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24 | JULY 2016 | GENengnews.com | Genetic Engineering & Biotechnology News enhancement but failed to achieve bioavailability of more than 2% (a good example is salmon calcitonin that is cur- rently 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 injec- tion) 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 suffcient exposure for maximum effcacy, typically required for the GLP-1s to be effective. "The combination of long exposure and permeation en- hancement makes this peptide and old oral delivery technol- ogy 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 effciently than larger peptides. Cara Therapeutics' opioid agonist, CR845, uses perme- ation-enhancement technology from Enteris, a peptide/pro- tein oral delivery specialty frm, and incidentally the com- pany 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 em- bracing the low systemic absorption of peptides. For exam- ple, peptides treating infammatory bowel disease and colitis where the disease exists on the inside of the gut wall. An added beneft for these gut-based diseases is that sys- temic 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 micronee- dles. Rani Therapeutics and an MIT consortium of engi- neers, separately, use this approach. Rani uses sugar-based microneedles that stabilize peptides within the microneedles, which are delivered through a stan- dard 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 avail- able 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 manufac- turability, 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 specifcity 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 indi- vidual molecules. The technique also allows the production of cyclic molecules and peptides containing backbone modi- fcations and unnatural amino acids. "We build into our libraries up-front the features that are critical for good bioavailability, stability, and potency of pep- tides," says Douglas Treco, president and CEO. "We can cre- ate libraries that allow selection of peptides that are structur- ally 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 con- taining all codon combinations for the desired length, tran- scribing 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 foating around in a test tube but you have to fsh 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 amplifed by polymerase chain reaction, sequenced, and the associated peptide is synthesized through conventional solid-phase syn- thesis. The process uses affnity 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 synthe- sis is signifcantly more effcient than re-introducing ampli- fed mRNA to ribosomes and having the organelles do the work. Ribosomal translation is not that effcient. "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-modifed building blocks, for example containing N-methylated spe- cies, replacing nitrogen with oxygen to create heterocyclic side-chains, or using fuorinated side-chains. Ra's technique allows the effcient construction of cyclic peptide libraries as well. "We're fnding peptides with extraordinary potency, sta- bility, and bioavailability coming right out of our libraries," says Treco. Production Issues Overcome Peptide production services, which all rely on automated synthesizers, are amply available to drug discovery and de- velopment organizations. Peptide Therapies Are Due for a Gut Check BIOPROCESSING 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. Continued from page 1