Christian Furlan Freguia Ph.D. Director of Research Synthetic Biologics
Michael Kaleko M.D., Ph.D. Senior Vice President of Research and Development Synthetic Biologics
From the Bottom Up
As discussed in Part 1 of this article, it is now clear that the gut microbiome participates in many human physiologic functions and is considered a target for therapeutic intervention. Part 2 of this article will briefly address some of the fundamental scientific questions that underpin such drug development.
First, what is a healthy microbiome? There is no definitive answer to this question. The single most important factor seems to be diversity. That is, how many different bacteria and other microbes are represented. More to the point, how many different genetic pathways are represented and with how much redundancy. The more pathways and types of organisms, the better. In fact, a permanent loss of diversity over the past few decades, due in part to improving hygiene and antibiotic use, is now offered as a potential explanation for the increasing prevalence of many Western diseases. That said, specific changes to microbial profiles, and even to some individual bacterial species, have been associated with increased risk for certain diseases.
Second, what factors shape an individual’s microbiome? The process starts at birth and potentially in utero. Thus, vaginal deliveries tend to seed a newborn with vaginal bacteria such as lactobacillus, while Caesarian deliveries tend to seed with skin bacteria. This could potentially lead to minor shifts in the more permanent microbiota profile, which is established during the first three years of life. A second factor in shaping the microbiota is diet. Interestingly, breast milk is known to contain polysaccharides that cannot be digested by the child. One speculation is that these nutrients have evolved to support specific gut microbial species.
For the most part, the adult microbiome of an individual is stable over time, even with minor dietary changes. On the other hand, Western diets create very different gut microbial communities than the diets of indigenous African tribes. And Westerners who switch to such an African diet will rapidly undergo a large shift in microbiota. A third factor affecting the microbiome is the host itself. The gastrointestinal (GI) tract has the unenviable task of recognizing and supporting commensal symbiotic bacteria while preventing colonization and infection by pathogens. The complexities of the host microbiota relationship are only now being elucidated. Many host factors are at play. The innate immune system through its TLRs and NOD receptors regulates the local GI environment. The gut epithelial cells and those in the submucosa produce many polypeptides that regulate the microbiota including, for example, defensins, intestinal alkaline phosphatase, cytokines, and secretory IgA. Similarly, the Gut Associated Lymphoid Tissue (GALT), a major component of the immune system, constrains the microbiota with both inflammatory and immunosuppressive T cells. Through these interactions, the host and the microbiota achieve a stable equilibrium. Not unexpectedly, following a fecal microbiota transplantation (FMT), some transplanted bacteria persist and others do not, which differs from patient to patient. A fourth factor affecting the nature of the microbiota is the microbial organisms themselves. These competitive and warring organisms also must reach a stable equilibrium. Many have evolved metabolic, adherence, quorum sensing, antimicrobial, and resistance functions to promote their own survival. Thus, it may not always be easy to introduce a new “therapeutic” organism into this environment.
Perhaps the most important factor that shapes the microbiome is the rampant use of antibiotics. While these miracle drugs have saved millions of lives during the past 80 years, the unintended consequence of antibiotic use is the slaughter of many commensal gut bacteria. After a course of antibiotics, it can take weeks for the microbiome to recover, and with each successive course, the recovery time gets longer. Eventually, some bacterial species may be lost forever. And these changes are particularly problematic in the elderly due to a natural, age-related decline in microbiome diversity.
In the short-term, antibiotic use can lead to an overgrowth of drug resistant pathogens. Some, like Clostridium difficile (C. diff), can cause serious GI infection while others can seed distant organs and cause secondary infections such as pneumonia. The long-term consequences of antibiotic-mediated microbiome changes are currently more speculative, but may include an increased risk for diseases such as obesity, diabetes, inflammatory bowel disease, and asthma. Biotech companies are developing technologies to protect the microbiota from antibiotics by eliminating them in the GI tract upstream of the colon. The most advanced technology, which is completing Phase II clinical trials, consists of an orally delivered beta-lactamase. This enzyme is designed to be released in the proximal small intestine to rapidly degrade penicillins and cephalosporins, the most commonly used antibiotics. A second strategy to remove antibiotics from the GI tract is via oral delivery of enteric-coated activated charcoal. Success with these strategies is expected to represent a major advance in shielding the microbiome from antibiotics and preventing overgrowth of C. diff as well as other antibiotic-resistant pathogens.
A third question critical to the development of microbiome-directed therapeutics is how does the microbiota regulate human physiology? Answers to this question are only beginning to emerge. The most common proposed mechanism, at least in the lay press, is that the microbiota can contribute to “leaky gut.” The digestive system has the amazing capacity to recognize and absorb nutrients while simultaneously blocking entry of bacteria and their inflammatory components such as endotoxin. But this gut barrier function is not perfect. And translocation of bacteria and their components into the circulation can lead to a state of chronic inflammation, which has been implicated as an exacerbating factor for many diseases. Among others, these include obesity, metabolic syndrome and type 2 diabetes, nonalcoholic steatohepatitis (NASH), type 1 diabetes, autoimmune diseases, and progression of HIV AIDS. Thus, strategies to tighten up the gut barrier could have a major impact on global health.
But there are other means by which the microbiota can modulate the host physiology. The microbial species release many metabolites into the GI tract, referred to as the metabolome. Some of the more well-studied components include short chain fatty acids such as propionate and butyrate, which can regulate local inflammation and serve as nutrients for the colonic epithelium. The microbial species can also generate secondary bile acids, which may play a role in regulating the germination of C. diff spores. Some bacterial species can generate tryptophan metabolites or even neurotransmitters such as GABA. Clearly, as the field of metabolomics advances, the complexities of the microbiome-host interactions will be revealed as will the means to utilize these pathways for therapeutic intervention. Finally, the gut microbiota can interact directly with the host nervous system through the myenteric plexus and the vagus nerve.
The next important question in the pursuit of microbiome-based therapeutics pertains to the basic science itself. Microbiome science was enabled by the advent of “Next Generation” DNA sequencing, with extraordinary increases in productivity and concomitant decreases in cost. But sequencing is not perfect. There is an error rate, host DNA sequences must be identified and removed, and the identification of bacterial species is only as good as the databases of genes in those bacteria. Additionally, microbiome studies frequently generate billions of data points, which require sophisticated methods for analysis. As the field advances, some of the fundamental hurdles will be to minimize the errors, standardize the technologies across laboratories, and improve the analytical computer algorithms. Moreover, since the data vary from person to person and longitudinally over time, the analyses require complex statistics.
A second basic science question pertains to the value of animal modeling. In nearly all biological fields, animal modeling is critical for the initial discoveries, developing and testing hypotheses, and establishing the preclinical parameters for human studies. Microbiome science is no exception. Many of the discoveries as well as proof that manipulation of the microbiome can actually alter host physiology have come from animal studies. That said, microbiome animal modeling is limited by the differences between human and animal microbiota, thus arguing for early testing in humans. As the field progresses, it will be critical to better understand the strengths, limitations, and predictive value of animal modeling for human successes. Failure to do so could result in some expensive clinical fiascos.
The final question addressed here is how can we utilize the microbiome to develop human therapeutics? The most obvious strategy is to deliver “bugs as drugs” starting with the decades-old use of probiotics. While the literature is replete with studies showing modest clinical benefits for a variety of indications, probiotics have not yet been proven to provide definitive treatments. In contrast, colonic or nasogastric tube administration of complex fecal solutions for FMT is rapidly and durably efficacious for the treatment of recurrent C. diff infection. And the full fecal mix may not be necessary. A report in Lancet as far back as 1989 demonstrated successful C. diff treatment with a mix of 10 bacteria. Clearly, such a reductionist approach with defined bacterial strains would facilitate pharmaceutical development.
Importantly though, C. diff infection remains the only indication for which FMT has been robustly successful. A second strategy, built on “bugs as drugs,” involves the use of genetically engineered organisms. For example, recombinant bacteria can be engineered for improved persistence in the GI tract or as drug delivery vehicles. Thus, surface expression of epithelial cell-binding proteins could improve bacterial persistence and engineering to secrete a therapeutic product could enable local and continuous drug delivery. These and other recombinant technologies are expected to broaden the potential utility of “bugs as drugs” and create an opportunity for new intellectual property, which is critical to attract industrial interest. A third strategy, designed to alter the ratio of bacteria in the GI tract, is to administer prebiotics, non-digestible compounds that feed the beneficial bacteria so they will bloom. Prebiotics can be used alone or in combination with probiotics.
It is reasonable to assume that small molecules or biologics will be identified that maintain or fortify commensal flora. For example, recent publications suggest that oral delivery of intestinal alkaline phosphatase may protect the microbiota from antibiotics. Alternatively, small molecules can be used to target specific bacterial pathways. By way of example, methane has been implicated as a cause of IBS with constipation. One potential therapeutic, currently in clinical trials, uses a formulation designed to deliver lovastatin throughout the GI tract, where it blocks methane production by the archaea, Methanobrevibacter smithii. Finally, as research continues to elucidate the mechanisms by which the microbiota regulate human physiology, we will likely find new druggable pathways for the development of traditional pharmaceuticals.
Many new microbiome-based therapeutics may not be covered by current regulatory guidelines. It is not intuitively clear what lot release specifications would be required for a product that can mutate during manufacturing, be contaminated by an exogenous bacterium, or propagate in the environment. Thus, companies developing such therapeutics will need to work closely with regulatory agencies to author new guidelines to address efficacy, safety, and quality standards.
The field of microbiome science is in its infancy and is sometimes compared to the “Wild Wild West.” But akin to the early days of the American West, there is tremendous excitement for this frontier of new opportunities.
Christian Furlan Freguia, Ph.D. (firstname.lastname@example.org), is director, research and Michael Kaleko, M.D., Ph.D. (email@example.com) is senior vice president, research & development at Synthetic Biologics, Inc.