Advanced Drug Delivery Reviews 106 (2016) 277–319
`
`Contents lists available at ScienceDirect
`
`Advanced Drug Delivery Reviews
`
`j o u r n a l h o m 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
`
`Intestinal permeation enhancers for oral peptide delivery☆
`Sam Maher a, Randall J. Mrsny b, David J. Brayden c,⁎
`
`a RCSI School of Pharmacy, Royal College of Surgeons in Ireland, St Stephen's Green, Dublin 2, Ireland
`b Department of Pharmacy and Pharmacology, University of Bath, UK
`c UCD School of Veterinary Medicine and Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland
`
`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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`Intestinal permeation enhancers (PEs) are one of the most widely tested strategies to improve oral delivery of
`therapeutic peptides. This article assesses the intestinal permeation enhancement action of over 250 PEs that
`have been tested in intestinal delivery models. In depth analysis of pre-clinical data is presented for PEs as
`components of proprietary delivery systems that have progressed to clinical trials. Given the importance of co-
`presentation of sufficiently high concentrations of PE and peptide at the small intestinal epithelium, there is an
`emphasis on studies where PEs have been formulated with poorly permeable molecules in solid dosage forms
`and lipoidal dispersions.
`
`© 2016 Elsevier B.V. All rights reserved.
`
`Article history:
`Received 29 April 2016
`Received in revised form 7 June 2016
`Accepted 9 June 2016
`Available online 16 June 2016
`
`Keywords:
`Oral peptide delivery
`Intestinal permeation enhancers
`Paracellular transport
`Transcellular
`Solid dose formulation
`Surfactants
`Emulsions
`
`Contents
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`1.
`2.
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`Introduction .
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`Therapeutic peptides .
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`Barriers to translation of PE-based oral peptide technologies .
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`Intestinal PEs
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`4.1.
`Paracellular PEs .
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`4.1.1.
`Paracellular PEs emerging from the study of toxins .
`4.1.2.
`Paracellular PEs that bind claudins
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`Paracellular PEs that target E-cadherin and Ca2 + .
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`4.1.3.
`4.1.4.
`Paracellular PEs that target occludin .
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`4.1.5.
`Paracellular PEs and cytoskeletal reorganisation .
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`Trancellular PEs .
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`4.2.1.
`Soluble surfactant PEs .
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`4.2.2.
`Insoluble surfactants .
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`Peptide hydrophobisation .
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`4.3.
`Non-surfactant PEs
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`4.4.
`4.5. Multiple modes of enhancement action .
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`Safety and regulation of PEs .
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`5.1.
`Transcellular enhancers and membrane perturbation .
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`5.2.
`The bystander absorption argument .
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`5.3.
`Are paracellular PEs safer than transcellular PEs? .
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`PE developability classification system .
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`4.2.
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`
`☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “SI: Oral delivery of peptides”.
`⁎ Corresponding author. Tel.: +353 17166013; fax: +353 17166204.
`E-mail address: david.brayden@ucd.ie (D.J. Brayden).
`
`http://dx.doi.org/10.1016/j.addr.2016.06.005
`0169-409X/© 2016 Elsevier B.V. All rights reserved.
`
`Grün. Exhibit 1093
`Grünenthal v. Antecip
`PGR2017-00022
`
`

`

`278
`
`S. Maher et al. / Advanced Drug Delivery Reviews 106 (2016) 277–319
`
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`Conclusions
`7.
`Acknowledgements
`References
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`1. Introduction
`
`Growth in global peptide markets has spurred development of
`technologies that enable oral delivery of poorly permeable drugs. Initial
`delivery strategies focused on inclusion of candidate excipients that
`protected the peptide from intestinal degradation and transiently
`altered intestinal permeability [1]. The majority of oral peptide delivery
`technologies that are currently in clinical trials use formulations with
`established intestinal PEs that have a history of safe use in man [2].
`Recent clinical data suggests that inclusion of PEs in oral formulations
`can safely assist absorption of selected potent peptides with a large
`therapeutic index. For example, primary endpoints were met in a
`Phase III trial of octreotide formulated in an oily suspension with a
`medium chain fatty acid salt, sodium caprylate (C8) [3]. In parallel, a
`new generation of PEs with more specific mechanisms of action are in
`preclinical research, and may confer improved safety and efficacy over
`those currently in development. This article summarises the progress
`of ~250 PEs that have been tested in preclinical intestinal delivery
`models (Tables 1, S1). An in-depth review of pre-clinical data is present-
`ed for PEs in proprietary delivery systems that have progressed to
`clinical development. The review by Aguirre et al. (this Issue [4]) evalu-
`ates the performance of technologies in clinical trials, of which most are
`enteric-coated solid dosage forms containing PEs. We focus here on how
`PEs alter intestinal permeability and on innovations that may further
`assist translation of safety and efficacy outcomes from pre-clinical
`models to man.
`
`2. Therapeutic peptides
`
`A drug delivery system that facilitates oral peptide administration
`has long been desired. There are ~55 therapeutic peptides marketed
`as parenteral formulations (based on a ~9 kDa cut-off in molecular
`weight (MW)) (Table 2) and a further 140 in clinical development [5].
`Compared to small molecules, peptides are attractive due to their spec-
`ificity, potency, efficacy, and low toxicity. Clinical potential of unmodi-
`fied injectable peptides can be hampered by a short plasma half-life
`(t1/2) due to labile moieties and higher manufacturing costs relative to
`small molecules. A breakdown of marketed peptide products indicates
`that injection routes (61%) are the most common, followed by topical
`(11%), nasal (9%), oral (9%) and ophthalmic (4%), noting that bioavail-
`ability is typically low and variable from non-injectable routes [6].
`Injection requirements are associated with lack of adherence to dos-
`ing regimens, hence the impetus towards long acting formulations that
`are administered less often. Thus, for glucagon-like-Peptide 1 (GLP-1)
`analogues, sub-cutaneous (s.c.) injection of exenatide has shifted from
`twice-a-day administration (Byetta®; Lilly, USA) to once weekly admin-
`istration (e.g. Bydureon®, Lilly). This was achieved by development of a
`microsphere-based controlled release system [7], whereas competing
`approaches have attempted to improve stability and reduce recognition
`by the reticuloendothelial system by conjugating lipid moieties to
`amino acid residues or by fusing the analogue to albumen. Although
`needle fabrication technology has improved in the last 20 years, injec-
`tions are still inconvenient in the longer term and can delay take-up
`and adherence to regimes necessitated by chronic diseases. In the case
`of type 2 diabetes (T2D), early initiation of insulin can slow the progres-
`sive destruction of pancreatic β-cells [8], but T2D patients frequently re-
`quire dose adjustments related to peripheral hypoglycaemia [9]. Oral
`insulin may reduce such risks because it is absorbed via the portal
`vein and therefore imitates pancreatic secretion to the liver [10]. This
`
`can also reduce two other side effects attributed to s.c. insulin in the
`periphery: weight gain and lipodystrophy [11].
`An oral peptide dosage form would likely reduce costs associated
`with sterile manufacture of injectables, cold chain, needle disposal,
`and staff/patient training, but these savings would be offset against
`the requirement for higher doses compared to injection. A commercial
`driver for oral peptides is life cycle extension and increased revenue
`from branded medicines based around new patents. Development of
`oral delivery systems for approved injectable peptides has the benefit
`of known pharmacology for the active pharmaceutical ingredient
`(API), good safety profiles (at least for the injected route) and
`established analytical detection methods. The most clinically-
`advanced oral peptide formulations are being developed for diabetes
`(insulin, GLP-1 analogues), osteoporosis (salmon calcitonin, sCT;
`teriparatide (PTH 1–34)), and acromegaly (octreotide). Anti-diabetic
`peptides account for ~40% of peptides in commercial oral peptide deliv-
`ery programmes and Table S2 details selected patents filed on oral insu-
`lin over the last 30 years. Synthesis of injectable anti-diabetic peptides
`with long plasma t1/2 values is also contributing to investment in oral
`peptide delivery systems (e.g. t1/2 = 160 h for the GLP-1 analogue,
`semaglutide, Novo-Nordisk, Denmark [12]), as they may yield better
`oral pharmacokinetic (PK) data than short-acting counterparts.
`Competition between GLP-1 analogues makes oral formulation a key
`battleground [5].
`Development of non-injected dosage forms has had some commer-
`cial successes,
`including oral desmopressin (DDAVP®, Ferring,
`Switzerland), oral cyclosporin (Neoral®, Novartis, Switzerland) and
`nasal calcitonin (Miacalcin®, Novartis). The suitability of commercially
`available peptides for oral reformulation depends on their physico-
`chemical properties (MW, solubility), chemical complexity, therapeutic
`considerations (route/frequency of administration, therapeutic index)
`and cost-effectiveness. Peptides typically exhibit high aqueous solubili-
`ty and low permeability, properties that unofficially place them in the
`Biopharmaceutics Classification system (BCS) Class III. Nevertheless,
`some peptides with cationic and anionic functional groups exhibit com-
`plicated pH-dependent solubility, where solubility is high in acidic con-
`ditions at pH values below their isoelectric point (pI), and is relatively
`low at pH values at and above their pI. Many basic molecules rely on
`acid/base phenomena for dissolution within the stomach and subse-
`quent absorption across the duodenum and jejunum, so peptides with
`low intrinsic solubility are problematic. For example, insulin dissolves
`in dilute acid but not at neutral pH, which could manifest as poor disso-
`lution in the small intestine. Peptides that have a MW N6000 Da do not
`have any appreciable intestinal permeability when delivered orally, this
`makes insulin (5808 Da) especially challenging, with difficulty decreas-
`ing in the order of teriparatide (4118 Da) N exenatide (4187 Da) N sCT
`(3532 Da) N octreotide (1019 Da). In addition, there is a correlation
`between MW and susceptibility to proteolysis [13].
`An ideal oral candidate peptide should therefore have a low MW,
`high potency, enzymatic/chemical stability (e.g. cyclised peptides, D-
`substituted amino acids), a high therapeutic index and be of relatively
`low cost to synthesise. Desmopressin (MW 1069 Da) contains stable
`amino acids; it has an oral bioavailability (F) of only 0.17%, so high
`potency is its key attribute [14]. Prandial insulin is more challenging
`because it requires three relatively high mealtime doses to reach the re-
`quired plasma levels per day. The s.c. insulin dose required for manage-
`ment of Type 1 diabetes (T1D) of 0.5–0.8 IU/kg per dose (1.2–1.9 mg); if
`normalised for an oral system designed for an oral F of 10%–20%, a dose
`level of 6–20 mg would be required. A recent oral insulin clinical study
`included 8 mg (240 IU) insulin three times daily [15], whereas
`
`

`

`S. Maher et al. / Advanced Drug Delivery Reviews 106 (2016) 277–319
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`279
`
`exenatide is injected at a dose of 10 μg and has been tested orally at
`15-fold higher doses using the same technology [16].
`
`3. Barriers to translation of PE-based oral peptide technologies
`
`Peptides have poor oral bioavailability due to peptidase sensitivity
`and low intestinal permeability. They may be sensitive to gastric pepsin
`and acid- dependent destabilisation of disulphide bridges, hydrogen
`bonding and electrostatic interactions, although stomach-related break-
`down can largely be overcome by enteric coating (e.g. Eudragit®,
`Evonik, Germany; Kollicoat® (BASF, Germany) [17]. Enteric coating ex-
`cipients exhibit pH-dependent dissolution due to deprotonation of
`weakly acidic functional groups at high pH values. Oral peptide formu-
`lations that are enterically coated must be administered pre-prandially
`to avoid premature release in the stomach when buffered by food. Gas-
`tric emptying time is therefore a consideration for peptides like insulin
`that require absorption to coincide with ingestion of a meal. In the
`fasted state, capsule dosage forms are consistently found in the small in-
`testine 1 h post administration [18]. The lag time between dose and food
`intake is an important therapeutic consideration for peptides that re-
`quire prandial administration (e.g. insulin), but less so for peptides
`like exenatide and octreotide. Requirement for pre-prandial administra-
`tion also raises concerns around adherence, when the dosage form must
`be administered in complex regimes over an indefinite period. Applica-
`tion of Eudragit® coatings without inclusion of excipients that address
`peptide degradation and poor permeability ultimately will not increase
`oral F [19,20].
`Upon leaving the stomach the peptide is vulnerable to proteolytic
`degradation in the lumen, brush border membrane, and in the cytosol
`of small intestinal enterocytes. The pancreas can produce over 40 g of
`proteolytic enzymes [21] delivered in 2.5 L of pancreatic juice per day
`[22]. Large linear peptides including insulin, sCT, glucagon and secretin
`are sensitive to human intestinal fluid (HIF), while higher stability is
`noted for short and structurally-confined peptides with stable bonds
`(e.g. octreotide, cyclosporin and desmopressin) [13]. PEs can also have
`a dual benefit in inhibiting regional proteolysis, examples being sodium
`glycocholate [23] and ethylenediaminetetraacetic acid (EDTA) [24]).
`However, any PE that is a peptide may itself be sensitive to proteolysis,
`examples being zonula occludens toxin (ZoT) and the C-terminal frag-
`ment of Clostridium perfringens enterotoxin (C-CPE).
`Many PEs are surfactants, so it is possible to protect the peptide in li-
`poidal dispersions including microemulsions (e.g. Macrulin™, Provalis,
`UK) (Section 4.2.2.3). A leading PE-based technology appears to offer
`peptidase inhibition by non-covalent complexation of the peptide
`with a carrier (e.g. sodium salcaprozate, Eligen®, Emisphere, USA)
`(Case 14: Eligen®). Inclusion of peptidase inhibitors like aprotinin can
`improve oral peptide delivery, however established excipients with
`similar properties are less risky in terms of toxicology. Acidifying organ-
`ic acids including citric acid (CA) and tartaric acid lower the optimal pH
`for proteolysis and can benefit oral peptide formulation, since if they
`reach a pH of 1–2 units below the isoelectric point, they can improve
`solubility (e.g. insulin). If however, the pH remains at the isoelectric
`point for the peptide, the solubility of the peptide will be low, and it
`may be sensitive to secreted bicarbonate. Acidifiers can also interfere
`in the dissolution and enhancement action of anionic PEs, some of
`which exhibit low dissolution and poor enhancement action at pH
`values below their pKa. For example, the pKa of another lead PE, sodium
`caprate (C10) (the sodium salt of the medium chain fatty acid, capric
`acid) is 6.5; should an acidifier decrease pH to 5.5, over 90% of the mol-
`ecule will exist as an insoluble oil. Further, the hydrophilic-lipophilic
`balance (HLB) of capric acid is 4.8, which is lower than C10 (HLB: 21.8)
`and ultimately well below the HLB considered optimal for permeation
`enhancement [2]. The reduction in luminal pH by co-encapsulated acid-
`ifiers can also decrease the dissolution of enteric-coated dosage forms,
`and efforts to overcome this include separate coating of granules prior
`to tableting to prevent such interactions. In addition, the cationic charge
`
`imparted on many therapeutic peptides (e.g. sCT, pI=10.1) in acidified
`conditions can increase entrapment in mucus by electrostatic complex-
`ation. Finally, some acidifiers chelate metals, which can reduce proteol-
`ysis due to removal of peptidase co-factors. Ca2+ is also an important
`component in epithelial tight junction (TJ) formation, and some studies
`suggested that CA can also increase intestinal permeability via chelation
`(Table 1), although this hypothesis was challenged recently in an in vitro
`insulin permeability study in rat tissue mucosae where the data sug-
`gested that the main role of CA in oral peptide formulations is to reduce
`peptidase activity [25].
`Protease inhibitors in oral peptide dosage forms include soybean
`trypsin inhibitor (SBTI)) [26], aprotinin [23], ovomucoids [27], EDTA
`[24], sodium glycocholate [28] and camostat mesilate [23]. Some im-
`proved absorption of peptides to an extent [23,28]; a combination of
`an enteric coating with aprotinin significantly improved oral peptide
`bioavailability in rats [20]. A safety argument against the use of inhibi-
`tors is that impaired dietary protein digestion may occur. However, in-
`hibitors may provide localised protection where the dosage form
`dissolves and not throughout the GI tract. Nevertheless, agents like
`aprotinin target ubiquitous biological functions, which raises concerns
`regarding suitability for oral peptides. SBTI was included in oral
`exenatide formulations at concentrations as high as 125 mg/dose in
`clinical trials [26]. Despite the GRAS status of soy protein isolate [29],
`purified SBTI can cause pancreatic hyperplasia and carcinoma in rats
`[30,31] and systemic absorption is undesirable. To this end, retention
`in the intestine has focused on conjugation to non-absorbed polymers
`(e.g. chitosan–aprotinin [32], chitosan–EDTA [33]). It is noteworthy
`that pancreatic peptidases are responsible for only 20% of the degrada-
`tion of ingested proteins, with the brush border enzymes accounting for
`the majority [34]. Therefore, inhibitors need also to access the brush
`border for optimum efficacy. One example was the protection at the
`brush border membrane achieved for metkephamid by inhibiting ami-
`nopeptidase N with puromycin, thereby improving oral F in rats from
`0.5% to 3.5% [35]. Ovomucoids also inhibit intestinal serine proteases
`and are commonly isolated from egg white of avian species [27]. Despite
`successful peptidase inhibition, the apparent permeability coefficient
`insulin across rat jejunal mucosae was decreased by
`(Papp) of
`ovomucoids, so peptidase inhibition is not predictive of improved flux
`per se [36]. Combining peptidase inhibitors with PEs may therefore
`achieve improved permeability compared to either approach alone [37].
`Mucous can decrease the rate and extent that peptides diffuse to the
`intestinal epithelium. The estimated (variable) mesh pore diameter of
`porcine jejunal mucous ranges between 200 and 2000 Å [38], much
`larger than the molecular radius of most candidate peptides for oral
`delivery (e.g. insulin b2 Å). Nevertheless, diffusivity (D) of peptides
`through mucous is affected by viscosity (ranging between 1000 and
`10,000-fold greater than water at low shear [39]) and the MW of the
`peptide (approximating the dissolved peptide as a sphere) according
`to the Stoke-Einstein equation (D = RT/6ηπrN). Poor diffusivity is di-
`rectly related to residence time in the small intestinal lumen, which de-
`pends on the peptide surface charge at a given luminal pH, the potential
`for non-covalent bonding, and susceptibility to proteolysis [40,41]. Dif-
`fusivity measurements for cyclosporine and desmopressin were compa-
`rable at selected concentrations across porcine GI mucous, although
`they have comparable MW but different lipophilicity [42]. The capacity
`of mucous to reduce diffusivity may in part account for why mucolytics
`such N-acetylcysteine (NAC), either alone or in combination with non-
`ionic surfactant PEs (e.g. polyoxyethylene octyl phenyl ether (Triton®
`X-100, Dow Chemicals, USA) improve fluorescent dextran-4kDa (FD4)
`and sCT bioavailability in rat intestinal instillations [43,44]. Despite
`NAC having established safety in man, with approvals in respiratory
`and abdominal conditions, there is little interest in combining PEs
`with NAC or other mucolytics. In part, this relates to high variability in
`mucous production, the requirement for high concentrations of muco-
`lytics in the formulation, and because disulphide bond reduction can
`also degrade certain peptides [45]. The capacity of mucous to complex
`
`

`

`280
`
`S. Maher et al. / Advanced Drug Delivery Reviews 106 (2016) 277–319
`
`Ref
`
`[209]
`[488]
`[489]
`[489]
`[211]
`[203]
`[490]
`[408]
`[208]
`[212]
`[210]
`[491]
`[283]
`[492]
`[492]
`[287]
`[284]
`[288]
`[494]
`[161]
`[495]
`[495]
`[279]
`[161]
`[497]
`[25]
`[201]
`[202]
`[227]
`[227]
`[497]
`[498]
`[498]
`[499]
`[245]
`[500]
`[501]
`[86]
`[231]
`[241]
`[504]
`[503]
`[136]
`[88]
`[504]
`[500]
`[20]
`[505]
`[265]
`[209]
`[208]
`[266]
`[185]
`[257]
`[506]
`[506]
`
`Representative peptide/metric
`In situ: flux (fosfomycin)
`In vitro: flux (FD-10)
`Ex vivo (rabbit): flux (insulin)
`In vivo (rabbit): AUC (insulin)
`In vivo: RH (insulin)
`In vivo: PK/PD (heparin)
`In situ: flux (PABA)
`In vivo: PK/PD, flux (insulin)
`In situ: PK/PD (insulin)
`In situ: PK/PD (calcitonin)
`In vivo: flux, PK/PD (insulin)
`In vivo: PK/PD (insulin)
`In vitro: TEER, flux (mannitol)
`Ex vivo: flux (LY)
`In situ: F (rhodamine123)
`In situ: F (insulin)
`In situ: AUC,F (erythropoietin)
`In situ: AUC, F (erythropoietin)
`In situ: F (lansoprazole)
`In situ: AUC (LMWH)
`In situ: F (gentamicin)
`In situ: AUC (vancomycin)
`In situ: AUC (LMWH)
`In situ: AUC (LMWH)
`In vivo: F (gentamicin)
`In vitro: flux (insulin)
`In vivo: PK/PD, F (sCT)
`Ex vivo: flux (FD-4)
`In vitro: TEER, flux (mannitol)
`In vitro: TEER, flux (FD-4)
`In vitro: flux (tiludronate)
`In vitro: flux (ranitidine)
`Ex vivo: flux (tiludronate)
`Ex vivo: flux (Phenol red)
`Ex vivo: flux (ebiratide)
`In situ: AUC(phenol red)
`In situ: F (carboxyfluorescein)
`In situ: AUC, flux (azetirelin)
`In situ: PA (hCT)
`In vivo: F (azetirelin)
`In vitro: flux (FD-4)
`In vitro: flux (FD-4)
`In vitro: flux (FD-4)
`In vitro: flux (PEG 4000)
`Ex vivo: flux (inulin)
`In situ: AUC (phenol red)
`In situ: AUC (insulin)
`In vivo: AUC,(norfloxacin)
`In vivo: flux (trypan blue)
`In vivo: flux (fosfomycin)
`In situ: PK/PD (insulin)
`In situ: F (cefmetazole)
`In situ: AUC (insulin)
`In vitro: Papp (mannitol)
`Ex vivo: S (DPH)
`In vivo: F (cefoxitin)
`
`Concentration & dose
`
`In situ: 1%
`In vitro: 0.1%
`Ex vivo: 5%
`In vivo: 1%
`In vivo: 1%
`In vivo: 500 mg/kg
`In situ: 1%
`In vivo: 3% w/w
`In situ: 5%
`In situ: 0.5%
`In vivo: 3% w/w
`In vivo: 3%
`In vitro: 1% w/v
`Ex vivo: 0.1% v/v
`In situ: 0.1% v/v
`In situ: ―
`In situ: 94 mg/kg
`In situ: 50 mg/kg
`In situ: 170 mg
`In situ: 30 mg/kg
`In situ: 1 mL/kg
`In situ: 1.06 g/kg
`In situ: 50 mg/kg
`In situ: 30 mg/kg
`In vivo: 0.6 mL
`In vitro 5 mg
`In vivo: 10 mg
`Ex vivo:
`In vitro: 0.1% w/v
`In vitro: 0.1% w/v
`In vitro: 0.025% w/v
`In vitro: 0.1% w/v
`Ex vivo: 0.025% w/v
`Ex vivo: 20 mM
`Ex vivo: 20 mM
`In situ: 20 mM
`In situ: 20 mM
`In situ: 2.5 mM
`In situ: 10 mM
`In vivo: 2.5 mM
`In vitro: 1 mM
`In vitro: 1 mM
`In vitro: 0.25%
`In vitro: 0.25%
`Ex vivo: 50 mM
`In situ: 20 mM
`In situ: 1% w/v
`In vivo: 1:1
`In vivo: ―
`In vivo: 1% w/v
`In situ: ―
`In situ: 0.25 mL/kg
`In situ: 50 mM
`In vitro: 1 mM
`Ex vivo: ―
`In vivo: ―
`
`Enhancement
`In situ: ―
`In vitro: 520-fold
`Ex vivo: ―
`In vivo: ―
`In vivo: RH = 55%
`In vivo: ―
`In situ: 19-fold
`In vivo: ―
`In situ: 3-fold
`In situ: ―
`In vivo: ―
`In vivo: 118-fold
`In vitro: 34-fold
`Ex vivo: no effect
`In situ: F = 22.82%
`In situ: F = 0.25%
`In situ: 21-fold
`In situ: 12-fold
`In situ: F = 28.1%
`In situ: 4-fold
`In situ: F = 55.3%
`In situ: ―
`In situ: ―
`In situ: ―
`In vivo: F = 22.4%
`In vitro: no effect
`In vivo: F = 1.8%
`Ex vivo: ―
`In vitro: 9-fold
`In vitro: 26-fold
`In vitro: 3-fold
`In vitro: 19-fold
`Ex vivo: 7-fold
`Ex vivo: no effect
`Ex vivo: 7-fold
`In situ: 4-fold
`In situ: F = 57%
`In situ: 9-fold
`In situ: 4-fold
`In vivo: F = 43.5%
`In vitro: 6-fold
`In vitro: 6-fold
`In vitro: 53-fold
`In vitro: 29-fold
`Ex vivo: ―
`In situ: 2-fold
`In situ: 55-fold
`In vivo: no effect
`In vivo: ―
`In vivo: ―
`In situ: 3-fold
`In situ: F = 18.2%
`In situ: 15-fold
`In vitro-
`Ex vivo: ―
`In vivo: 26-fold
`
`Table 1
`Leading PEs tested in oral delivery of poorly permeable drugs and transport markers.
`
`Enhancer
`
`Mode
`
`Actions
`
`Model
`
`C12E9
`
`Transcellular Membrane fluidity
`
`Caprylocaproyl PEG 8 glycerides
`
`Transcellular
`
`Fluidic dispersion Membrane fluidity
`
`Citric acid
`
`Paracellular
`
`Intracellular ATP
`
`Dodecyl-β-D-maltopyranoside (DDM)
`
`Multimodal Membrane fluidity
`
`EDTA
`
`Paracellular
`
`Ca2+ chelation
`PKC activation
`
`Glyceryl monocaprate
`
`Transcellular Membrane fluidity
`
`Laurylocarnitine
`
`Multimodal
`
`Membrane fluidity
`―
`
`In situ (rat): intestinal loop
`In vitro: Caco-2
`Ex vivo (rabbit): Ussing
`In vivo (rabbit):rectal instillation
`In vivo (dog): suppository
`In vivo (rat): gavage
`In situ (rat): perfusion
`In vivo (dog):suppository
`In situ (rat): rectal instillation
`In situ (rat): rectal
`In vivo (dog): suppository
`In vivo (rat): suppository
`In vitro: Caco-2
`Ex vivo (rat): ileum
`In situ (rat): closed loop
`In situ (rat): colon
`In situ (rat): jejunal patches
`In situ (rat): jejunal
`In situ (rat): duodenal
`In situ (rat): duodenal
`In situ (rat): colon
`In situ (rat): Ileal
`In situ (rat): jejunal
`In situ (rat): intraduodenal
`In vivo (Dog): oral
`In vitro: Caco-2
`In vivo (rat): oral
`Ex vivo (rat): Ussing
`In vitro: Caco-2
`In vitro: Caco-2
`In vitro: Caco-2
`In vitro: Caco-2
`Ex vivo (rat): perfusion
`Ex vivo (rat): Ussing
`Ex vivo (rat): colon
`In situ (rat,) loop
`In situ (rat): loop
`In situ (rat): colon
`In situ (rat): closed loop
`In vivo (dog): oral
`In vitro: Caco-2
`In vitro: Caco-2
`In vitro: Caco-2
`In vitro: Caco-2
`Ex vivo (rat): colon
`In situ (rat,) loop method
`In situ (rat): ligated loop, colon
`In vivo (rabbit): oral
`In vivo (rat): rectal, microenema
`In vivo (rat): jejunum
`In situ (rat): rectal
`In situ (rat): rectal
`In situ (rat): rectal loop
`In vitro: Caco-2
`Ex vivo (rat): BBM
`In vivo (rat): rectal
`
`

`

`S. Maher et al. / Advanced Drug Delivery Reviews 106 (2016) 277–319
`
`Ex vivo (rats): S-G diffusion cell
`Ex vivo (rats): S-G diffusion cell
`In vivo (rat):oral (microcapsule)
`In vivo (dog): oral (EC capsule)
`In vitro: Caco-2
`Ex vivo (rat) Ussing
`Ex vivo (rat) Ussing
`In vitro: Caco-2
`In vitro: Caco-2
`Ex vivo (rat): Ussing (jejunal)
`Ex vivo (rat): Ussing (jejunal)
`In vitro: Caco-2
`In situ (rat): instillation
`In situ (rat): intubation
`In vivo (pig): oral (EC capsule)
`Ex vivo (rat): Ussing
`Ex vivo (rat): Ussing
`Ex vivo (rat): BBM
`In vivo (rat): rectal
`Ex vivo (rats): S-G diffusion cell
`Ex vivo (rats): S-G diffusion cell
`In vitro: Caco-2
`In vitro: Caco-2
`In vivo (rat): oral (microcapsule)
`In vivo (dog): oral (capsule)
`In vitro: Caco-2
`In vitro: Caco-2
`Ex vivo (rat): Ussing
`Ex vivo (human): Ussing
`In vitro: Caco-2
`In vitro: Caco-2
`In vitro: Caco-2
`In vivo (rat): oral
`In vitro: Caco-2
`In situ (rat): perfusion
`In situ (rat): perfusion
`In vitro: Caco-2
`In vivo (rat): instillation
`In vivo (human): oral
`In vivo (human): oral
`In vitro: Caco-2
`In situ (rat): oral
`In vitro: Caco-2
`In vivo (rabbit): oral
`In vitro: Caco-2
`In vivo (rat): suppository
`In vitro: Caco-2
`In situ (rat): instillation
`In situ (rat): colon
`In vivo (rat): oral
`In vivo (dog) oral
`In vivo (rat): suppository
`In vitro: Caco-2
`In vivo (human): suppository
`In situ (rat): suppository
`In vivo: (rabbit) suppository
`Ex vivo (rat): inverted sac
`Ex vivo (rat): everted colon
`In situ (rat): colon)
`
`Ex vivo: flux (LY)
`Ex vivo: TEER
`In vivo: F (DMP 728)
`In vivo: F (DMP 728)
`In vitro: TEER, flux (FD-40)
`Ex vivo: I sc (Cl)
`Ex vivo: flux (FD-4)
`In vitro: TEER, flux (mannitol)
`In vitro: TEER, flux (FD-4)
`Ex vivo: flux (mannitol)
`Ex vivo: flux (FD-4)
`In vitro: flux (FD-4)
`In situ: F (buserelin)
`In situ: F (octreotide)
`In vivo: F (octreotide)
`Ex vivo: Isc (Cl−)
`Ex vivo: flux (FD-4)
`Ex vivo: fluorescence polarization
`In vivo: F (cefoxitin)
`Ex vivo: flux (LY)
`Ex vivo: TEER
`In vitro: TEER, flux (ruthenium red)
`In vitro: flux (PEG 4000)
`In vivo: F (DMP 728)
`In vivo: F (DMP 728)
`In vitro: TEER, flux (mannitol)
`In vitro: flux (mannitol, Ca2+)
`Ex vivo: flux (FD-4)
`Ex vivo: flux (FD-4)
`In vitro: TEER, flux (mannitol)
`In vitro: TEER, flux (FD-40)
`In vitro: TEER, flux (fluorescein)
`In vivo: PA (insulin)
`In vitro: flux (insulin)
`In situ: AUC (GLP-1)
`In situ: AUC (extendin-4)
`In vitro: flux (insulin)
`In vivo: F (insulin)
`In vivo: AUC (GLP-1)
`In vivo: AUC (PYY3−36)
`In vitro: TEER, flux (mannitol)
`In situ: PK/PD (heparin)
`In vitro: TEER, flux (insulin)
`In vivo: flux (heparin)
`In vitro: TEER, flux (mannitol)
`In vivo: AUC (sodium ampicillin)
`In vitro: flux (mannitol)
`In situ: flux (FD-4)
`In situ: AUC (cefmetazole)
`In vivo: AUC, F (DMP 728)
`In vivo: F (DMP 728)
`In vivo: AUC (sodium ampicillin)
`In vitro: flux (mannitol)
`In vivo: F (cefoxitin sodium)
`In situ: AUC (acyclovir)
`In vivo: flux (gentamicin)
`Ex vivo: flux (CsA)
`Ex vivo: flux (inulin)
`In situ: flux (inulin)
`
`Ex vivo: 10 mM
`Ex vivo: 2 mM
`In vivo: 8 mg/kg
`In vivo: 2 mg/kg
`In vitro: 100 μM
`Ex vivo: 0.5%
`Ex vivo: 0.5%
`In vitro: 0.1% w/v
`In vitro: 0.1% w/v
`In vitro: 0.1% w/v
`In vitro: 0.1% w/v
`In vitro: 2.5% w/v
`In situ: 1% w/v
`In situ: 10% w/v
`In vivo: 40% (70 mg)
`Ex vivo: 0.5%
`Ex vivo: 0.5%
`Ex vivo: ―
`In vivo: ―
`Ex vivo: 5 mM
`Ex vivo: 1 mM
`In vitro: 0.2 mM
`In vitro: 0.2 mM
`In vivo: 8 mg/kg
`In vivo: 2 mg/kg
`In vitro: 500 μM
`In vitro: 100 μM
`Ex vivo: 0.5%
`Ex vivo: 0.5%
`In vitro: 0.75 mM
`In vitro: 100 μM
`In vitro: 200 μM
`In vivo: 5 mM
`In vitro: 60 μM
`In situ: 0.5 mM
`In situ: 0.5 mM
`In vitro: ―
`In vivo: ―
`In vivo: 2 mg
`In vivo: 1 mg
`In vitro: 50 mg/mL
`In situ: 300 mg/kg
`In vitro: 55 mM
`In vivo: 120 mg/kg
`In vitro: 50 mg/mL
`In vivo: 20 μmol/kg
`In vitro: 10 mM
`In situ: 100 mM
`In situ: 0.25% w/v
`In vivo: 40.2%
`In vivo: 40.2%
`In vivo: 20 μmol/kg
`In vitro: 120 mM
`In vivo: 0.5 g
`In situ: 4%
`In vivo: 0.18 mM
`EX vivo: 1% w/v
`Ex vivo: 0.25%
`In situ: 0.25%
`
`Ex vivo: 20-fold
`Ex vivo: ―
`In vivo: F = 6.9%
`In vivo: F = 17%
`In vitro: ―
`Ex vivo: ―
`Ex vivo: ―
`In vitro: 143-fold
`In vitro: 153-fold
`Ex vivo: 9-fold
`Ex vivo: 20-fold
`In vitro: 363-fold
`In situ: 16-fold
`In vivo: 15-fold
`In vivo: F = 0.5%
`Ex vivo: ―
`Ex vivo: ―
`Ex vivo: ―
`In vivo: 34-fold
`Ex vivo: 18-fold
`Ex vivo: ―
`In vitro: 20-fold
`In vitro: no effect
`In vivo: F = 14.6% (6-fold)
`In vivo: F = 20.5% (2-fold)
`In vitro: 10-fold
`In vitro: ―
`Ex vivo: 13-fold
`Ex vivo: 8-fold
`In vitro: TEER b10%
`In vitro: ―
`In vitro: ―
`In vivo: 79-fold
`In vitro: ―
`In situ: no effect
`In situ: 2-fold
`In vitro: 3-fold
`In vivo: F = 3.1%
`In vivo: 2-fold
`In vivo: 2-fold
`In vitro: 27-fold
`In situ: ―
`In vitro: 10-fold
`In vitro: no effect
`In vitro: 2-fold
`In vivo: 6-fold
`In vitro: 8-fold
`In situ: 14-fold
`In situ: 10-fold
`In vivo: F = 6% (3-fold)
`In vivo: F = 17.7%
`In vivo: 5-fold
`In vitro: 2-fold
`In vivo: 2.5-fold
`In situ: 2-fold
`In vivo: ―
`Ex vivo: 9-fold
`