Advanced Drug Delivery Reviews 101 (2016) 108–121
`
`Contents lists available at ScienceDirect
`
`Advanced Drug Delivery Reviews
`
`j o u r na l h om e p a g e : w w w . e l s e v i e r . c o m / l o c a t e / a d d r
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`Oral delivery of macromolecular drugs: Where we are after almost
`100 years of attempts☆
`Elena Moroz 1, Simon Matoori 1, Jean-Christophe Leroux ⁎
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`ETH Zurich, Department of Chemistry and Applied Biosciences, Institute of Pharmaceutical Sciences, Vladimir-Prelog-Weg 3, 8093 Zurich, Switzerland
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`a r t i c l e
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`i n f o
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`a b s t r a c t
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`Article history:
`Received 15 November 2015
`Received in revised form 11 January 2016
`Accepted 18 January 2016
`Available online 27 January 2016
`
`Keywords:
`Oral drug delivery
`Oral bioavailability
`Permeation enhancers
`Protease inhibitors
`High molecular weight
`Biologics
`
`Contents
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`Since the first attempt to administer insulin orally in humans more than 90 years ago, the oral delivery of
`macromolecular drugs (N1000 g/mol) has been rather disappointing. Although several clinical pilot studies
`have demonstrated that the oral absorption of macromolecules is possible, the bioavailability remains generally
`low and variable. This article reviews the formulations and biopharmaceutical aspects of orally administered
`biomacromolecules on the market and in clinical development for local and systemic delivery. The most
`successful approaches for systemic delivery often involve a combination of enteric coating, protease inhibitors
`and permeation enhancers in relatively high amounts. However, some of these excipients have induced local
`or systemic adverse reactions in preclinical and clinical studies, and long-term studies are often missing.
`Therefore, strategies aimed at increasing the oral absorption of macromolecular drugs should carefully take
`into account the benefit–risk ratio. In the absence of specific uptake pathways, small and potent peptides that
`are resistant to degradation and that present a large therapeutic window certainly represent the best candidates
`for systemic absorption. While we acknowledge the need for systemically delivering biomacromolecules, it is our
`opinion that the oral delivery to local gastrointestinal targets is currently more promising because of their
`accessibility and the lacking requirement for intestinal permeability enhancement.
`© 2016 Elsevier B.V. All rights reserved.
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`1.
`2.
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`3.
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`2.2.
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`Introduction .
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`Local delivery
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`2.1.
`Peptides
`Vancomycin and fidaxomicin .
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`2.1.1.
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`Linaclotide .
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`2.1.2.
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`Plecanatide and derivatives .
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`2.1.3.
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`Antibodies
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`2.2.1.
`Antibody delivery .
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`Genetically modified antibody-secreting bacteria .
`2.2.2.
`Oral enzyme therapy .
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`2.3.
`Cytoprotective/anti-inflammatory proteins .
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`2.4.
`Nucleic acids
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`2.5.
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`Systemic delivery .
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`3.1.
`Peptides
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`3.1.1.
`Octreotide
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`3.1.2.
`Desmopressin .
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`3.1.3.
`Cyclosporine
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`Abbreviations: ADH, anti-diuretic hormone; AON, antisense oligonucleotide; 5-CNAC, N-(5-chlorosalicyloyl)-8-aminocaprylic acid; EDTA, ethylenediaminetetraacetate; FDA, Food and
`Drug Administration; GC C, guanylyl cyclase C; GI, gastrointestinal; GLP-1, glucagon-like peptide 1; IBD, inflammatory bowel diseases; ICAM-1, intercellular adhesion molecule 1; MG, my-
`asthenia gravis; NDA, New Drug Application; SNAC, N-(8-[2-hydroxybenzoyl]amino)caprylic acid; tkRNAi, transkingdom RNA interference platform.
`☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “Understanding the challenges of beyond-rule-of-5 compounds”
`⁎ Corresponding author at: ETH Zurich, HCI H 301, Vladimir-Prelog-Weg 3, 8093 Zurich, Switzerland. Tel.: +41 44 633 73 10; fax: +41 44 633 13 14.
`E-mail address: jleroux@ethz.ch (J.-C. Leroux).
`1 These authors contributed equally to this manuscript.
`
`http://dx.doi.org/10.1016/j.addr.2016.01.010
`0169-409X/© 2016 Elsevier B.V. All rights reserved.
`
`Grün. Exhibit 1097
`Grünenthal v. Antecip
`PGR2017-00022
`
`

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`3.2.
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`4.
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`Acyline
`3.1.4.
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`Calcitonin .
`3.1.5.
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`Semaglutide
`3.1.6.
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`Proteins
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`Insulin .
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`3.2.1.
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`Nucleic acids
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`3.3.
`Safety of bioavailability-increasing excipients .
`4.1.
`Protease inhibitors
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`4.2.
`Permeation enhancers .
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`Conclusions .
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`5.
`Acknowledgments
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`References .
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`E. Moroz et al. / Advanced Drug Delivery Reviews 101 (2016) 108–121
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`1. Introduction
`
`The first attempt to deliver insulin orally in humans was undertaken
`as early as 1922, only 1 year after the discovery of insulin by Drs. Fred-
`erick Banting and Charles Best [1], when increasing doses of insulin
`were given orally to a single diabetes patient. The results were negative,
`and already in this first study, the critical challenges of oral protein de-
`livery became apparent: poor and variable absorption, and low efficacy
`compared with subcutaneous injection. Although the interest and ef-
`forts in the oral delivery of biomacromolecules have intensified over
`the past two decades, safely and effectively delivering high-molecular-
`weight substrates via the oral route remains highly challenging for for-
`mulation scientists [2,3].
`The gastrointestinal (GI) tract is a hostile environment for biomacro-
`molecules because it is evolutionarily optimized to break down nutri-
`ents and deactivate pathogens. The highly acidic pH in the stomach
`results in the protonation of proteins and their unfolding, which ex-
`poses more motifs that are recognized by protein-degrading enzymes
`[4]. The enzymes in the stomach (pepsin) and small intestine (e.g.,
`chymotrypsin, amino- and carboxypeptidases, RNases and DNases)
`cleave proteins and nucleic acids into smaller fragments and single
`units [4]. In the colon, enzymatic fermentation processes further de-
`grade biomacromolecules [4]. Because therapeutically active biomacro-
`molecules are equally affected by these processes, the fraction surviving
`these degradation processes is generally low and variable, especially in
`the presence of food [5]. The macromolecular drug needs to overcome
`multiple barriers designed to prevent the entry of dietary and bacterial
`antigens in order to reach the systemic compartment. To access the ep-
`ithelial cell layer, the biomacromolecule firstly needs to diffuse through
`the mucus layer covering the intestinal epithelium [5]. The latter is an-
`other important barrier, as the tight junctions which seal the epithelial
`cells restrict the paracellular transport (i.e., the passage between cells)
`to small molecules and ions (b600 Da) [6]. The transcytotic pathway
`(i.e., the passage across the cell in an endocytotic vesicle) is mediated
`by luminally expressed endocytotic receptors (e.g., vitamin B12 recep-
`tor, transferrin receptor), and therefore necessitates conjugation to the
`respective ligands in order to be exploited in drug delivery [7]. Another
`access point to the systemic compartment is the phagocytotic M-cells of
`Peyer's patches which sample luminal antigens and can take up partic-
`ular substrates in the low micrometer range [8]. However, the propor-
`tion of M-cells in the gut epithelium is small and varies greatly
`between species, which complicates predictions of absorption in
`humans based on animal data [9].
`Not surprisingly, only six biomacromolecules have been approved
`by the Food and Drug Administration (FDA) for oral delivery: two local-
`ly and two systemically delivered peptides, one locally delivered non-
`peptidic macrocycle, and one locally delivered protein mixture. How-
`ever, several orally applied formulations of proteins, peptides, and
`nucleic acids are currently under clinical evaluation. Often, these formu-
`lations contain at least one of the following excipients (Fig. 1): an enter-
`ic coating and/or protease inhibitors to prevent drug degradation and
`
`permeation enhancers to enable paracellular transport of macromole-
`cules [10,11]. Mechanistically, absorption enhancement can be achieved
`by mechanically disrupting tight junctions or the plasma membrane,
`lowering mucus viscosity, and modulating tight junction-regulating sig-
`naling pathways [2]. Additional strategies for the oral delivery of
`biomacromolecules under clinical development include buccal delivery,
`utilizing carrier-mediated transcytosis, and local delivery to GI targets.
`The overwhelming majority of currently approved oral drugs and
`clinical candidates exhibit a molecular weight of b1000 Da [12]. Above
`this threshold, low bioavailability, inter- and intraindividual variability,
`food effects, and long-term safety concerns of bioavailability-enhancing
`excipients remain important challenges of oral delivery despite clear
`advances in knowledge after nearly 90 years of trial and error. In this re-
`view, we address orally applied biomacromolecular therapeutics
`(N1000 Da) already marketed or under clinical investigation for local
`or systemic delivery with an emphasis on the drug formulations and
`the biopharmaceutical aspects. The oral delivery of vaccines will not
`be covered in this manuscript, and the readers are referred to other re-
`cent reviews for more information on this topic [13–16].
`
`2. Local delivery
`
`Macromolecular drugs that act on GI targets increasingly move into
`focus because local delivery avoids the challenges of reaching the sys-
`temic compartments. Advantages of locally delivering high-molecular-
`weight drugs include fewer restrictions regarding drug size and a
`potentially more favorable safety profile due to minimal systemic
`exposure, reduced immunogenicity, and the absence of permeation-
`enhancing excipients [17,18]. To conserve the therapeutic activity in
`the GI tract, several formulation strategies have been employed, such
`as enteric/colon-targeting capsules that protect against the harsh GI en-
`vironment, supplementation with sacrificial proteins that compete for
`degradation, and hindering enzymatic access using polymer conjuga-
`tion or protective antibodies which bind to known cleavage epitopes
`[4,17,19]. The ailments that are targeted by locally acting macromole-
`cules include inflammatory diseases (Crohn's disease, ulcerative colitis),
`metabolic disorders (e.g., exocrine pancreas insufficiency), constipation,
`and infections (Table 1) [4,17].
`
`2.1. Peptides
`
`2.1.1. Vancomycin and fidaxomicin
`Isolated in 1953 from a soil sample in the jungle of the island Borneo,
`the antibiotic vancomycin is produced by the bacterium Amycolatopsis
`orientalis [20]. Vancomycin is a glycosylated tricyclic heptapeptide
`(1449 Da) which contains modified amino acid residues (e.g., chlorinat-
`ed tyrosine) [20–22]. Due to its highly hydrophilic (log P − 3.1) and
`large cyclic structure, vancomycin is only marginally absorbed and me-
`tabolized in the GI tract [23,24]. Intravenously applied vancomycin was
`initially approved by the FDA to treat penicillin-resistant bacterial infec-
`tions in 1958, and it is still indicated for severe infections caused by
`
`

`

`110
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`
`Fig. 1. Strategies for oral delivery of macromolecular drugs that act on local or systemic targets. In buccal or sublingual delivery, the drug targets the buccal or sublingual mucosa, which
`avoids degradation pathways in the GI tract. Often, permeation enhancement is necessary to cross the multilayered buccal epithelium. Mucoadhesive bacteria that secrete the desired
`protein in situ are a novel means of achieving sustained release in the oral cavity. To enhance stability against degrading enzymes, macromolecular drugs can be chemically modified
`by polymer conjugation (proteins), backbone and base modifications (nucleic acids), and cyclization as well as by introducing D-amino acids (peptides). Sacrificial proteins, protease
`inhibitors, and enteric coatings can also be included in the formulation to further improve GI resistance. To achieve meaningful systemic exposure, absorption enhancement by tight
`junction-disrupting excipients is often needed. A novel approach is the in situ production and secretion of therapeutic biomacromolecules by genetically modified bacteria.
`
`susceptible strains of methicillin-resistant staphylococci, Clostridium dif-
`ficile-associated diarrhea, and staphylococcal enterocolitis and for pa-
`tients who are allergic to beta-lactam antibiotics. Vancomycin is
`formulated as gelatin capsules containing 125 or 250 mg of active com-
`pound (package leaflet). In 2011, a non-peptidic macrocyclic macrolide
`antibiotic fidaxomicin (1058 Da, Dificid®, Cubist Pharmaceuticals) with
`minimal systemic absorption and GI metabolism was approved for the
`treatment of C. difficile infections. Fidaxomicin showed lower relapse
`rates compared with vancomycin. In view of its much higher costs,
`fidaxomicin treatment may be most suitable for patients who are at
`highest risk of relapse [25]. Further cyclic peptidic compounds in clinical
`trials for the local treatment of C. difficile infections include the
`lipopeptide surotomycin (1681 Da, phase III, Cubist Pharmaceuticals/
`Merck), the lipoglycodepsipeptide ramoplanin (2554 Da, NTI-851,
`phase IIb, Nanotherapeutics), the thiazolyl peptide LFF-571 (1367 Da,
`phase II but currently removed from pipeline, Novartis), and the
`lantibiotic NVB302 (2115 Da, phase I, Novacta Biosystems Limited)
`[26,27].
`
`2.1.2. Linaclotide
`Linaclotide (1525 Da) is a truncated derivative of Escherichia coli
`heat-stable (ST) enterotoxin that consists of 14 amino acid residues
`and three disulfide bonds [28]. It acts as a guanylyl cyclase C (GC
`C) agonist locally in the small intestine (Fig. 2) [28–30]. In 2012, it re-
`ceived FDA approval (Linzess®, Forest Labs LLC) for the treatment of
`chronic idiopathic constipation and irritable bowel syndrome with con-
`stipation. After oral administration, linaclotide is minimally absorbed,
`and its plasma concentrations are below the limit of quantification
`[31]. Because linaclotide is stable in the stomach, it is formulated as
`hard gelatin capsules containing 0.145 or 0.290 mg of the active com-
`pound (package leaflet). However, it is cleaved in the small intestine
`to a 13-amino acid active metabolite [31], which is eventually degraded
`in the intestine [32].
`
`2.1.3. Plecanatide and derivatives
`The GC C agonist plecanatide (1682 Da, Synergy Pharmaceuticals) is
`a synthetic analog of uroguanylin, a naturally occurring GI regulator of
`
`GC C signaling, with one amino acid substitution that results in stronger
`receptor binding [33]. It is a bicyclic peptide consisting of 16 amino acid
`residues with two disulfide bonds [33]. Plecanatide completed the clin-
`ical phase III for the treatment of chronic idiopathic constipation, and it
`is currently being evaluated in phase III for irritable bowel syndrome
`with constipation. It showed a statistically significant increase in the
`number of complete spontaneous bowel movements per week com-
`pared with placebo (approximately 20% of durable responders at a
`dose of 3 mg per day vs. 12% in the placebo group). In comparison to
`linaclotide, plecanatide showed reduced diarrhea incidence (10% vs.
`20%) [34]. In addition to plecanatide, Synergy Pharmaceuticals is devel-
`oping a more stable analog, dolcanatide (SP-333), to treat opioid-
`induced constipation. Dolcanatide has two D-amino acid substitutions
`at C- and N-terminus in order to improve stability in simulated gastric
`and intestinal fluids (www.synergypharma.com). Dolcanatide has com-
`pleted a randomized, double-blind, placebo-controlled phase II clinical
`trial in patients with constipation who take opioid analgesics for chronic
`pain (NCT01983306). Despite the increased stability in the intestinal
`environment, the dose of dolcanatide needed to achieve a therapeutic
`effect was comparable with that for plecanatide. Furthermore, the
`dolcanatide-mediated activation of GC C ameliorated inflammation in
`a colitis mouse model, promoting its development in the treatment of
`ulcerative colitis [35].
`
`2.2. Antibodies
`
`2.2.1. Antibody delivery
`The vast majority of clinically tested antibodies with GI luminal tar-
`gets inactivate undesired molecules, such as bacterial toxins, cytokines,
`viruses, and virulence factors [17]. In the treatment of inflammatory
`bowel diseases (IBD), capturing TNF-α in the intestinal lumen is a
`promising alternative to systemic treatment with anti-TNF-α antibodies
`(e.g., adalimumab, infliximab and certolizumab pegol) with regard to
`systemic immunosuppression, development of neutralizing antibodies,
`and needle-free administration [36–38]. AVX-470 (Avaximab™-TNF,
`Avaxia Biologics) is an orally administered polyclonal antibody which
`targets luminal TNF-α in the intestine [39]. It is produced by purifying
`
`

`

`Table 1
`Macromolecular treatments for local GI therapy in clinical development (as of January 2016, source: Thomson Reuters Integrity®, ClinicalTrials.gov, and company press releases).
`
`E. Moroz et al. / Advanced Drug Delivery Reviews 101 (2016) 108–121
`
`111
`
`Type
`
`Generic and trade
`name, molecular
`weight
`
`Modification/Formulation
`
`Indication
`
`Vancomycin (Vancocin®),
`1449 Da
`Fidaxomycin (Dificid®),
`1058 Da
`Surotomycin,
`1681 Da
`Ramoplanin, 2554 Da
`LFF-571, 1367 Da
`NVB302, 2115 Da
`
`Linaclotide (Linzess®),
`1525 Da
`
`Cyclic structure, modified amino
`acid residues; gelatin capsule
`
`Non-peptidic macrocycle; tablet
`
`Cyclic structure
`
`Cyclic structure
`Cyclic structure, thiazolyl peptide
`Cyclic structure, thioether bonds
`
`Cyclic structure; gelatin capsule
`
`Peptide
`
`Clostridium difficile-associated diarrhea
`(CDAD) and staphylococcal enterocolitis
`including methicillin-resistant strains
`
`CDAD
`
`CDAD
`
`CDAD
`CDAD
`CDAD
`Chronic idiopathic constipation (CIC)
`and
`irritable bowel syndrome with
`constipation (IBS-C)
`
`Plecanatide, 1682 Da
`
`Cyclic structure
`
`CIC and IBS-C
`
`Dolcanatide (SP-333),
`1682 Da
`
`Cyclic structure, two D-amino
`acid substitutions
`
`Opioid-induced constipation (OIC) and
`ulcerative colitis (UC)
`
`Development
`phase (NCT#)
`
`Marketed
`
`Marketed
`
`Phase III (NCT01597505,
`NCT01598311)
`Phase IIb (N.A.)
`Phase II (NCT01232595)
`Phase I (N.A.)
`
`Marketed
`
`CIC phase III completed in 2015
`(NCT01919697)
`IBS-C phase III ongoing
`(NCT02493452)
`OIC phase II completed in 2014
`(NCT01983306)
`UC phase Ib ongoing
`(N.A.)
`Phase II ongoing
`(N.A.)
`Phase Ia completed in 2014
`(N.A.)
`
`Antibody
`
`Enzyme
`
`AVX-470 (Avaximab™-TNF),
`broad
`
`AG014, 47.8 kDa
`
`Creon® (porcine protease,
`amylase, lipase)
`
`Protein-secreting
`bacteria
`
`AG013, 6.5 kDa
`
`AG011, 18 kDa
`
`Mongersen (GED-0301),
`6952 Da
`
`Alicaforsen, 6368 Da
`
`Nucleic acid
`
`CEQ508
`
`N.A: unknown or non-existent NCT number.
`
`Polyclonal antibody;
`enteric capsule
`Lyophilized Lactococcus lactis secreting
`certolizumab; enteric capsule
`
`Pediatric UC
`
`Inflammatory bowel disease
`
`Enteric capsule
`
`Exocrine pancreatic insufficiency
`
`Marketed
`
`Oral rinsing solution containing 2.0 × 1011
`colony-forming units (CFU)/15 mL of
`Lactococcus lactis secreting human trefoil
`factor 1
`Lactococcus lactis secreting
`anti-inflammatory
`cytokine IL-10; enteric capsule in
`combination
`with enema
`
`UC
`
`Chemotherapy- or radiation-induced
`oral mucositis
`
`Phase Ib completed in 2012
`(NCT00938080)
`
`Phase IIa completed in 2009,
`failed
`(NCT00729872)
`
`Phase II completed in 2014
`Phase I ongoing
`(NCT02367183)
`
`Phase III ongoing
`(NCT02525523)
`
`Phosphorothioate AON; enteric coating
`
`Moderate to severe Crohn's disease
`
`UC, pouchitis
`
`Phosphorothioate AON; nightly enema
`with
`hydroxypropyl methylcellulose
`Suspension of genetically modified E. coli
`expressing
`invasin, listeriolysin, and short hairpin
`RNA targeting
`β-catenin
`
`Familial adenomatous polyposis
`
`Phase I/II ongoing since 2011
`(N.A.)
`
`total antibodies from the milk of dairy cows that are immunized with
`human TNF-α, which results in approximately 0.3–0.9% (m/m) TNF-α-
`specific and mainly IgG1 subtype antibodies (www.avaxiabiologics.
`com). Providing early immunity to neonates, high amounts of colostrum
`antibodies seem to be partially resistant to GI digestion in adults with
`fully developed digestive systems [40–42]. AVX-470 is developed as an
`enteric-coated capsule for treating pediatric ulcerative colitis and is cur-
`rently in phase II of clinical trials. According to the company's website,
`a phase Ib trial (NCT01759056) in 36 ulcerative colitis patients showed
`that the treatment resulted in consistent encouraging trends across mul-
`tiple disease parameters (colonic TNF-α levels, endoscopic index of se-
`verity, serum C-reactive protein levels) after four weeks, and no allergic
`reaction or human anti-bovine antibodies in serum were observed. Bo-
`vine immunoglobulin was detected in stools and displayed TNF-α-bind-
`ing activity, suggesting that the large excess of decoy antibodies in the
`formulation (N99%) protected a fraction of the anti-TNF-α antibodies.
`
`2.2.2. Genetically modified antibody-secreting bacteria
`Intrexon's (formerly ActoGeniX) approach for luminal antibody
`therapy consists of delivering attenuated bacteria to the GI tract,
`where the antibody is secreted in situ. Food-grade Lactococcus lactis
`was genetically modified to secrete certolizumab, an anti-TNF-α
`Fab fragment (47.8 kDa). In an open-label clinical trial in healthy vol-
`unteers, lyophilized bacteria administered in enteric capsules were
`recovered in both the small and large intestine by endoscopic sam-
`pling, and the antibody was secreted by living bacteria in the colon
`lumen (www.dna.com). The treatment was safe and well-tolerated.
`In addition, preclinical studies demonstrated the treatment's efficacy
`in multiple colitis models and showed that L. lactis adhere especially
`to the inflamed mucosa, which increases the concentration of the an-
`tibody in the close proximity of target cells [43]. However, questions
`remain regarding the concentration variability and the intestinal sta-
`bility of the secreted antibody.
`
`

`

`112
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`E. Moroz et al. / Advanced Drug Delivery Reviews 101 (2016) 108–121
`
`Fig. 2. Schematic representation of the mechanisms of action, primary structure, and diarrhea incidence of the GC C agonists linaclotide, plecanatide, and dolcanatide. The sequences of
`these peptides are based on the physiological agonist uroguanylin and the structurally similar heat-stable Escherichia coli enterotoxin (ST) but include sequence adaptations such as
`unnatural D-amino acids (dN = D-asparagine, dL = D-leucine). Lines above and below the peptide sequences denote the disulfide bonds. Upon binding of an activating ligand, GC C
`converts guanosine triphosphate (GTP) to cyclic guanosine monophosphate (cGMP), increasing the intracellular cGMP concentration and activating the cystic fibrosis trans-membrane
`conductance regulator (CFTR). The resultant efflux of chloride ions out of the epithelial cell leads to a water influx into the intestinal lumen, which normalizes bowel movements.
`Extracellular cGMP further inhibits intestinal nociceptors. Thus, GC C agonists improve constipation and alleviate chronic abdominal pain in patients suffering from irritable bowel
`syndrome with constipation. The incidence of diarrhea, a common treatment-emergent adverse event associated with excessive treatment response, is also indicated for the three
`peptides (dose and regimen in parentheses). The information was collected from [29–34] and the website of Synergy Pharmaceuticals.
`
`2.3. Oral enzyme therapy
`
`2.4. Cytoprotective/anti-inflammatory proteins
`
`Enzyme replacement therapy via the oral route is an important ther-
`apeutic option in metabolic disorders in which a certain enzymatic ac-
`tion in the GI tract is absent. Numerous oral enzyme products are on
`the market for a variety of disorders such as exocrine pancreatic insuffi-
`ciency due to pancreatitis, cystic fibrosis and other conditions, and lac-
`tose,
`fructose, sucrose and histamine intolerance (e.g., Creon®,
`Lacteeze®, Sucraid®, DAOSiN®) [4]. Whereas the only FDA-approved
`enzymatic drug is pancreatin (a mixture of bovine or porcine pancreatic
`amylase, proteases, and lipases), the other oral enzyme products are
`generally under the legal framework of dietary supplements, which
`usually do not require thorough efficacy studies.
`Because these therapeutic enzymes are inactivated in the stomach
`by low pH and pepsin, one or a combination of stabilization strategies
`are generally needed. Creon® (Abbvie), for instance, consists of pan-
`creatin formulated in enteric-coated capsules, whose dissolution is
`triggered by the pH increase in the small intestine. An uncoated formu-
`lation of pancreatin (Viokace®, Aptalis Pharma) which is used to treat
`exocrine pancreatic insufficiency is co-administered with proton
`pump inhibitors to decrease gastric degradation. As an exception,
`sacrosidase and the acid lactase (tilactase) produced by Aspergillus
`oryzae are intrinsically stable and active in acidic conditions [4], and
`they do not require sophisticated delivery systems. However, average
`residence time of lactase in the stomach might be insufficient for com-
`plete digestion of high amounts of lactose [44]. Therefore, a formulation
`of a lactase, which is active in neutral to basic pH of the small intestine,
`was recently developed as a capsule containing enteric-coated pellets
`(Lactosolv®, Sciotec Diagnostic Technologies) [44]. The same approach
`has been successfully applied for other enzyme deficiencies (Xylosolv®
`for fructose and DAOSiN® for histamine intolerance). As an alternative,
`the administration of probiotics that secrete β-galactosidase, which is
`capable of lactose hydrolysis, has been proposed; however, different
`clinical trials in patients with lactose intolerance gave inconsistent effi-
`cacy results [45]. Oral formulations of polymer-conjugated enzymes
`and enzymes in enteric-coated capsules are under preclinical investiga-
`tion for other pathologies such as celiac disease and phenylketonuria [4,
`46–50].
`
`As a consequence of chemotherapy and radiation, cancer patients
`often develop a complication called oral mucositis in which the mouth's
`mucosal lining is destroyed and ulcers form. AG013 (Intrexon Corpora-
`tion) is a bacteria-based system which delivers a mucosa-healing pro-
`tein for the treatment of oral mucositis. The protein, called human
`trefoil factor 1, is involved in mucosal healing and tissue protection via
`multiple mechanisms, including inhibiting apoptosis during cell migra-
`tion and stabilizing the protective mucus layer [51]. The formulation
`consists of an oral rinsing suspension containing attenuated (i.e., unable
`to replicate due to thymidylate synthase deficiency [51]), genetically
`engineered L. lactis which adhere to the buccal mucosa and secrete the
`mucosa-healing factor locally for up to 24 h after administration [52].
`A phase Ib study in head and neck cancer patients undergoing chemo-
`therapy produced positive results, showing a 35% reduction in the dura-
`tion of oral ulcers [51].
`A similar in situ system based on L. lactis which secretes the anti-
`inflammatory cytokine interleukin-10 (AG011, Intrexon) was proposed
`for ulcerative colitis. However, a combination of enema and enteric
`capsules containing interleukin-10-secreting L. lactis did not result in
`mucosal healing in a phase IIa, double-blind, placebo-controlled study
`with 60 ulcerative colitis patients [53]. Recently, another genetically
`modified strain of L. lactis was engineered to deliver the endogenous
`serine protease inhibitor elafin. The expression of this anti-
`inflammatory peptide is lowered in patients with active celiac dis-
`ease, and elafin treatment was effective in decreasing inflammation
`and intestinal permeability in a mouse model of celiac disease [54].
`
`2.5. Nucleic acids
`
`Antisense oligonucleotides (AONs) are short, single-stranded, syn-
`thetic sequences of nucleotides which can hybridize with complemen-
`tary mRNA and consequently decrease the expression of the encoded
`protein [55]. Several GI tract pathologies hold attractive targets for
`local nucleic acid therapy (e.g., IBD or familial adenomatous polyposis)
`[56,57]. Because nucleic acids are readily degraded by the RNases
`and DNases in the GI tract and depurinated in the acidic stomach
`
`

`

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`
`113
`
`environment, they require stability-enhancing chemical modifications
`of the backbone or bases [58]. Various modifications of the ribose ring
`(e.g., 2′O-(2-methoxyethyl) or 2′F-ANA) not only exhibit enhanced nu-
`clease resistance but also show higher affinity to complementary
`mRNAs and thus improved knockdown efficacy [59,60].
`Mongersen (GED-0301, Celgene) is a 21-mer phosphorothioate AON
`targeting the mRNA of the Smad7 protein, which is overexpressed in
`IBD mucosal tissues and leads to suppression of anti-inflammatory
`TGF-β signaling [61]. Mongersen acts on epithelial and lamina propria
`cells, and its bioavailability after oral administration is minimal [62].
`An enteric-coated oral formulation of mongersen was evaluated in
`166 patients with active Crohn's disease in a phase II trial [61]. Partici-
`pants received 10, 40, or 160 mg of mongersen once daily for 2 weeks.
`In the mongersen groups that received 40 and 160 mg, significantly
`more patients reached clinical remission, the primary end point, after
`four weeks of treatment (55% and 65%, respectively), compared with
`10% in the placebo group. According to the company, follow-up data
`of this trial (e.g., on mucosal healing by endoscopy) will be presented
`in the future.
`Alicaforsen (Atlantic Healthcare) is a 20-mer phosphorothioate AON
`targeting the mRNA of intercellular adhesion molecule 1 (ICAM-1),
`which recruits immune cells to the site of inflammation and is
`overexpressed in the gut epithelium of IBD patients [63]. Although a
`four-week treatment with injectable alicaforsen fell short of inducing