Peptide Therapies Are Due for a Gut Check

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July 1, 2016 (Vol. 36, No. 13)

Peptide Drugs Are Not Only Diverse and Manufacturable, They Are Also More Available Via the Oral Route

Peptide drugs have gone in and out of fashion during the last 30 years. With manufacturability and discovery largely solved, success will depend in large part on overcoming issues related to formulating and delivering these promising agents in oral form.

Christopher A. Rhodes, Ph.D., president and CEO at consulting firm Drug Delivery Experts, notes that successful oral peptide drugs have evolved by using both old and new technologies.

Classically, the way to improve bioavailability of peptides has been through permeability enhancement. These techniques disrupt the bilayer in the gut, which permits drugs to pass rapidly into the vast vasculature underneath. “The problem with those methods is they deliver a pulsatile dose,” Dr. Rhodes says.

This works for some drugs but not those that require long exposure time to achieve therapeutic effectiveness. He notes that top pharmaceutical companies have

applied permeability enhancement but failed to achieve bioavailability of more than 2% (a good example is salmon calcitonin that is currently in Phase III and has been under development by Tarsa Therapeutics, and predecessor companies for almost 20 years).

But Novo Nordisk’s semaglutide, the oral GLP-1 analog currently in Phase II, utilizes this technology (as well as injection) effectively because the drug has a long circulating half life—more than one week. Despite less than 1% of the drug entering the bloodstream, its in vivo stability means that the drug need not be dosed exactly at the time of the meal to provide sufficient exposure for maximum efficacy, typically required for the GLP-1s to be effective.

“The combination of long exposure and permeation enhancement makes this peptide and old oral delivery technology combination very promising,” says Dr. Rhodes, noting that sponsor Novo Nordisk plans to take weekly injectable and oral dosage forms of semaglutide into Phase III.

Permeation Enhancement

Permeation enhancement has been the principal strategy for turning poorly absorbed drugs into successful products. It works adequately well for some newer peptide drugs, which tend to be lower in weight (1000 to 2000 Da) and thereby absorb more efficiently than larger peptides.

Cara Therapeutics’ opioid agonist, CR845, uses permeation-enhancement technology from Enteris, a peptide/protein oral delivery specialty firm, and incidentally the company that holds the drug delivery technology that is used in the Tarsa Therapeutics program. The Cara drug recently completed Phase II testing for acute and chronic pain.

“There’s nothing special about permeation enhancing technology,” notes Dr. Rhodes. It has been around for some time. It can be useful if used with the right peptide properties.

A newer strategy (but still old technology) involves embracing the low systemic absorption of peptides. For example, peptides treating inflammatory bowel disease and colitis where the disease exists on the inside of the gut wall.

An added benefit for these gut-based diseases is that systemic exposure is minimal, thereby minimizing side effects. Ironwood Pharmaceuticals’ linaclotide, an example of this strategy, has been approved for irritable bowel syndrome. Blood levels of linaclotide are undetectable.

The newest approach to oral delivery of biologics comes from a device-based approach, namely dissolving microneedles. Rani Therapeutics and an MIT consortium of engineers, separately, use this approach.

Rani uses sugar-based microneedles that stabilize peptides within the microneedles, which are delivered through a standard oral delivery gelatin capsule. The capsule has an enteric coating that opens in the upper gastrointestinal tract at pH 5–7, which pushes needles into the intestinal wall. As they dissolve they release the drug. Bioavailability is around 50%, which while lower than the 90% provided by subcutaneous injection, is an order of magnitude better than the best available permeability enhancer. Rani uses this technology with parathyroid hormone, a GLP-1 analog, interleukin 17, and a half dozen other biologics.

“Rani appears to have solved two of the three major hurdles for oral biologics delivery, stability in the GI tract (sugar formulation is quite stable), and limited permeability of biologics,” according to Dr. Rhodes.

Microneedles are still an early-stage idea whose manufacturability, safety, and tolerability must still be demonstrated.

Solving the Numbers Game

Ra Pharma has developed a technology that adds a new dimension to the concept of peptide libraries, enabling it to develop drug candidates with the diversity and specificity of antibodies and the bioavailability of small molecules. Rather than simply generating collections of short peptides from natural amino acids, the company creates huge assemblies representing every possible combination of amino acids for a given peptide length.

For example a 10-mer using all combinations of the 20 natural amino acids can be represented by 10 trillion individual molecules. The technique also allows the production of cyclic molecules and peptides containing backbone modifications and unnatural amino acids.

“We build into our libraries up-front the features that are critical for good bioavailability, stability, and potency of peptides,” says Douglas Treco, president and CEO. “We can create libraries that allow selection of peptides that are structurally and physicochemically more related to drugs and useful natural products.”

The technology, pioneered by Massachusetts General Hospital researcher, Nobel Laureate, and Ra co-founder Jack Szostak, Ph.D., uses ribosomes to make peptides containing natural and unnatural amino acids. 100 trillion-member peptide libraries in a test-tube are possible. “The numbers are much higher than you could expect from phage display or any other technology,” Treco comments.

The process begins by constructing DNA libraries containing all codon combinations for the desired length, transcribing the DNA to RNA, and feeding that product to the ribosomes.

The trick is to get ribosomes to translate mRNA into a peptide and physically link it to the original mRNA. One trillion mRNA molecules equals 1 trillion peptides. “They’re all floating around in a test tube but you have to fish out the ones you want,” Treco notes.

This is done by exposing the assembly of new peptide-RNA complexes to an immobilized, physiologically relevant target. RNA molecules that bind to the target are amplified by polymerase chain reaction, sequenced, and the associated peptide is synthesized through conventional solid-phase synthesis. The process uses affinity to pull out putatively active peptides, based on no a priori knowledge of what the peptide looks like.

To produce peptides to assay and study, chemical synthesis is significantly more efficient than re-introducing amplified mRNA to ribosomes and having the organelles do the work. Ribosomal translation is not that efficient. “Plus, one of our goals was to avoid biological manufacturing,” points out Treco.

The question of “unnatural” amino acids here is relevant. Ra focuses largely on side-chain and backbone-modified building blocks, for example containing N-methylated species, replacing nitrogen with oxygen to create heterocyclic side-chains, or using fluorinated side-chains. Ra’s technique allows the efficient construction of cyclic peptide libraries as well.

“We’re finding peptides with extraordinary potency, stability, and bioavailability coming right out of our libraries,” says Treco.

Automated room-temperature peptide synthesizers and microwave peptide synthesizers provided by Biotage come in various sizes and evince different degrees of sophistication, from entry-level to high-throughput configurations.

Production Issues Overcome

Peptide production services, which all rely on automated synthesizers, are amply available to drug discovery and development organizations.

Biotage manufactures room-temperature peptide synthesizers and microwave peptide synthesizers in manual, semi-automated, and fully automated formats. John Urh, regional market manager for the Americas, notes that instrument sales are flat or at best growing slowly. Worldwide sales of peptide synthesizers totals between 300 and 400 units annually. Considering the number of organizations with peptide drug programs, and assuming an approximately 50/50 academic/industrial split, those numbers aren’t so bad.

Most experts interviewed in this article note an uptick of interest in peptide drugs, particularly at large drug companies. Merck has established a peptide effort; Novartis and AstraZeneca have stepped up their programs, according to Urh.

One factor that may underestimate overall interest, particularly at universities and research institutes, is the presence of cheap labor (i.e., post-docs and grad students), which makes synthesis by hand through traditional solution chemistry more attractive than instrument-based synthesis.

On the topic of unnatural amino acids, Urh mentions beta-peptides as growing in popularity. These molecules contain an extra carbon between the carboxylate and amino groups. “They can be formed more easily into cyclic peptides,” Urh says, with enhanced pharmacologic and delivery properties.

Also popular are “lollipop” structures composed of a cyclic molecule with a polypeptide tail. Oxytocin, which is used to induce labor, is just one example of a commercially successful lollipop peptide.

Thermo Fisher Scientific’s peptide production business may not be well known to readers, but it operates at the forefront of custom, research-use synthesis services. Peptide synthesis is part of the firm’s mass spectrometry reagents unit and serves a broad range of projects —about half in industry, half in academic or institute organizations.

“We’re seeing a spike in immunology and drug discovery applications, both from academic and industrial organizations,” says Kelsey Song, project specialist.

Thermo’s strength is stable isotope peptides which, with the availability of exquisitely sensitive MS, have replaced radionuclides in a majority of life science applications. The company’s HeavyPeptide™ AQUA products show high accuracy in quantitation studies. The workhorse isotope is 13C, whose presence is easily discerned in peptides and their metabolites from the much more abundant 12C. The company also provides labeled or unlabeled peptide libraries and individual high-purity peptides.

Capabilities include more than 400 modifications of standard peptides, including L-amino acids, sidechain alterations, and CAM and Phospho variants. CAM adds stability to unoxidized cysteine residues, while Phospho phosphorylates oxygen-containing amino acids such as threonine, tyrosine, and serine.

Ya Chen, Ph.D., a scientist at peptide specialty firm LifeTein, explains that with the growing pipeline of peptide drugs, diagnostics, and research reagents business is booming. The well-known problem of degradation in the gut limits the effectiveness of many peptide drugs, but modifying side chains serves as a good work-around for some molecules. “Modified peptides have better resistance to both chemical and enzymatic degradation,” says Dr. Chen.

Aside from common side chain modifications (“unnatural” amino acids), LifeTein produces enantiomers which, according to Dr. Chen, are not in high demand these days. Other mods include phosphorylation or glycosylation on threonine and serine, cyclic peptides, and fluorescent tags for following peptides inside cells.

LifeTein is a research-only provider, not a cGMP shop. For drug development that is further along the regulatory pathway Bachem, GenScript, and PolyPeptide offer their services.

LifeTein has overhauled the entire peptide product process and developed a proprietary method that delivers purified peptides of typical length in 3–5 days versus several weeks. High-purity product takes about one week. “Our record is 159 residues, which took a month,” says Dr. Chen.

LifeTein, a custom peptide synthesis service company, deploys solid-phase peptide synthesis and hybrid technology to generate long peptides. As this image indicates, the company stays on top of peptide production runs by making use of automated liquid- handling systems.

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Related questions

01What is the concept of the immune self, and how has it evolved over the decades?

Adaptive immunity is the ability of specific lymphocytes to differentiate between self and non-self (foreign) antigens and defend the body by selectively destroying non-self-peptides. This concept is possibly the most crucial factor in several immunological medical domains and is increasingly being explored across cancer immunotherapy, vaccine design, pathogen identification, and autoimmune disorders (including allergies). A growing body of literature elucidates the importance of peptides, short amino acid chains linked via peptide bonds, in providing the adaptive immune system with the information required to effectively distinguish between self and non-self particles. This has resulted in the proposal of the ‘immune self’ concept, which postulates that self-similarity is a fundamental determinant of immune recognition. First introduced by Frank MacFarlane Burnet in 1949, the immune self-concept and its sister, the self-nonself theory, have substantially evolved over the decades. Initially driven by observations from Medawar’s early transplantation experiments, Nils K. Jerne (1974; eigen-behavior theory), Polly Matzinger (1994; danger theory), and most recently, evidence from research conducted independently by Waldmann, Mitchison, and Janeway has refined the immune self-concept from ‘all body elements are self, and foreign elements are non-self’ to the most recent ‘infectious non-self (foreign and usually harmful) versus noninfectious self (safe) elements.’

Source: www.news-medical.net ↗

02A peptide identified in a fungus found in northern European pine forests possesses as much power as penicillin as well as vancomycin, according to an international team of researchers.

Reporting in the October 13 issue of Nature, a team from Denmark-based biotech company Novozymes, and researchers from Georgetown University Medical Center and the David Geffen School of Medicine at UCLA, say they have isolated "plectasin," the first defensin ever found in fungi. The research was performed at Novozymes laboratories in Denmark. Defensins are peptides, miniature protein molecules that are produced by a wide range of animals to protect themselves against infection. Humans have defensins in their white blood cells and in their skin, for example, but it is believed that this new fungal defensin, plectasin, is more potent and targets certain bacteria more specifically. Indeed, when plectasin was tested in the laboratory and in animals, it proved to be highly effective against the bacteria Streptococcus pneumoniae, and Streptococcus pyogenes, including strains that are now resistant to conventional antibiotics. These bacteria are responsible for such diseases as meningitis, community-acquired pneumonia, strep throat, life-threatening sepsis, and flesh destroying skin infections. The discovery of plectasin has implications for the development of defensins as a treatment against many common, and deadly, infections, and may initiate a new era of antibiotic discovery and development, said study co-author Michael Zasloff, M.D., Ph.D., Professor in the Departments of Surgery and Pediatrics at Georgetown University Medical Center. Zasloff says that the field of antibiotic development has not changed much since 1929 when Alexander Fleming realized that the fungal "bread mold" Penicillium, which had landed by chance in a Petri dish produced a substance that eliminated colonies of staphylococcal bacteria. "Most antibiotics used by humans are produced by fungi and certain soil bacteria," he said. "Using our existing tools of discovery, we have failed to uncover any new classes of antibiotics from these sources over the past decade. However, by utilizing a new genetic approach that allowed the team to discover plectasin, we now know that a whole class of antibiotics has been overlooked." "This finding (plectasin), and the existence of about 200,000 additional species of fungi, opens up a vast universe to explore for novel peptide antibiotics," said co-author Robert Lehrer, M.D., Distinguished Professor of Medicine at the David Geffen School of Medicine at UCLA. Plectasin, if proven safe and effective in humans, could be on the market by 2012, said Lehrer. Zasloff and Lehrer are known internationally as experts in antimicrobial peptides - the class of antibiotics that plectasin falls within - and in this study they collaborated with Novozymes, a Danish biotech company that led the research. Zasloff and Lehrer are the only two scientists from U.S. universities on the team of 20 researchers who co-authored the research paper. All life forms have to defend themselves against microbial invaders - bacteria, fungi, viruses - and to do this, they produce antimicrobial defensin peptides. In humans, defensins are made by specific white blood cells and immune cells that later engulf foreign invaders, and by the skin and mucous membranes, in order to kill microbes before they invade protective barriers. Researchers believe that fungi have a similar system of defense, especially since these plant-like organisms live off rotting matter, said Zasloff. "They must compete with other organisms, like bacteria and viruses, which also want to consume the same meal. In addition, they need to defend themselves from being eaten by the microbes which surround them." But he said no one had been able to find defensins in fungi using traditional research techniques, which involved growing fungi in liquid cultures and then testing the culture to see if it contained any antibiotic molecule. The research team instead used the latest genetic science to search for the defensins they thought fungi must have. Selecting the Pseudoplectania nigrella species of fungus may have been serendipitous, Lehrer said, but the Novozymes team used state-of-the-art biotechnology to intercept ,and interpret its genetic messages and exhibited tremendous skill in producing plectasin efficiently, economically, and in large amounts." "I started working on antimicrobial peptides over three decades ago, said Lehrer, and my laboratory first described human defensins in 1985. So, the discovery of plectasin makes me feel like a grandfather." Further examination revealed that this defensin, plectasin, resembles defensins found in spiders, scorpions, dragonflies and mussels - thus suggesting that the defensins found in insects, molluscs and fungi arose from a common ancestral gene, the researchers say. Based on this information, the scientists now believe that defensins appeared in living things more than a billion years ago. The investigators then turned to the National Center for Antimicrobials and Infection Control, the Danish equivalent of the U.S. Centers for Disease Control, to test plectasin in the laboratory for antimicrobial activity against a broad spectrum of bacteria. It showed potent activity against several species of Gram-positive bacteria, and was especially active against S. pneumoniae (the leading cause of pneumonia), including all known clinical strains and those that are now resistant to conventional antibiotics. "That is important because increasing bacterial resistance to conventional antibiotics threatens the future of many antibiotics in current use," Zasloff said. "In mouse studies, plectasin showed extremely low toxicity, and was as effective as vancomycin and penicillin in curing the animals of experimental peritonitis (inflammation of the lining of the abdominal cavity, which can be deadly) and pneumonia caused by S. pneumoniae, the researchers report. "Although the precise mechanism by which plectasin exerts its antimicrobial activity is still under investigation, it may work by a mechanism that is very different from traditional antibiotics, Zasloff said. "As a group, defensins exhibit activity against many types of bacteria, fungi, protozoa, and even viruses. It is entirely possible that fungal defensins will be discovered that could be developed against all of these human pathogens," Zasloff added.

Source: www.news-medical.net ↗

03What are functional peptides?

Conventional pharmacological studies on spices have traditionally focused on secondary metabolites like polyphenols, alkaloids, and terpenes. More recently, food science research has also examined spice proteins and their enzymatic hydrolysates, using proteomic methods such as liquid chromatography–tandem mass spectrometry (LC-MS/MS) to identify short bioactive peptide sequences released from larger precursor proteins.6 Once released during food processing, fermentation, or gastrointestinal digestion, these functional peptides can act as metabolic regulators, antimicrobials, or antioxidants.1 Functional peptides refer to specific protein fragments that, once released from their parent proteins, exert biological activities.1,2 In the context of foods, these activities are most often demonstrated using in vitro biochemical or cell-based assays, and their physiological relevance depends on bioavailability and dose.2 Unlike intact proteins, which can have the potential to be allergenic or difficult to absorb due to their complex tertiary structures, functional peptides may exhibit improved bioaccessibility, and some small peptides can cross the intestinal epithelial barrier via peptide transport systems. However, absorption efficiency varies substantially by peptide sequence and digestive conditions.6 Nutriomics and mechanistic investigations have established that the bioactivity of a peptide is dictated by its physicochemical properties, particularly its amino acid composition, molecular weight, and net charge. For example, the presence of hydrophobic amino acids like proline, leucine, and valine often correlates with high antioxidant and enzyme-inhibitory activity.2,3 Smaller peptides, typically those less than three kilodaltons (kDa) in size, exhibit greater stability against proteolytic degradation in the gastrointestinal tract.3 Moreover, cationic peptides are particularly effective as antimicrobial agents through their electrostatic interactions with bacterial membranes.3

Source: www.news-medical.net ↗

04What roles does the system play?

The endogenous opioids and their receptors are widely distributed throughout the central and peripheral nervous systems, particularly the parts of these systems that regulate pain, emotion, reward, stress responses, motivation, drug addiction, and autonomic control. The differential expression and location of the various receptor subtypes across different neurons account for the wide range of opioid-related behaviors. The activation of µ-opioid receptors is mainly known for playing a role in pain relief. Still, research has also indicated it may be involved in behaviors related to survival, such as appetite and reproduction. The activity of µ-opioid receptors is also known to play a critical role in responses to social stimuli by modulating responses to social rejection or social acceptance, for example. Activation of the δ-opioid receptors and κ-opioid receptors is also known to be involved in pain modulation. Also, studies have shown that NOP activation is involved in pain mechanisms and several behaviors related to psychological stress. Alterations in the endogenous opioid system are suspected to be involved in Parkinson's disease, seizures, neuroprotective mechanisms, and depression.

Source: www.news-medical.net ↗

05What was this study about?

It has been noted in around 20 percent of the world population suffers from some form of pain or the other. In many individuals, pain may be relieved initially with pain medications, but soon tolerance develops, and there is a decrease in the efficacy of pain relievers. One of the main symptoms of IBS seen commonly in many sufferers is chronic abdominal pain. Professor Lewis said, "All pains are complex, but gut pain is particularly challenging to treat and affects around 20 percent of the world's population. Current drugs are failing to produce effective pain relief in many patients before side effects limit the dose that can be administered." Professor Brierley echoed this statement saying, "Internal organs have a complex network of sensory nerves that have a wide array of voltage-gated ion channels and receptors to detect stimuli... The hypersensitivity of these nerves in disease often contributes to the development of pain."

Source: www.news-medical.net ↗

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Longevity, Performance & Obesity Research

A research peptide formulation developed to investigate metabolic regulation, mitochondrial function, and nutrient-sensing pathways.

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