and Sperandio, 2007, Cogan et al., 2007).

Studies have also implicated the gut microbiota in modulating the sensation of pain. Treatment with antibiotics can lead to pain hypersensitivity (hyperalgesia) in the intestines, and it has been shown that this hypersensitivity can be reduced with the use of probiotics (Verdú et al., 2007). Certain probiotics have also been shown to have antihyperalgesic effects in animal models of pain such as acute inflammatory hyperalgesia (Rousseaux et al., 2007). It is hypothesized that this antihyperalgesic effect of probiotics is related to the engagement of endogenous opioid systems. At the same time, however, studies in germ-free mice have shown that the presence of the gut microbiota is necessary for the development of a hyperalgesic state, perhaps because in the normal gut, the presence of bacteria stimulates the production of interleukin-10, a cytokine involved in suppressing the inflammatory response (Amaral et al., 2007). Further research will be needed to reconcile these somewhat contradictory results.

Integration of the microbiota into the gut-brain axis. (Modified with permission from Collins S, Bercik P. 2009. Gastroenterology 136: 203-214.)

A number of other lines of research suggest a direct connection between the gut, activities of the brain, such as mood and behavior, and the gut microbiota. Initial intestinal infection with Toxoplasma gondii, followed by localization to the brain, has been reported to have direct effects on the performance of rats in behavioral tests (Berdoy et al., 2007). Epidemiologic studies in humans show that negative emotions are often associated with the development of acute GI infections, and conversely, chronic GI inflammation has multiple effects on mood, including symptoms of depression and fatigue. Risk factors for the development of irritable bowel syndrome include adverse life events, depression, and neuroticism. A recent study of the neurobiological and immunomodulatory effects of a probiotic strain of Bifidobacteria infantis in rats suggests that this probiotic could have antidepressant effects in this model (Desbonnet et al., 2008). These findings illustrate the intimate nature of the connections between gut, brain and gut microbiota, and suggest that the uses of probiotics might someday go well beyond maintenance of intestinal health alone.

Novel oral targeting vaccine and therapy using probiotics

The internal cavity of the gut, where the microbiota resides, is known as the lumen. Lining this cavity is a layer of cells and mucus, called the mucosa, which includes epithelial cells and multiple types of immune cells, such as T-lymphocytes, B-lymphocytes, and dendritic cells. These immune cells can mount localized or systemic immune responses to antigenic materials found in the gut. Dendritic cells play a key role in this process by sampling the lumen, capturing antigens (including whole or component parts of bacteria, viruses, and parasites), and presenting them to immune cells downstream to elicit immune responses.

Mansour Mohamadzadeh and his coworkers at Northwestern University and North Carolina State University are developing methods to harness these properties of gut dendritic cells in the development of oral vaccines. Delivery of vaccines by mouth rather than injection is more efficient, less likely to provoke a dangerous hypersensitivity reaction, and may facilitate vaccine use in developing countries. Mohamadzadeh's team is making use of probiotics as the delivery vehicle to carry such vaccines into the gut.

In their system, Lactobacillus acidophilus and Lactobacillus gasseri have been engineered to carry a fusion protein that consists of the vaccine antigen linked to a protein that binds to dendritic cells with high affinity (Mohamadzadeh et al., 2009, Mohamadzadeh et al., 2010). Once introduced into the gut, these bacteria release the fusion protein, which binds to and is taken up by dendritic cells. The antigenic part of the fusion protein is then presented to T-cells, provoking mucosal immunity resulting in systemic immune responses. Preliminary studies of this system show that it performs quite well in mice, protecting them from a challenge with anthrax that kills control mice who did not receive the fusion protein. Their studies show that, among other beneficial effects, the mice who receive the fusion protein produce large amounts of antibodies against the anthrax antigen.

Mohamadzadeh is also testing this method as a means of delivering tumor specific antigens for the treatment of breast and skin cancer, and as a means of developing vaccines against viral infections such as influenza and HIV. In another project, he and his colleagues are producing altered Lactobacillus acidophilus NCFM strains that may also be used to treat GI disorders that are associated with aberrant immune activity, such as inflammatory bowel disease. These studies will lead to a better understanding of the mechanisms of gut-mediated immunity, its relationship to the gut microbiota, and the possibilities of probiotic delivery systems in the development of new vaccines for humans.

Speakers:
Justin Sonnenburg, Stanford University
Colin Hill, University College Cork, Ireland
Glenn Gibson, University of Reading, UK

Highlights

• Simplified animal models of the gut microbiota can be used to investigate the complex intestinal ecosystem, as well as help to identify mechanisms by which pre- and probiotics exert their effects.
• The probiotic organism Lactobacillus salivarius produces a bacteriocin that protects mice from listeriosis by directly killing the infectious organism Listeria.
• A continuous, multicompartmental culture system that replicates the human gut can be used for in vitro investigation of the mechanisms of action of pre- and probiotics and their effects on the gut microbiota.

Understanding and altering the intestinal microbiota

One of the difficulties with attempting to manipulate the gut microbiota is the scarcity of knowledge about how it functions. Researchers would like to know in a systemic way how the microbiota changes with changes in the host diet, how it adapts to the introduction of new species or to the loss of species, and what role is played by host genetics in its composition and function. The Human Microbiome Project has generated large amounts of important sequence data that identifies the many bacterial species and genes involved; however, data on how this complex ecosystem functions has lagged quite a bit behind.

Justin Sonnenburg and his group at Stanford University are working to develop a simplified model system in which such questions can be addressed more readily. The team has been studying carbohydrate metabolism, an aspect of metabolism in which gut microbes are quite active. A complex ecosystem exists in the distal gut in which numerous bacterial species compete to break down and utilize the carbohydrates that we ingest, particularly the more complex plant carbohydrates that we are poorly equipped to metabolize on our own.

Starting with germ-free animals, they are colonizing mice with simplified microbial communities and looking at how the genetics of both host and microbes affect function. Although 10 bacterial divisions and thousands of species are represented in the human and mouse gut microbiota, more than 90% of the microorganisms belong to the Bacteroidetes and Firmicutes divisions of bacteria, so one way to reduce complexity is by working with representatives of these two groups.

For their initial experiments, they studied the genetics and metabolism of a single species Bacteroides thetaiotaomicron (B. theta) colonized in germ-free mice. This organism is a prominent member of the human microbiota that has the advantage of being easily cultivated outside the body. The genome of B. theta is largely geared toward carbohydrate metabolism, including ≥241 enzymes that metabolize many different types of sugars, and at least 64 enzymes that are devoted specifically to the digestion of plant polysaccharides, out of a total genome of 4779 genes. This compares to fewer than one hundred such enzymes in humans, who have on the order of 25,000 genes.

Figure adapted from Sonnenburg JL et al. PLoS Biology, 2006. B. thetaiotaomicron and B. longum bacteria, when occuping the same cecal space (co-colonized), show differential expression of Glycoside Hydrolase and Polysaccharide Lyase genes. The columns marked "B. theta" and "B. longum" above show the expression of these same genes for bacteria that were colonized in separate mice ceca. These mice are called "mono-associated." Green indicates a deviation below the mean level of expression, and red indicates deviation above the mean level for mono-associated mice. Results indicate that B. theta expands expression of glycoside hydrolases in co-colonization with B. longum — a greater variety of genes associated with digestion of plant polysaccharides show expression in co-colonized B. theta.

Taking a functional genomics approach, Sonnenburg and his group have examined how the expression of these genes changes when different foods are introduced into the mouse host's diet (Sonnenburg et al, 2005). They have also added another species to the system, Bifidobacterium longum, and are studying this organism's gene expression as well, to find out how these two species compete for and adapt to the ecological niches that are available under different conditions (Sonnenburg et al., 2006). They are also testing the effects of the prebiotic inulin in the system. Inulin, a dietary polysaccharide that is found in many plants, has been shown to alter the human gut microbiota, in some cases by expanding the population of Bifidobacterium species (Sonnenburg et al., 2010). These studies will help to elucidate the mechanisms of pre- and probiotics, and to identify whether changes in gut microbiota are the cause or the result of a given disease state. The insights derived from these experiments should someday allow more precise manipulation of the human gut microbiota, which will be important once a better understanding of its optimal content has been achieved.

Bacteriocin production as a probiotic trait to combat infection

Certain probiotics are thought to assist humans and animals in fighting infections, but the mechanisms by which they do so are still unknown. Potential mechanisms that have been proposed include improving the barrier function of the gut, improving the ability of the immune system to fight infection, competition between "good" and "bad" microbes for ecological niches within the gut, and direct antagonism, in which a probiotic species directly kills an infectious species of bacteria. Understanding which mechanism is at work in a given situation will be critical for selecting and using the appropriate probiotic for each infectious disease condition.

Colin Hill and his coworkers at University College Cork, Ireland, are studying probiotics that act via a mechanism of direct antagonism. In the course of evolution, one of the ways that bacterial species have developed to compete amongst each other is by killing their rivals with peptides known as bacteriocins. Many gut bacteria secrete these compounds, which have very specific abilities to kill some bacterial species and not others. There is significant evidence to suggest that probiotics can prevent infectious diarrhea, particularly Clostridium difficile associated diarrhea, which is very dangerous and difficult to treat effectively (Hickson et al., 2007). Many of the probiotic strains used in these studies belonged to the Lactobacillus genus, species of which are known to produce bacteriocins, so Hill and his group set out to investigate the role of these peptides in the anti-diarrheal effect.

As a model system, they are studying listeriosis in mice. Listeria is a rare but important systemic foodborne pathogen that has a high rate of fatalities, often targeting individuals who are immunocompromised. The pathogenesis of listeriosis is well understood in mice. Hill and his group screened probiotic strains used in humans for their ability to protect mice from Listeria infection and death, and have identified a particularly effective strain, Lactobacillus salivarius UCC118. They have developed multiple lines of evidence to show that the bacteriocin produced by this strain protects mice by killing Listeria almost as soon as these microorganisms are introduced (Corr et al., 2007). They are also using this system to study the effects of another bacteriocin, thuricin, which is known to be active against C. difficile (Rea et al., 2010).

These types of experiments will provide important knowledge on the specific interactions between benign and pathogenic strains of gut bacteria, eventually allowing more targeted use of probiotic strains for specific types of infections. This knowledge could also be used to allow scientists to produce and use bacteriocins themselves. These potential new biotechnological agents could be designed as highly targeted antimicrobial therapies that kill infectious bacteria with a greatly reduced effect on beneficial gut flora, compared to the antibiotics that are used currently.

Models for studying efficacy in probiotics

A culture model of the large intestine. (Photo courtesy of Glenn Gibson)

As in many areas of clinical research, probiotics studies will progress more quickly and cost-effectively with the development of model systems that can be used to test new ideas before they are tried in humans. Such models can be particularly challenging to develop for pre- and probiotics because the gut and the gut microbiota are such highly complex systems. Glenn Gibson of the University of Reading, UK, provided an overview of current in vitro systems for studying the properties of these potential therapies. These models include both batch culture and continuous fermentation systems in which colonies of gut microbes can be grown and the effects of pre- and probiotics investigated. A number of laboratories have developed complex, multistage fermentation systems that are intended to replicate different areas of the human GI tract.

Gibson and his coworkers have developed one such system, a continuous, multi-compartmental culture model whose properties have been validated against the gut contents of sudden death victims. The system consists of several culture vessels whose contents replicate the sequence of volumes and pHs found in different areas of the human large intestine. This system is inoculated with mixed fecal bacteria from human volunteers and allowed to run continuously to set up an in vitro model of this area of the gut. Gibson and his group can add pre- or probiotics to the system to study their actions as well as their persistence in a human gut-like environment.

Among other things, they are investigating a prebiotic which consists of a mixture of galactooligosaccharides, known as GOS, and has been shown to help patients with irritable bowel syndrome. Addition of GOS to the model system had a considerable effect on the distribution of bacterial species, including a large increase in Bifidobacterium species. This increase has also been observed to occur in healthy human volunteers and in individuals with irritable bowel syndrome who ingest GOS (Depeint et al., 2008, Silk et al., 2009). The in vitro system thus appears to replicate the human system, and could be used to elucidate the mechanisms by which GOS raises Bifidobacterium content and alleviates irritable bowel symptoms. Ultimately, studies of pre- and probiotics must be done in humans before it will be possible to make health or disease related claims for these agents, but the information provided by in vitro models will provide valuable guidance on what might or might not be fruitful when tried in humans.

Speakers:
Duane Charbonneau, Procter and Gamble Company
Kevin Donato, Hospital for Sick Children, Toronto
Patrick Veiga, Danone Research
Howard Young, National Cancer Institute
Arthur Ouwehand, Danisco
Guy Rousseau, Hopital du Sacre-Coeur de Montreal

Innovation in probiotics research

In this session, researchers presented briefly on ongoing research projects designed to probe the mechanisms by which pre- and probiotics work, and intended to help move these agents along from benchtop to marketplace.

Duane Charbonneau of Procter and Gamble Company reported on his work developing the strain Bifidobacterium longum subsp. infantis 35624 as a probiotic for use in treating infection and irritable bowel syndrome. This probiotic dramatically reduces Salmonella colonization in mice. Reported studies in humans with irritable bowel have been hampered by difficulties with the formulation: capsules with larger amounts of bacteria did not dissolve in the gut as well as those with smaller amounts, so results have been difficult to interpret (Whorwell et al., 2006).

Kevin Donato of the Hospital for Sick Children, Toronto, is studying the effects of probiotics on epithelial barrier function in the gut, using cultured intestinal cells. Normally, tight junctions between the epithelial cells in the mucosal layer of the GI tract prevent leakage of fluids, but pathogenic bacteria such as the notorious E. coli strain O157:H7 or pro-inflammatory cytokines can break down these junctions, causing watery diarrhea and dangerous fluid loss. Donato and his coworkers are investigating the molecular details by which the bacterium Lactobacillus rhamnosus GG (LGG) prevents the colonization of pathogenic bacteria and influences immune responses in the human gut. When tested in intestinal cell tissue culture, LGG attenuates barrier dysfunction, apparently by reducing the attachment of pathogenic bacteria and modulating pro-inflammatory epithelial cell signaling (Donato et al., 2010).

Patrick Veiga of Danone Research reported on clinical studies of the effects of a fermented milk product (yogurt) containing Bifidobacterium lactis DN-173 010 on symptoms in humans who have irritable bowel syndrome with constipation (Agrawal et al., 2009). They are using genetic analyses to monitor changes in the gut microbiota after consuming the probiotic yogurt. Instead of large global shifts in microbial species they found only a few species that changed in abundance with ingestion of the probiotic product. They also found that survival of the ingested probiotic strain, as monitored in fecal samples, varied from person to person. They are conducting ongoing research to find out what causes this variation and how is it might be related to whether or not individuals with irritable bowel syndrome experience relief from symptoms after consumption of the specific probiotic product.

Howard Young of the National Cancer Institute is studying the potential of probiotic strains as delivery systems for therapeutic compounds, including immunomodulatory peptides and proteins. A series of studies have suggested that interferon β-1α might be beneficial as a treatment for ulcerative colitis in humans, although other studies have suggested that treatment with this protein has actually caused ulcerative colitis to develop in patients being treated for multiple sclerosis and hepatitis. It was thought that delivering the interferon molecule locally in the gut might make it a more effective treatment for colitis, potentially free of systemic effects. However, mice inoculated with a probiotic Lactobacillus acidophilus strain expressing interferon β-1α were rendered more susceptible to experimentally induced colitis rather than protected from it (McFarland et al., 2010). Young and his coworkers are continuing to investigate the molecular mechanisms of this unexpected result.

Arthur Ouwehand of Danisco is studying the effects of probiotics on metabolic syndrome, which in humans is characterized by elevated blood triglycerides, high blood pressure, high blood glucose, increased waist circumference, and reduced HDL-cholesterol (the so-called "good" cholesterol). Clinical evidence suggests that this disease is related to high levels of inflammation, promoted by a high fat diet. One hypothesis for the mechanism by which a high fat diet causes this syndrome is that increased fat in the gut breaks down its barrier function, allowing lipopolysaccharides (LPS), which are highly inflammatory compounds produced by Gram negative bacteria, to enter the blood and tissues, promoting systemic inflammation. Ouwehand and his colleagues are testing whether the probiotic Bifidobacterium lactis 420 can protect mice fed a high fat diet from developing metabolic syndrome. They have found that this probiotic reversed diabetic changes and reduced fat mass in mice, while at the same time reducing blood levels of LPS and markers of inflammation. B. lactis also counteracted changes in the gut microbiota that were induced by the high fat diet. These promising studies will need to be confirmed in humans.

Guy Rousseau of the Hopital du Sacre-Coeur de Montreal is studying the ability of probiotics to relieve the depression that often ensues following a myocardial infarction (MI). Epidemiologic studies show that over 20% of patients who suffer an MI develop major depression, and this depression is associated with a 3 to 4 fold increase in subsequent mortality. This phenomenon has been hypothesized to be related to the release of pro-inflammatory substances and increased cell death, or apoptosis, in certain areas of the brain. Rousseau and his colleagues have shown that prophylactic intake of a combination of two probiotics, Lactobacillus helveticus R0052 and Bifidobacterium longum R0175, reduced post-MI apoptosis in the brains of rats, when given before and after an experimentally induced MI (Girard et al., 2009). Compared to control animals, the animals given the probiotics showed a reduction in post-MI symptoms of depression, as measured by behavioral tests, as well as reduced post-MI increase of interleukin-1β, an inflammatory cytokine. Probiotics also restored the integrity of the intestinal barrier in these animals. These probiotic strains have been associated with reduced stress in humans, suggesting that post-MI depression may be a stress-related condition.

Speakers:
Cary Frye, International Dairy Foods Association
Cara Fiore, Office of Vaccines Research and Review, FDA
Dan Merenstein, Georgetown University Medical Center
Patricia Hibberd, Massachusetts General Hospital, Boston

Highlights

• Probiotics may be foods or biological drugs. When used to prevent, treat, or cure a human disease these live biotherapeutic products (probiotics used as biological drugs) are regulated by FDA's Center for Biologics Evaluation and Research.
• Clinical research on the use of probiotics to prevent, treat, or cure a human disease requires the investigator to submit an Investigational New Drug application to the FDA before clinical studies can begin.
• There is no full agreement between researchers and the FDA as to whether the establishment of a study protocol on probiotics warrants the need to file an Investigational New Drug Application.

Major regulatory challenges

Probiotics are subject to a wide range of potential types of regulation depending on their intended use. Probiotics may be regulated as conventional foods, dietary supplements, or drugs. Claims for probiotics could potentially range from structure/function claims (e.g., “helps support the immune system”), to health claims (i.e., that the product reduces the risk of a specific disease). A probiotic that is intended for use in diagnosing, curing, mitigating, treating, or preventing a human disease is considered a drug. Clinical trials to evaluate probiotics for these uses require an Investigational New Drug application (IND). The last session of the meeting brought together experts in these regulatory matters with clinical investigators to discuss the challenges and pitfalls involved.

Cary Frye of the International Dairy Foods Association discussed the regulation of probiotics from the perspective of food labeling claims. Manufacturers are understandably eager to communicate the health benefits of their products to consumers but must follow certain regulations and guidelines.

Two types of food labeling claims, those that mention nutrient content (such as "a good source of calcium"), and health claims (such as "reduces the risk of osteoporosis") must receive FDA clearance before they can be put on product labels. Health claims must be based on FDA's review and approval of a health claim petition or notification of an authoritative statement from a scientific body of the U.S. government or the National Academy of Sciences, or they must be qualified if the science is not yet conclusive. FDA guidance, published in 2009, describes the standards of evidence that health claims must meet. In addition, FDA guidance published in 2008 describes what is needed to substantiate labeling claims for probiotics that are administered as dietary supplements. Currently there are no FDA recognized health claims for probiotics, largely because conclusive scientific evidence is lacking.

Structure/function claims, which focus on the maintenance or support of body structures or functions in healthy individuals rather than disease prevention or treatment, are not subject to FDA approval but statements made in product labeling must be truthful and not misleading. Frye shared the examples that the claims: "Helps maintain healthy intestinal flora" or "Helps support immune function" are considered structure/function claims, not disease claims; where as "Helps individuals using antibiotics to maintain normal intestinal flora" or "Protective against the development of diarrhea" would be viewed as an unauthorized health claim or drug claim. Similarly, dietary guidance that is put on food labels, such as "diets rich in dairy foods, fruits, and vegetables reduce the risk of some chronic diseases" does not need to be approved by the FDA prior to use but must be accurate and substantiated. In all cases, the burden of proof for product labeling rests with the manufacturer to have accurate substantiation supported by competent and reliable scientific evidence.

Probiotics manufacturers face substantial challenges in navigating these regulatory requirements. They must have a good working knowledge of the regulations involved, and word their package labeling so that the claims align with available evidence. Often they themselves will need to develop the necessary scientific evidence for claims they wish to make, by performing or funding the necessary clinical studies. Novel types of claims may also require manufacturers to seek expert counsel or ask the FDA to provide specific guidance on how to substantiate them. These rules and regulations are sometimes difficult to follow but provide needed protection for the consumer.

Current CBER / U.S. FDA regulatory issues using live biotherapeutic products

Manufacturers or clinical researchers who wish to evaluate live biotherapeutic products (LBPs) to prevent, treat, or cure a human disease should submit an an Investigational New Drug application, or IND, to the Center for Biologics Evaluation and Research at the U.S. FDA. Cara Fiore spoke as a representative of the Office of Vaccines Research and Review (OVRR), the FDA office responsible for the regulation and oversight of live biotherapeutic products (live microorganisms that are not vaccines and are intended to prevent, treat, or cure a human disease).

Individuals or companies who wish to study the use of probiotics for the treatment of a disease must follow a regulatory pathway similar to that followed for live vaccines to prevent infectious diseases. This pathway begins with an IND that must be submitted before studies in humans can begin. The IND must contain sufficient information for the FDA to evaluate how the product is made, to ensure that safe, high-quality manufacturing processes are used. In addition, data from preclinical animal toxicology studies may be required to demonstrate that it is safe to proceed with human clinical studies.

Fiore said that many of the INDs received in OVRR are placed on "clinical hold" because they do not include sufficient data about the product to assess the risks to subjects in the proposed study. If the institution or individual performing the research is not the company that makes the product, researchers may have difficulty obtaining proprietary manufacturing information. However, there are ways to overcome this difficulty, such as cross-referencing a Master File (MF) provided by the product manufacturer. The information in this MF can be reviewed by the FDA but is not available to the IND holder. However, this requires that the manufacturer establish a MF. Fiore advised investigators to request a pre-IND meeting with the FDA to discuss the information required for an IND submission and avoid a clinical hold.

Probiotic products must go through the same process as other products intended to prevent, diagnose, mitigate, treat, or cure a human disease.

Once an IND is in effect, probiotics that are intended for use as biological products must follow the same regulatory pathway as other biological products or drugs, including Phase 1, 2, and 3 clinical studies. After safety and efficacy of the product have been demonstrated, the manufacturer files a Biologics License Application (BLA) which permits them to market the product for the specific indication in the specific population. When the BLA is approved, the FDA may require post-marketing safety studies. In addition, the manufacturer may conduct additional studies to support post-approval labeling changes such as new indications or changes to the dose or dosing regimen. This process is intended to ensure that all biological products licensed for use in the U.S. are safe and effective.

Probiotic foods: developing and implementing quality clinical trials

Probiotics occupy an unusual position in the drug approval process because in many cases they are components of foods that have been consumed for centuries without apparent ill effects. Some researchers, including Dan Merenstein of the Georgetown University Medical Center, would argue that testing these substances for safety slows down the FDA approval process unnecessarily. Merenstein has conducted a number of randomized trials of probiotics in healthy children, and obtained NIH funding for a study on whether consuming probiotics, in the form of a commercially available probiotic, reduces diarrhea in children who are taking antibiotics. Because this study would investigate the ability to cure or mitigate a disease condition, the NIH center that funded his grant, the National Center for Complementary and Alternative Medicine (NCCAM), required that he ask CBER at the FDA if he was required to submit an IND application. CBER indicated that the study was required to be conducted under an IND.

Merenstein’s previous studies in children did not require INDs because, according to the stated aims of the studies, they were intended to support only structure/function claims. Outcomes that were assessed in these studies included the prevention of daycare absences and reduction of parental reports of loose stools. Merenstein is preparing a study comparing amoxicillin, prednisolone, and neti-pots for the treatment of sinusitis. This planning grant is supported by NIAID, which also required Merenstein to contact the FDA to inquire about a potential IND. Drug studies are under the aegis of the Center for Drug Evaluation and Research (CDER) at the FDA, rather than CBER. CDER, under the auspices of an investigator initiated IND, exempted the study from an IND process because of the long standing uses of the therapies involved.

Merenstein documented the long and arduous process involved in obtaining FDA approval for the diarrhea study. His grant application for funding for a clinical trial was submitted to the NIH in October 2006 and NIH approved it for funding in June 2007. Soon thereafter, CBER informed him that the study would have to be conducted under an IND. After multiple rounds of communication, the IND was approved in November 2008. NCCAM issued the grant award in August 2009, more than two years after indicating its intent to award the grant. As part of the process, the FDA required, and NIH funded, an initial safety study of the yogurt with probiotics in healthy adults, even though the product was equivalent to one currently on the market as a food and had been ingested by many people. However, this requirement is in keeping with current FDA general recommendations that drugs should be tested in adults before they can be tested for pediatric uses.

The FDA "needs to take a realistic approach to probiotic foods," Merenstein said, that includes CBER exemptions for INDs where feasible. In addition, he suggested that the NIH needs to fund more patient oriented clinical trials in this area, in addition to Phase 1 safety studies and basic science studies of the microbiome and probiotic mechanisms. He predicted that the U.S. will fall behind considerably in probiotics studies if such changes are not made.

Therapeutic probiotics: designing and implementing quality clinical trials

Patricia Hibberd of Massachusetts General Hospital is another researcher who has been involved in designing and conducting studies on probiotics for a number of years. As such, she is also well acquainted with the challenges of conducting research in this area.

Hibberd began by presenting an overview of current probiotics trials all over the world, based on data available at the public website clinicaltrials.gov. From 2005 through 2010, there were 63 probiotics trials in the U.S., compared to 84 in Europe, 39 in Asia, and 25 in the rest of the world. She noted a trend towards more trials outside the U.S., and more in healthy individuals. She also noted that many trials of probiotics have been of poor quality, including many with problems concerning the underlying biologic rationale, experimental design, safety, and measurement of appropriate outcomes. The Cochrane Review, which conducts rigorous analyses of clinical evidence, has raised questions about the quality of trials in most areas where probiotics have been tested. One difficulty in gauging the quality of the evidence is that these analyses aggregated studies on many disparate types of probiotics, most likely of necessity since clinical trials are still few and far between.

Hibberd described her personal experiences with IND submissions for two NIH-supported trials on the use of probiotics to prevent infection. One IND eventually had to be withdrawn due to a number of issues, including minimal assistance from a manufacturer. The other IND had strong support from the manufacturers but was placed on clinical hold when the FDA requested a Phase 1 study in healthy adults before the pediatric study could be done. In July 2009, after revisions to the study, the hold was lifted and the Phase 1 study in healthy adults was able to proceed. Hibberd seconded Merenstein's advice to seek a pre-IND meeting, and emphasized the importance of getting the manufacturers' cooperation as well.

Hibberd emphasized the critical role of NCCAM in guiding these studies and encouraging cooperation with other organizations to improve the intended research, in this case including the Human Microbiome Project and the U.S. Department of Agriculture. However she noted that cooperation between the NIH and the FDA in these studies has led to considerable delays due to the multiple layers of oversight involved. She also found that it was difficult in some cases to get peer reviewers of NIH grants to accept the need for the Phase 1 safety studies that the FDA required, jeopardizing grant approval.

Does the completion of an IND lead to higher quality studies of probiotics? Hibberd noted that completing an IND confers benefits, including assistance with product quality issues and other valuable advice from the FDA, but also creates substantial challenges, including the time involved and the need to reconcile FDA guidance with other institutional requirements. In addition, it remains to be seen whether completing an IND improves the credibility of clinical trials of probiotics, either with health care professionals or the general public. In the end, "quality remains the responsibility of the investigator," she said, whether or not an IND is filed. High quality research in humans remains the goal, and will ultimately determine whether probiotics are accepted in the mainstream to improve human health and treat diseases.