Formulation Strategies to Improve Oral Peptide Delivery:
A Patent Review
Sam Maher1, Aoife Duffy1, Ben Ryan1, David J. Brayden2†
1
School of Pharmacy, Royal College of Surgeons in Ireland, Dublin 2, and
UCD School of Veterinary Science and UCD Conway Institute, University
College Dublin, Belfield, Dublin 4, Ireland
2
†
Author to whom all correspondence should be addressed:
David J. Brayden
Room 214, Veterinary Sciences Centre
UCD, Belfield, Dublin 4, Ireland
Tel: +353 1 7166013
Fax: +353 1 7166219
Email: david.brayden@ucd.ie
1
ABSTRACT
Delivery of peptides by the oral route greatly appeals due to commercial, patient
convenience and scientific arguments. While there are over sixty injectable peptides
marketed worldwide and many more in development, most delivery strategies do not yet
adequately overcome the barriers to oral delivery for peptides, proteins and
macromolecules. Peptides are sensitive to chemical and enzymatic degradation in the
intestine and are poorly permeable across the small intestinal epithelium due to suboptimal physicochemical properties. A successful oral peptide delivery technology will
protect selected potent peptides from pre-systemic metabolism and improve epithelial
permeation to achieve a target bioavailability with acceptable intra-subject variability.
This review assesses the current status of oral peptide delivery with an emphasis on
patented formulations that have recently progressed to clinical evaluation.
2
Table of contents
1. Introduction
2. Impediments to effective oral peptide delivery
3. Strategies to improve oral peptide delivery
3.1 Prodrug and structural modification approaches
3.2 Peptide-targeted ligand conjugates
3.3 Carrier complexation
3.4 Permeation enhancers
4. Preclinical technologies in oral peptide delivery
4.1 Tight junction openers
4.2 Cell penetrating peptides
4.3 Colonic delivery of peptides
4.4 Microemulsion, micelle and liquid dispersion approaches
4.5. Oral nanoparticle delivery systems
5. Future pespective
6. Conclusions
Executive summary
Table 1
Figure 1
References
Websites
Patents
3
1. Introduction
Therapeutic peptides are a successful drug class with attributes of high potency,
significant efficacy and low toxicity. There are over 60 peptides licensed for clinical use
worldwide as defined, in most cases, by compositions of less than 50 amino acids. The
market for synthetic peptides alone has increased from $5 billion in 2003 to currently
over $13 billion [1]. There are 100 peptides in clinical development and over 400 are in
pre-clinical research [2]. In the period of 2009-2011, peptides represented 11% (8
licenses) of all FDA approvals, which is an impressive statistic, given that far fewer
peptides than small molecules are in discovery programs [3]. The growing success of
injectable peptides is in spite of poor serum stability, rapid plasma clearance and high
manufacturing costs compared to small molecules. The benefits of the oral route
outweigh those of parenteral, which is reflected in the fact that two thirds of all
pharmaceutical dosage forms are oral products [4]. The formulation of a peptide in an
oral dosage form could reduce the costs associated with sterile manufacture of injectables
and remove the need for them to be administered by healthcare providers. These
theoretical gains are balanced by inevitable compromises in percentages of oral
bioavailability compared to those of injections. Furthermore, the cost aspects of oral
formulations also depend on the required peptide dose level, which is a function of the
efficiency of the delivery system, peptide potency, dosing regimen, and cost of
manufacture. The selection of a suitable peptide for oral formulation is a key commercial
decision. For example, selecting a complex, high molecular weight (MW), narrow
therapeutic index peptide, manufactured by a costly recombinant approach, requiring
multiple daily oral administrations would be a poor choice. On the other hand, incentives
4
include increased revenue from new oral formulation patents that increase market share
by providing a more convenient route of delivery. The requirement for needles can
reduce patient compliance for the small cohort of patients with needle phobia, and an oral
format may address this. For most, advances in needle technology make daily or weekly
injections of biologics a relatively minor inconvenience, therefore arguments based solely
on parenteral-to-oral switching convenience is a weak justification. From a therapeutic
perspective, oral delivery of some peptides represent a more physiological route
compared with injectable formats; for example, a bolus injection can predispose diabetics
to hyperinsulinemic hypoglycaemia, whereas insulin entering the portal vein from an oral
delivery system imitates pancreatic secretion to the liver target [5]. In addition, type 2
diabetics are reluctant to switch from oral metformin and sulphonylureas to insulin
injections due to the psychological and lifestyle changes involved. Basal or meal-time
oral insulin could be administered earlier in diabetes progression leading to better patient
outcomes, thus providing a scientific rationale.
The most widely studied oral peptide formulations target delivery of therapeutics for
diabetes, osteoporosis and to a lesser extent, acromegaly. In 2013, 382 million people
worldwide were diagnosed with diabetes and the disease caused 5.1 million Deaths [201].
Statistics for the US alone show that 22.3 million people were diagnosed with diabetes in
2012 at an approximate national cost of $245 billion [6]. Antidiabetics are the third
highest grossing class of drug with global sales of $39 billion in 2011 [202]. Some
specialist drug delivery companies have also extended their oral delivery platforms from
peptides to other poorly permeable macromolecules such as low molecular weight
5
heparins (LMWHs) for thrombotic disorders (e.g. Eligen®, Emisphere, USA [7]) and
nucleic acids for inflammatory diseases (Isis Pharmaceutical, USA [8]). The main
peptides in oral clinical development however include insulin, exenatide and glucagonlike peptide-1 (GLP-1) analogues, salmon calcitonin (sCT), octreotide, parathyroid
hormone (PTH), PTH (1-33), opioid analogues, and human growth hormone (hGH) [9].
Most formats in clinical development are new oral formulations of established
parenterally-administered peptides, so oral formats are piggy-backing on established
peptide pharmacology, analytical assays and preclinical animal models of disease.
The concentration of peptide in oral dosage forms is necessarily higher than those
delivered by injections, and this is only feasible by making improvements in costeffective peptide synthesis (e.g. Biocon, India [10] [301]). For example, a Phase III oral
clinical trial of a sCT formulation from Tarsa (USA) using the PeptelligenceTM
technology contained 0.8mg (4800 IU) of sCT, over 20 and 40 times higher concentration
than the respective marketed nasal and parenteral preparations (Miacalcin®, Novartis,
Switzerland) [11]. The higher concentration of peptide in an oral format partcompensates for loss during absorption. Low oral bioavailability is usually accompanied
by wide inter/intra-subject variation leading to variable efficacy. An exception is the
highly potent, cheap, and stable oral peptide with a wide safety margin, desmopressin
(Ferring, Switzerland), which has oral bioavailability of only 0.17% [12]. In this review,
we outline selected strategies used to overcome the barriers to oral peptide delivery with
particular emphasis on patented dosage forms that have reached clinical development.
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2. Impediments to effective oral peptide delivery
The key challenges encountered in oral peptide delivery are pre-systemic degradation,
poor permeability across the intestinal epithelium and first pass metabolism [13].
Peptides are susceptible to conformational instability, chemical instability and enzymatic
degradation at various stages in their life cycle, including formulation, under storage, and
during systemic absorption. Bioactivity can be lost in gastric secretions as a result of
reduction of disulphide bridges and hydrolysis. The gastric milieu is typically avoided by
enteric coating the solid dosage form with weakly acidic polymer films that delay release
by dissolving above their pKa in a pH-dependent fashion. Examples include cellulose
derivatives (e.g. cellulose acetate phthalate) and co-polymers of poly methacrylate (e.g.
Eudragit®, Evonik, Germany). Different Eudragit® preparations dissolve at values
ranging from pH 5.5 up to pH 7 depending on the relative ratio of each block of the
copolymer; they are therefore suitable to avoid the acidic environment of the stomach and
proximal duodenum (reviewed in [14]). Thus, all of the oral peptide formulations in
clinical trials outlined here have enteric coatings designed to undergo initial dissolution at
the pH (>5.5) of duodenal and jejunal fluid.
The most common form of peptide degradation is proteolysis of peptide bonds by rennin
and pepsin in the stomach and by pancreatic peptidases in the small intestine (reviewed in
[15]). Insulin is substantially degraded within 30-60 minutes in the presence of pepsin,
trypsin and chymotrypsin [16]. Overcoming proteolytic degradation in the jejunum is a
more difficult proposition as the peptide must undergo dissolution and is highly
vulnerable in the soluble form to peptidases in the lumen, brush border membrane and in
7
the cytosol of epithelia, as well as by hepatocytes prior to reaching the systemic
circulation. It is difficult to completely inhibit the actions of luminal proteases, but
inhibition in the region where the formulation dissolves is a possibility, for example by
addition of an acidifier (e.g. citric acid or tartaric acid [302]) to lower the pH optimum for
serine protease activity. An alternative approach to protect peptides in the jejunum is
inclusion of protease inhibitors from natural foodstuffs (e.g. soybean trypsin inhibitor
[303]). Caution must be taken when selecting a protease inhibitor as many are not
considered safe for use as excipients, and inhibition of such a ubiquitous biological
function can be risky. For example, aprotinin (TrasylolTM, Bayer, Germany) has
significant pharmacology and safety issues [17]. Likewise, soybean trypsin inhibitor
caused pancreatic hyperplasia and carcinomas in rats [18, 19]. Attempts have therefore
been made to retain such inhibitors in the GI lumen in order to avoid systemic toxicity.
For example, conjugation of aprotinin to chitosan formed polymer-inhibitor conjugates
that could inhibit enzymatic degradation of an associated peptide, and the likelihood of
systemic absorption of the high molecular weight conjugate is minimal [20]. Colloidalprotease conjugates in liposome and microemulsion formats also inhibited protease action
and improved peptide bioavailability, although little is known about the relative safety of
the immobilized forms [21, 22]. Encapsulation within nano- or micro-particulates
provides increased luminal protection for peptides, but the payload may need to be
released in advance of reaching the epithelial surface, as few nanocomplexes can
sufficiently permeate the epithelium. Non-covalent complexation of peptide with a carrier
has also been shown to sterically protect cargoes from luminal digestion (e.g.
salcaprozate sodium, Emisphere Inc., USA [7]). Covalent chemical modification can also
8
provide steric protection from proteases without diminishing peptide therapeutic action
(e.g. PEGylated insulin [23]).
Peptides must then reach intestinal epithelium, the first layer of which is an exogenous
mucus gel layer containing proteases and antibodies [24] which together reduces the rate
of diffusion to the epithelial surface. Attempts to overcome mucoadhesion have focused
on incorporation of mucolytics [25] or use of hydrophilic PEGylated nanoparticles, which
avoid entrapment in mucus glycoprotein meshes [26]. A contrary approach is to exploit
mucoadhesion to increase the residence time of the dosage form in the small intestine,
however there is a variable fluid volume that region and the contents of the lumen are in
continuous motion caused by peristalsis [13, 27]. Both factors can influence the rate and
extent of dissolution, and ultimately causes erratic absorption especially in drugs with
low oral bioavailability. The high rate of mucus turnover in humans makes mucoadhesion
approaches questionable for oral peptide delivery. Slowing the rate of transit using
motility inhibitors to inhibit muscle contraction has also been suggested, but is not
practical for chronic therapy.
If the peptide successfully negotiates the mucous gel layer, the next barrier is the
epithelial surface (Figure 1). The routes available for peptides to permeate this cell layer
include facilitated transport across epithelial cell using endogenous transport mechanisms
(e.g. receptor mediated endocytosis) or passive movement across the epithelial tight
junctions (TJs). The lipophilic acyl chains of enterocyte phospholipids restrict the passive
transcellular flux of hydrophilic peptides. The LogP OCTANOL:WATER coefficient, the
9
number of hydrogen bond donors and acceptors, and molecular weight of the peptide are
useful in predicting intestinal permeability [28]. The optimal Log P value for passive
permeation is in the range of -0.4 to 3.5 [13], but most peptides have values outside this
and consequently permeate the jejunum via peptide transporters, via tight junctions, or
not at all [29]. The precise binding of peptides to target receptors make it difficult to
modify or reduce the number of amino acids that impact on hydrogen bonding, molecular
weight and LogP. Approaches to altering peptide LogP include alkylation via
hydrolysable bonds. Recently, there has been renewed interest in using permeation
enhancers to improve peptide flux across the intestinal epithelium as they do not involve
chemical modification of the peptide. These modulate paracellular permeability either
through opening tight junctions (TJs) [30], by mildly perturbing the intestinal wall to
facilitate transcellular permeation [31], or via a combination of both. Some permeation
enhancers appear to complex non-covalently with peptides, which according to a
controversial theory is thought to temporarily alter solubility and facilitate shuttling
across the intestinal epithelium [32].
Slow or differential dissolution of the components of a dosage form that contain a
delivery vehicle could limit the rate and extent of peptide absorption. Slow dissolution
reduces the concentration gradient across the intestinal epithelium, which reduces the rate
of diffusion, thereby prolonging residence time in the small intestinal lumen and
extending the period that the peptide is exposed to proteases. At the same time, gradual
dissolution may reduce the efficacy of the delivery system. Instead, contemporaneous and
immediate exposure of the epithelium to the enhancer, sodium caprate (C10) was more
10
effective in improving absorption of cefoxitin [33] or 4 kDa FITC-dextran (FD4) [34]
than when the agents were presented at different times. One way to ensure the peptide
and other additives reach the epithelial surface at the same time is to formulate as liquidfilled or semi-solid dispersions (e.g. Chiasma Pharmaceutical, Israel [304]), although
there may be some physical and chemical stability issues. Some patents also disclose
stability and oral bioavailability of peptides in solution formats with polyprotic alcohols
(Novo-Nordisk A/S, Denmark [305]). Another strategy involves inclusion of a
disintegrant that facilitates more rapid dissolution of the peptide and enhancers (e.g.
PeptelligenceTM technology, Unigene Laboratories, USA: [306, 307]).
Should the peptide transverse the intestinal epithelium and diffuse through the relatively
porous basement membrane and lamina propria, the peptide must permeate the
endothelium of blood and/or lymphatic vessels. Tight junctions between capillaries in the
sub-mucosa are relatively leaky and have fenestrations of 20-100nm [35], which permit
flux of peptides in to the capillary lumen. However, if the peptide is encapsulated in an
intact nanostructure (>100nm), permeation across the endothelium must also be
considered a barrier to absorption; this is of relevance considering that 35% of all
publications relating to oral peptide delivery over the past 5 years are based on
nanostructures [36]. The final barrier to oral peptide bioavailability is the liver, where
first pass metabolism takes place. For example, 8% of cyclosporine, a relatively stable
cyclic peptide, is inactivated by hepatic metabolism, although this is less than the ~40%
loss caused by intestinal P-glycoprotein/cytochrome P450 interplay [37]. In some cases,
peptides elicit their primary biological actions on the liver and therefore, first-pass
11
metabolism is not a consideration. Insulin for instance, is naturally secreted from the
pancreas into the hepatic portal vein and hence oral insulin mimics physiological delivery
aimed at its liver target.
3. Strategies to improve oral peptide delivery
Significant research effort has focused on improving peptide stability and permeation by
chemical modification, non-covalent complexation [32], conjugation to targeting ligands
and use of permeation enhancers.
3.1 Pro-drug and structural modification approaches
Structural modification, including the formation of prodrugs, is a successful drug delivery
approach that is commonly used to improve the physicochemical properties of drugs,
including peptides and proteins. PEGylation increases serum half life of peptides,
proteins and antibodies that are delivered parenterally [38]. Desmopressin contains a
deaminated amino terminal and contains a more stable D-arg at position eight which are
modifications that make the peptide degrade more slowly than vasopressin, and increases
its lipophilicity, both of which improve oral bioavailability. Substitution with D-ala and
D-leu in enkephalin also improved oral bioavailability by 20-fold in rats when delivered
with the aminopeptidase inhibitor, amastatin [39]. An alkylated form of exendin-4 has
also been disclosed, where a long chain fatty alcohol at position 20 (Lys) or position 32
(lys) increases the lipophilicity [305]. Although the effect of this modification on oral
bioavailability of exendin-4 has not yet been disclosed, the conjugate had good solubility
in blends of ethanol and propylene glycol.
12
In 2005, Biocon acquired Nobex Corp. (USA) and the intellectual property of the
PEGylated alkylated oral insulin conjugate, IN-105 [308-312]. Insulin is covalently
modified at position B29 with PEG using an acyl chain linker, which improves stability
of the peptide to proteolysis without significantly altering pharmacological activity [40].
In some patent filings, iterations of this structure contain hydrophobic alkyl chains linked
by hydrolysable bonds to the surface of PEG [312]. Biocon have since filed a number of
patents that include approaches to improve the efficiency of conjugate synthesis, as well
as new insulin polymer conjugates (e.g. aryl carbamate prodrugs) and sCT-polymer
conjugates in novel oral formulation approaches [313-316]. Pre-prandial administration
of IN-105 to type 2 diabetics reduced post-prandial glycaemic excursions, although there
was considerable intra-patient variability [41]. In a subsequent Phase III trial, IN-105 did
not adequately reduce glycated haemoglobin (HbA1c) levels, and therefore failed to meet
its primary endpoint. Despite the inclusion of surface alkyl chains to IN-105, the overall
advantage of such a conjugation seems to be to reduce proteolysis rather than to enhance
permeability. Biocon patents also embody a broad range of absorption promoters that
improved permeation of IN-105 in rodent and canine models [317]. These include
sodium cholate, the carboxylate of the primary bile salt cholic acid (a component of the
GRAS-listed and US Pharmacopeia (USP)-allowed excipient, sodium choleate (which
comprises taurocholate, glycocholate, deoxycholate, and cholate), and C10. Bristol-Myers
Squibb (BMS, USA) and Biocon® have announced a licensing agreement whereby BMS
have the option to develop and market IN-105 in the event that certain milestones are
13
reached [203]. Biocon have also filed patents for calcitonin-polyalkylene glycol
conjugates which are in early development [318, 319].
3.2 Peptide-targeted ligand conjugates
An alternative modification approach is to conjugate the peptide to an intestinal epithelial
receptor-targeted ligand that can shuttle the peptide across the epithelium. A number of
potential receptor targets have been identified including transferrin, vitamin B12, PepT1,
folate, biotin and FcRn. There are issues with the capacity of peptide-ligand conjugates to
adequately improve peptide permeation due to low or variable receptor expression,
possible saturation, the impact of diet, and instability of the conjugate in the intestine
[42]. Another candidate of commercial interest researched by Biorexis Technology Inc.
(USA) involved conjugation of peptide to modified transferrin leading to more
favourable pharmacokinetics in preclinical studies [320]. Others have combined insulintransferrin conjugates in a complexation hydrogel format and demonstrated increased
resistance to enzymatic attack [43]. Targeting approaches using ligand coated
nanoparticles are reviewed in Section 4.1.
3.3 Carrier complexation
Physical complexation of peptide to a carrier is an alternative to chemical modification.
Ionic complexation is widely used to formulate small molecules and it is estimated that
50% of all drugs are formulated as salts [44]. In the case of peptide delivery, ionic
complexation was used to form nanocomplexes of sCT with trisodium polyphosphate and
oral bioavailability of sCT in rats was improved due to in part to inhibition of proteolysis
14
[45]. Mediplex Corp. (South Korea) has developed a non-covalent complexation
approach using sterols, bile acids and/or hydrophobic small molecules [321]. The
dihydroxyl bile acid derivative, Nα-deoxycholyl-L-lysine-methylester (DCK), is one of
their representative carriers that when admixed with insulin, significantly increased its
oral bioavailability [46]. Eligen® ( Emisphere, USA) is a group of low molecular weight
carriers that represent the main complexation approach that remains in clinical
development. The main patent from over 20 years ago disclosed the capacity of
cyclohexanepropanoic acid to improve oral delivery of heparin and sCT following oral
gavage in rats [322]. A number of patents have been filed describing carriers that have
been evaluated clinically including SNAC (N-(8-[2-hydroxybenzoyl]amino)caprylic acid)
or salcaprozate sodium [323], 5-CNAC (N-(5-chlorosalicyloyl)-8-aminocaprylic acid)
[324], 4-CNAB (4-[(4-chloro-2-hydroxy- benzoyl)amino]butanoic acid) [325] and SNAD
(N-(10-[2-hydroxybenzoyl]-amino) decanoic acid) [326]. Of their large portfolio of small
carriers, the acylated amino acid, SNAC is the most widely studied. It improved oral
bioavailability of peptides and macromolecules in many preclinical and clinical studies
[7]. These include GLP-1 and Peptide YY (3-36) [47], insulin [48], hGH, sCT and PTH.
Well studied candidates with this delivery platform are unfractionated heparin and
LMWH) [49, 50]. A recent patent assigned to Oramed Limited (Israel) included SNAC as
an additive to improve oral macromolecule absorption [327]. In another patent filing,
Emisphere found that disodium salts and various solvents improved the efficacy of
Eligen® carriers including SNAC [328]. It is not yet clear whether improved
enhancement will translate to better performance of Eligen® in clinical assessment for
oral peptides, where its most advanced Eligen® formulation is for vitamin B12. A
15
number of patents exist for another of Emisphere’s carriers, 5-CNAC which is licensed to
Novartis. The capacity of 5-CNAC to deliver peptide via the oral route (e.g. PTH [51]
[324] and sCT [52]) has been outlined in pre-clinical studies in monkeys, rats and dogs,
and an enteric coated oral formulation has completed Phase II and III trials [53, 54]. A
Phase I trial for osteoporosis showed an enteric coated oral formulation with 5-CNAC
was safe and had greater relative bioavailability than the nasal format (Miacalcin®;
Novartis) [55]. Other Phase I [56] and Phase II [57, 58] trial data for this formulation
showed early promise and helped to improve understanding of the pharmacokinetics and
pharmacodynamics of oral sCT [55]. Novartis and Nordic Biosciences undertook a
multicentre Phase III clinical trial of the sCT/5-CNAC oral formulation in 4500
postmenopausal women [53]. This dosage form failed to meet its primary endpoint and
key secondary endpoints. A recent proof-of-concept trial for oral delivery of PTH with 5CNAC had absolute bioavailability of less than 1%, but the variability was four times
higher than that of the commercial s.c. format (Forsteo®, Eli Lilly, USA) [204].
3.4 Permeation enhancers
The inclusion of permeation enhancers that alter the properties of the intestinal
epithelium by either paracellular or transcellular permeation is a delivery strategy that
was first attempted with macromolecules over fifty years ago. The ability of such
additives to increase permeation of peptides across the intestinal wall has been widely
studied, although has not yet been successful in oral peptide delivery. Many groups have
focused on paracellular permeation enhancers to opening of epithelial TJs. FDA
guidelines pertaining to development of pharmaceutical excipients states that “if the
16
excipient is found to be pharmacologically active, this information can influence
subsequent development” [205], so steps to regulatory approval are likely to be complex
for novel TJ modulators as part of oral peptide formulations. For example, the side effects
of the excipient Cremophor ®EL, rather than paclitaxel, were responsible for
discontinuation of early Phase I trials of the anti-cancer agent, so development of a
formulation with new excipients comes with a significant risk [59]. The majority of
permeation enhancers embodied in the more clinically advanced proprietary oral peptide
dosage forms are therefore simple, relatively inert substances with similar properties to
many established excipients or food additives. Enhancement at relatively high doses in
vivo is mechanistically related to mild and reversible transmucosal perturbation which,
depending on the amount of the additive, can be accompanied by mild superficial
mucosal injury.
One of the most widely studied permeation enhancers is C10, the soluble sodium salt of
the medium chain fatty acid, capric acid (reviewed in [60] [329, 330]). The current
clinical progress with C10 is largely due to it being a major component of gastrointestinal
permeation enhancement technology (GIPETTM; Merrion Pharma, Ireland), an enteric
coated (Eudragit® L) solid dosage form [31, 61]. In clinical trials, this matrix tablet
formulation improved oral bioavailability of zoledronic acid, alendronate, acyline,
heparin, as well as insulin, sCT and GLP-1. Numerous other pre-clinical and proof-ofconcept clinical studies have reported the absorption enhancing effects of GIPET™ [62].
One such study carried out in healthy adults with LMWH showed an increase of 3.97.6% in oral bioavailability compared with the parenteral format [62]. In 2008, Merrion
17
entered a licensing agreement with Novo Nordisk A/S (Denmark) to develop oral insulin
(NNI953) and oral GLP-1 (NN9926). Phase I trials yielded promising results and Phase
II trials are planned [206, 207]. The use of C10 as a vehicle for the oral delivery of nucleic
acids was separately examined by ISIS Pharma (USA) who reported oral bioavailability
as high as 13 % with a proprietary oligonucleotide in man [8]. C10 has a history of use in
rectal antibiotic suppositories previously marketed in Sweden and Japan (Doktacillin,
Meda, Sweden). While it has been widely shown to increase permeability of
macromolecules in pre-clinical delivery models, it was not until it was formulated in
GIPETTM that significant enhancement of peptide bioavailability was observed in man.
There have been contradicting reports on the mode of action of C10 because of the
estimated concentrations required to enhance permeation in vivo. There is convincing
evidence that ~10mM enhances paracellular flux in cell culture monolayers [60]. In one
recent study, C10 decrease cell permeability by reducing tricellulin expression in
tricellular tight junctions and claudin-5 in bicellular tight junctions [63]. As C10 is a
soluble anionic mild surfactant, higher concentrations are capable of increasing the
fluidity of the plasma membrane and can lyse and solubilise phospholipid bilayers into
mixed micelles, a feature that increases transcellular permeation . Damage to the gut wall
caused by higher concentrations of C10 is temporary and reversible [30, 64], but there is
debate on the true free concentration of C10 presented at the intestinal wall in vivo as a
result of slow dissolution, dilution and spreading along the small intestinal lumen.
18
Another medium chain fatty acid with established permeation enhancement action is
sodium caprylate (C8) [65-67]. Chiasma (Israel) has developed ‘transient permeability
enhancer’ (TPE) technology to deliver an oral version of the 1020 Da MW niche
octapeptide, octreotide (Octreolin®), for which they have received orphan status
designation from the FDA [331-334]. The enteric coated formulation contains C8 in
hydrophilic microparticles mixed with castor oil, medium chain glyceride (MCG) and/or
caprylic acid to yield an oily suspension [335]. In a Phase I trial, relative oral
bioavailability of octreolin was similar to s.c. injection (Sandostatin®, Novartis) and
there were no adverse safety reports. In four cross over open label trials involving 75
healthy volunteers, Octreolin® containing 20mg of octreotide had comparable
pharmacokinetic data to s.c. injection of Sandostatin® (0.1mg) [68]. Octreolin®
demonstrated significant basal growth hormone secretion (49%) and GHRH-stimulating
GH secretion (80%) both in a single-dose study in humans [69]. These data suggest that
liquid or semi-solid suspensions could be an appropriate alternative to admixed solid
dosage forms. Chiasma has signed a licensing agreement with Roche (Switzerland) for
the development of Octreolin® and this formulation is currently in Phase III trials for
acromegaly [70]. TPE formulations also improve insulin absorption in rats suggesting
that it could be useful in delivering peptides larger than octreotide [335]. TPE can protect
peptides from proteolytic degradation and is thought to increase paracellular permeation
across the gut wall by altering TJ integrity [71]. Increased permeation of BCS Class III
and Class IV drugs by C8 in rat everted sacs was accompanied by changes in membrane
fluidity of brush border membrane vesicles and release of protein from the cells,
indicators of mucosal perturbation consistent with transcellular enhancement [72]. The
19
safety of twice daily administration of octreolin® in monkeys was evaluated over 28
days, with no treatment-related pathology [71]. Oral Octreolin® demonstrated a
comparable safety profile to Sandostatin® in healthy volunteers suggesting that TPE is
well tolerated during repeat daily dosing [68]. Chiasma also determined the MW range of
enhancement with TPE. It increased permeation of 4-10 kDa markers (FD4 and FD10)
and to a lesser extent 20-40kDa markers (FD20 and FD40), but not FD70 in a rat model
[208]. While such studies do not exclude the potential for inadvertent permeation of
lipopolysaccharide (LPS), they suggest that large luminal bystanders are less likely to be
absorbed upon ingestion of the dosage form (see Section 5). In rodents, TPE administered
0-60 minutes ahead of FD4 was progressively less effective at boosting absorption; this
was also the case with C10 [34], and it further argues that contemporaneous release of
enhancer and payload is a fruitful approach, and confirms that enhancement is rapid and
reversible since the MCFA is itself absorbed quickly. Oshadi Drug Administration Ltd
(Israel) has developed a formulation with similarities to Octreolin®. The vehicle consists
of inert silica nanoparticles with an adsorbed polysaccharide (either branched or
unbranched), and a peptide (insulin), which are suspended in a naturally occurring (e.g.
sesame, olive) or synthetic (e.g. silicone) oil phase [336], and their formulation improved
oral delivery of insulin in a rodent model [337]. The safety and preliminary efficacy of
this dosage form has been evaluated in diabetic subjects, although the data has not been
disclosed.
The most advanced oral peptide formulation in clinical development is that of Tarsa
Therapeutics (USA) under licence from Unigene Laboratories Inc. (USA). The oral
20
peptide technology, PeptelligenceTM was recently acquired by Enteris BioPharma (USA),
and is an enteric coated solid dosage form containing citric acid, tartaric acid or vitamin
C in vesicles, which provides buffering capacity to reduce the local pH to prevent peptide
degradation by proteases [73]. In some Unigene patents, the formulation also includes a
permeation enhancer: lauroyl carnitine (LCC), palmitoyl carnitine (PCC), or sodium
taurodeoxycholate [306, 338]. By presenting citric acid in vesicles, this enables
dissolution of the Eudragit® enteric coating at the designated duodenal pH value. The
formulation also contains an antioxidant and a disintegrant to ensure rapid dissolution.
Study of a panel of formulations containing important components of PeptelligenceTM
were found to improve oral bioavailability of sCT in dogs [74].
The efficacy of Taras’s oral sCT dosage form was recently compared with Miacalcin®
nasal spray in a Phase III trial of women with post-menopausal osteoporosis (ORACAL)
[11]. Oral sCT reached its primary end points and significantly increased bone mineral
density and reduced bone mass density compared to nasal sCT and placebo. Of interest is
that permeation enhancers do not appear to be present in Tarsa’s sCT oral formulation
and it seems that a balance has been drawn between achieving a target oral bioavailability
of 1% and the regulatory issues that might be associated with permeation enhancers in the
first possible submission of PeptelligenceTM to the FDA. Whether another sCT
formulation will ever be marketed is highly unlikely however, after a 2012 European
Medicine’s Agency recommendation against its further use in osteoporosis after a small
increased relative risk of cancer was highlighted for sCT following 5 years therapy in
post-menopausal osteoporotic women [209]. Efficacy of sCT in improving bone mineral
21
density is relatively weak in any case and there are now more effective alternative
therapeutic options.
A Phase I trial of a PeptelligenceTM formulation of CR845 kappa
opioid receptor peptide agonist (CR845, Cara Therapeutics, USA) yielded a mean oral
bioavailability of 16% [210]. In a subsequent comprehensive Phase II trial comparing
oral PTH(1-31) with Forsteo® in 97 postmenopausal women, the oral format reached the
primary endpoint of a 2.2% elevation in bone mineral density at the lumbar spine [75].
Unigene also evaluated their oral PTH formulation alongside Forsteo® in a Phase II trial
[76]. The oral PTH (5mg) formulation achieved a plasma AUC∞ of 83 pg·h/mL
compared to 46 pg·h/mL with a sub-cutaneous (s.c.) injection (20µcg). These
PeptelligenceTM formulations contain a carnitine as an enhancer since a higher oral
bioavailability is required compared to sCT.
LCC and PCC have many of the ideal properties of an intestinal permeation enhancer
[77]. The optimal range of effectiveness of the acylcarnitines is within the range of C12C16, where an optimal hydrophilic lipophilic balance (HLB) for effective enhancement is
evident [78]. The precursor of acylcarnitines, L-carnitine is listed on GRAS inventory,
and acylcarnitines have been widely studied [78-81]. PCC demonstrates higher efficacy
in improving cefoxitin bioavailability in rat rectal administrations than LCC [79] and had
a greater effect on membrane fluidity [78], yet LCC seems to be more evident in clinical
formulations of PeptelligenceTM. Unigene also list bile salts as enhancers in a number of
their patent filings [306]. Constituents of bile have also been licensed to improve buccal
macromolecule absorption. The Oralyn® spray developed by Generex (Canada) contains
bile salts and the technology (RapidMist™) improves meal time insulin absorption in
22
human subjects [82]. In initial enhancer classification systems, bile salts were defined as
Class II agents, having moderate efficacy and fast recovery, compared with medium
chain fatty acids listed as Class I (strong efficacy and fast recovery) [83]. The most
common bile acids that have been studied as intestinal absorption promoters are
deoxycholic acid, glychocholic acid, taurocholic acid and cholic acid, as well as their
soluble sodium salts. Because bile salts are present in the small intestine at mM
concentrations high enough to emulsify fats and increase intestinal permeability, there is
interest in harnessing their use as excipients for peptide delivery [84]. There are many
examples describing their capacity to improve absorption of poorly permeable molecules
[84, 85].
Intravail® (Aegis Therapeutics, USA) is a technology that was developed initially to
improve intra-nasal (i,n.) delivery of macromolecules [86], but more recently for delivery
of peptides via the oral route [87, 88] [338]. The main constituent of Intravail® is one or
more alkylsaccharides that include alkyl thiomaltosides, which are non-ionic surfactants
[339, 340]. The most widely published alkyl thiomaltosides is tetradecylmaltoside
(TDM), which improves both nasal and oral molecule permeation. TDM (0.25%)
increased the bioavailability of LMWH following oral gavage in rodents from 2.4% to
8.4% [89]. In a subsequent study, TDM (0.1% w/v) also improved bioavailability of sCT
in colonic instillations from 1% to 5%, but had little enhancement action in parts of the
small intestine [87]. Another of the Intravail® alkylsaccharides, dodecylmaltoside
(DDM), was also an effective enhancer in a rat model [86]. When a panel of alkyl
saccharides were evaluated in rectal instillations or by buccal delivery, DDM was
23
considered the most effective enhancer [90, 91]. The relative oral bioavailability of
octreotide acetate with DDM (0.3% w/v) was 4% when administered to mice by gavage
as an admixed solution compared to the s.c. route [92] [341]. Oral bioavailability of the
peptide, [D-Leu-4]-OB3, with Intravail® in mice was 52.2%, 47.3%, 37.8% and 12.9%,
relative to relative to the s.c., intra-peritoneal (i.p.), intra-muscular (i.m.) and i.n. routes
respectively [93]. Finally, oral administration to mice of the anti-cancer/anti uterine
hyperplasia peptide, AFpep, revealed an 8-fold increase in anti-estrogen activity [94].
CriticalSorbTM is an absorption-promoting formulation developed by Critical
Pharmaceuticals (UK) which has completed phase I trials for nasal delivery of hGH [95]
[342]. The formulation comprises the non-ionic surfactant, macrogol 15-hydroxysteate
and PEG (Solutol® HS15, BASF, Germany), which is normally used as an approved
solubiliser, stabiliser and dissolution enhancer for poorly soluble drugs (reviewed in
[96]). Solutol® improved oral bioavailability of cyclosporine by improving its solubility
in gastrointestinal fluid [97]. Initial evaluation of intestinal permeation enhancement of
CriticalSorbTM found it to have modest paracellular permeation enhancement action in
Caco-2 monolayers and across isolated rat colonic mucosae [98]. Solutol® HS15 is listed
in the USP-National Formulary (NF) and hence is an allowed excipient that could
potentially be used in oral peptide delivery, provided it is used within the dose range
recommended in the relevant compendium. Other specialist drug delivery companies
have also attempted to use food additives with patents filed for GRAS status and/or
Pharmacopoeia-listed additives to improve oral delivery of peptides [343]. Capsulin®
and Capsitonin® are oral formats of insulin and calcitonin using AxcessTM technology
24
which was first developed by Proxima Concepts (UK) and evaluated under licence by
Diabetology Ltd (UK) and Bone Medical (Australia), respectively. While the exact
formulations have not been disclosed, their patents embody simple excipients that boost
transcellular permeation. Examples given include the hydrophilic aromatic alcohols;
phenoxyethanol, benzyl alcohol and phenyl alcohol which are commonly used
antimicrobial preservatives [344]. Capsulin® (150-300IU) increased the glucose infusion
rate when measured in patients undergoing a glucose clamp technique [99] and it
increased serum insulin levels in patients [100]. Capsitonin® reached its primary
endpoint in a 2007 Phase II clinical trial where there was a 60% drop in the plasma Cterminal telopeptide (CTX-1) biomarker five hours post administration [211]. Bone
Medical Ltd. have also developed a PTH formulation termed CaPTHymone®, where the
oral formulation had bioavailability comparable to s.c. administered Forsteo®
(unpublished data). A more recent Axcess™ technology patent disclosed the use of the
hydrophobic aromatic alcohols, butylated hydroxyalcohol (BHA), butylated
hydroxytoluene (BHT), and propyl gallate in oral peptide delivery [345, 346]. This group
of excipients are antioxidants widely used to preserve emulsions. The Axcess patents
claims that these aromatic alcohols can be solubilised with sodium taurodeoxycholate or
the diethylene glycol monoethyl ether (Transcutol®, Gattefosse, France).
Oramed Pharmaceuticals (Israel) is developing oral peptide dosage forms for the
management of diabetes. While the exact components of Oramed formulations have not
been disclosed, patents indicate that they consist of a carrier, a protease inhibitor and an
absorption enhancer, each of which are listed in USP-NF and/or have GRAS status [303,
25
347]. The formulations are enteric coated capsules containing a vegetable, fish or
synthetic omega-3 fatty acid-lipoidal carrier, along with a protease inhibitor and EDTA to
assist peptide flux. EDTA is a versatile chelating agent widely used in the food, cosmetic
and pharmaceutical industries [101]. In addition to its permeation enhancement action,
the capacity of EDTA to chelate calcium and zinc means that it may also inhibit
proteolytic enzymes, most of which require divalent cations to function [102]. However,
given the high concentration of calcium in the intestinal lumen, protease inhibition might
be difficult to achieve even in the immediate region where the dosage form dissolves.
The safety of EDTA as a permeation enhancer is considered a drawback to its use, but in
most studies with EDTA where mucosal perturbation was detected, excessive doses were
used. EDTA has widespread use in topical, oral and parenteral formulations firstly as a
chelating agent at concentrations of 0.01-0.1% (w/v) [103] It is considered non-toxic and
non-irritant at allowable levels and it is listed in the USP-NF. In general, the Oramed
formulations were well tolerated in short and longer term exposure in man with only mild
gastrointestinal events.
Oramed oral formulations include those for exenatide (ORMD-0901 [348]) and insulin
(ORMD-0801, [347, 349]), and the latter has progressed to Phase II. Preliminary studies
of ORMD-0901 in cannulated dogs led to a decreased mean glucose AUC by 43%
compared with a 51% drop following injection [104]. The more advanced ORMD-0801
has been studied in both type 1 [105] and type 2 [106] diabetics. It improved the
absorption of exenatide as there was a 28% increase in the post-prandial peak serum
insulin concentration in patients receiving glucose [105]. Assessment of five undisclosed
26
ORMD-0801 insulin formulations yielded a reduction in baseline serum glucose of 1135% and of C-peptide levels by 6-14% in patients [104]. There were no adverse events in
six week repeat exposures of ORMD-0801 in a Phase II trial in type 2 diabetics [105]. In
conjunction with daily s.c. insulin, ORMD-0801 administered pre-prandially three times
daily was well tolerated and significantly reduced blood glucose levels [107]. The
greatest reduction in plasma C-peptides level was 14%, and this formulation was selected
for further study. In a combined total of 1444 administrations of the insulin (ORMD0801) and exenatide (ORMD-0901) formulations in 121 patients, only 8 mild reversible
hypoglycaemic events were reported. In 2013, Oramed initiated a Phase IIa trial of
ORMD-0801.
4. Preclinical technologies in oral peptide delivery
4.1 Tight junction openers
Several new technologies have improved permeability of peptides in pre-clinical studies.
Second generation paracellular permeability enhancers selectively alter the TJ by either
directly disrupting interactions between TJ proteins in adjacent epithelial cells or alter a
cell signalling process that leads to reversible disbandment of TJs (see Table I and
references [108-112]). A homologous 14-mer peptide sequence corresponding to the first
extracellular loop of occluden disrupts TJ formation increasing epithelial permeability
[113]. In MDCK monolayers, two occluden analogues targeting the second extracellular
loop of occluden led to a concentration-dependent decrease in TEER and an increase in
marker permeability [114]. The development of a short C-terminal peptide of the
Clostridium perfringens enterotoxin (C-CPE) and its structural analogues has yielded a
27
selective TJ modulator [110]. This fragment of CPE binds to the second claudin
extracellular loop domain perturbing homophilic interactions between neighbouring TJs.
C-CPE had comparable enhancement action to C10 in rat intestinal instillations, but was
400-fold more potent [115]. The Vibrio cholera toxin, zonula occluden toxin (zot) and its
structural analogues also increased paracellular permeability in vitro [116]. In order to
avoid non-specific toxicity associated with zot, the short zot analogue AT1002 and other
structural analogues were developed by Alba Therapeutics (USA) [117] [350, 351]. To
date, no zot analogue has been reported to be successful in any oral peptide clinical trial
in the 15-20 years since the initial patent filings. The evidence that specific TJ
modulators can match the enhancement action of established permeation enhancers with
a broader-based mechanism in vivo is not yet apparent [118]. Moreover, the latter class
have regulatory acceptability due to a long history of use in man and are cheap to
manufacture. If a new chemical entity permeation enhancer proves to be safe and
effective in oral preclinical studies, will major pharmaceutical companies be willing to
take the risk and invest?
4.2 Cell penetrating peptides
Cell penetrating peptides (CPPs) are at an early stage of development that could mature
into an effective strategy for oral peptide delivery [119]. Major CPPs include HIV
transactivator of transcription (TAT) peptide, polyarginine, polylysine, transportan and
penetratin. The exact mechanism by which CPPs facilitate internalisation and
translocation of macromolecules is not clear, it is thought to involve endocytosis and/or
channel formation [120]. Delivery of a target peptide can be brought about by
28
conjugation to the CPP which leads to internalisation of the cargo. In cases where
conjugation reduces the bioactivity of the therapeutic peptide, delivery may be brought
about by encapsulating the peptide in a nanostructure coated in the CPP. Furthermore,
there is also evidence that admixed formats of penetratin and a peptide can also improve
permeation across epithelia [121]. CPPs improved permeation of GLP-1 [122] and
insulin [123] in pre-clinical animal models. Toray Pharmaceutical Inc. (Japan) have
patented a group of short, synthetic, cationic CPPs (SEQ ID NO:1 and modified variants
such as SEQ ID NO:4) that can improve both nasal and intestinal bioavailability of
insulin [352]. Unigene synthesised a membrane translocator sequence derived from HIV
TAT peptide that when conjugated to sCT and delivered orally to dogs in a
PeptelligenceTM formulation, improved bioavailability [353]. The CPP sequence was
partially cleaved in the systemic circulation.
4.3 Colonic delivery of peptides
The regional delivery of peptide to the lower GI tract is an attractive alternative to enteric
coating that could improve oral peptide delivery. By comparison with the duodenum and
jejunum, the colon has reduced levels of luminal and brush border proteases making it a
more suitable environment for release of peptides that are sensitive to proteolysis.
Approaches currently employed to target the colon include enzyme digestible-, pH
sensitive, time-dependent and pressure- sensitive film coatings (reviewed in [124]). To
date, local colonic targeting with non-peptide prodrugs has had commercial success in the
regional treatment of inflammatory colitis (e.g. glucocorticoids and mesalazine), but
despite there being significant effort to target the colon for systemic absorption of insulin,
29
no similar peptide system has progressed [125]. Peptides targeted to the colon still require
an enhancer to boost permeation across the colonic epithelium, but at least the efficacy of
many permeation enhancers is often greater in the colon compared with the small
intestine [126, 127]. None of the peptide formulations in current clinical evaluation are
delivered in colon-targeted formulations. This is due to considerable inter- and intrasubject variability in GI transit that prevent selective release of cargo in the colon and
also delays of up to 6 hours to detect plasma levels [128]. In addition, the low fluid
volume and semi-solid nature of the luminal content in the colon can reduce the
dissolution rate. Inclusion of a disintegrant or super-disintegrant could help to reduce
variability associated with low fluid volume [129].
4.4 Microemulsion, micelle and liquid dispersion approaches
There have been a number of recent attempts to deliver water soluble peptides in liquid
dispersions to the small intestine [130, 131]. Microemulsions are nano-sized liquid-inliquid dispersions consisting of a mixture of two immiscible liquid phases that are
emulsified with a surfactant and a co-surfactant. Oil-in-water (o/w) microemulsions are
successfully used in delivery of poorly soluble small molecules such as ritonavir
(Norvir®, Abbott Laboratories, USA) and sequinavir (Fortovase® Roche, USA), but
their widespread use is somewhat limited by restrictions on levels of surfactants that can
be used [132]. A water-in-oil (w/o) microemulsion could protect a peptide from
enzymatic degradation in the intestinal lumen [133]. Microemulsions also reduce storage
instability of proteins compared with solutions [354]. Importantly, emulsifiers
(surfactants and co-surfactants) required to stabilise a microemulsion are capable of
30
increasing intestinal permeability [134]. It is also possible that such a lipoidal vehicle
could deliver certain peptides to lymphatic capillaries and enable them to reach the
systemic circulation via the thoracic duct. However, this route is suitable only for very
hydrophobic molecules and, although first pass metabolism is avoided, it has a low
capacity and is not a viable option for hydrophilic peptides. There are additional
challenges for oral delivery of hydrophilic peptides in microemulsions; dilution of a w/o
microemulsion in GI fluid could lead to physical instability in the form of phase
inversion. Microemulsion droplets can be stabilised by interfacial polymerisation leading
to the formation of more rigid nanostructures, but these can have difficulty in permeating
the intestinal wall since the polymerised emulsifier is not freely available to assist
transcellular permeation in the same way as those in fluidic emulsion droplets.
Furthermore, if the peptide is susceptible to chemical instability in an aqueous
environment, such as hydrolysis, this can necessitate optimisation of a non-aqueous polar
solvent as the disperse phase. A microemulsion of commercial interest was MacrulinTM
(Provalis, UK), a w/o dispersion prepared with oleoyl polyoxyl-6 glycerides (Labrafil®
M1994CS; Gattefosse, France) as the continuous phase, along with a dispersed water
phase, and stabilised with a surfactant/co-surfactant mix of lecithin and alcohol [135].
Intra-gastric delivery of insulin in this microemulsion to rats marginally improved
bioavailability compared to insulin solution [22]. Other peptides that have been
formulated in w/o microemulsions include leuprolide acetate [136] and Nacetylglucosaminyl-N-acetylmuramyl dipeptide [137].
31
Soligenix Inc. (USA) (formerly DorBiopharma Inc.) has developed reverse micelle
formulations to orally deliver peptides [355]. The hydrophilic micelle core protected the
peptide from degradation, and it is plausible that surfactants (e.g. short and medium chain
fatty acid esters or ethoxylated monoglycerides and diglycerides) used to form the
micelle may also assist permeation. The reverse micelles were stabilised by either
inclusion of synthetic/semi-synthetic polymer (e.g. gelatin, poly-lactide and or polyglycolide) or interfacial polymerisation in order to prevent phase inversion upon
dispersion in luminal fluid. Duodenal instillation of leuprolide to rodents in reverse
micelles had bioavailability of 28.2% versus 0.23% compared with s.c. injection of
leuprolide. Similar enhancement bioavailability was observed in dogs.
Astellas Pharmaceuticals Inc. (Japan) have disclosed a formulation that outlines the use
of aminoalkyl methacrylate copolymer E (Eudragit® EPO (Röhm GmbH, Germany)) in
combination with an acidifier and a surfactant in order to improve oral peptide
bioavailability [356]. The additives may inhibit proteolysis, increase the fluidity of the
mucus gel and/or improve permeability across the intestinal epithelium. Eudragit EPO®
is an excipient with a monograph in the US, EU and Japanese NF.
Novo-Nordisk A/S has recently disclosed data on liquid dispersions to improve oral
insulin delivery. The bioavailability of insulin and selected structural analogues in rats
was greatly improved when delivered in ethylene glycol [305]. Iterations of this patent
also describe dispersions consisting of additives such as mono- di- and tri- glycerides,
propylene glycol, non-ionic surfactants (e.g. poloxamer®), co-surfactants and
32
mucoadhesives (e.g. polycarbophil, polyvinylpyrrolidone and various thiomers). It is
thought that surfactants used in the preparation of such microemulsions can also increase
peptide permeation as was suggested in the formulation of a non-aqueous microemulsion
of insulinotropic peptide with polysorbate® 80 and oleic acid [357]. Another Novo
Nordisk patent highlights improved absorption of structurally modified insulin in a self
micro-emulsified delivery system as measured by reduction in serum glucose in nondiabetic rats and dogs [358]. This patent lists a broad range of additives and commercial
dispersion vehicles commonly used in drug solubilisation including Capmul® MCM
(Abitec Corp, USA), Witepsol® H15 (Sasol, Germany), Labrafil® M (Gattefosse).
Efforts have also been focused towards optimising chemical stability in such relatively
crude raw materials [359]. Instillation of hydrophobised insulin with propylene glycol,
Capmul® MCM C8/10 (Abitec Corp.), Capmul MCM C10 and in particular Capmul®
PG8 significantly increased plasma insulin concentration in rats [360]. Other iterations
involved mixing these dispersions with Labrasol® (Gattefosse, France) and Cremophor®
RH40 (BASF, Germany), which also improved absorption of hydrophobised insulin in
rodent instillations [361]. Self- (micro) emulsifying drug delivery systems (S(M)EDDS)
can improve permeation of hydrophilic molecules across the intestinal epithelium.
Capmul® MCM and Capmul® MCM C10 (Abitec, USA) are well defined mixtures of
mono- and di- glycerides of C8 and C10 that are normally used to deliver poorly soluble
molecules. Capmul® MCM improved absorption of the BCS Class III drug, ceftriaxone
in rodents and primates [138], and increased mannitol permeability by 100-fold in rabbit
ileum and colon [139]. Morphological assessment of rabbit colonic mucosae indicated
Capmul® MCM causes some membrane perturbation [140].
33
Polyoxylglycerides are mixtures of mono- di- and tri- esters of glycerol and mono- and di
esters of PEG, and are listed in the European Pharmacopoeia (EP) and USP-NF; they
function as penetration enhancers, dissolution agents, emulsifiers and sustained release
agents. This group of widely used group of SEDDS includes Labrafil® M2125 CS,
Labrasol®, and Gelucire® 44/14 (all from Gattefosse, France; reviewed in [141, 142]).
Labrafil® M 2125 CS comprises polyoxyethylated glycolized glycerides, corn oil and
dehydrated ethyl alcohol that is used to formulate a coarse cyclosporine emulsion in soft
gelatin capsules (Sandimmun® (Novartis; Switzerland) , which forms a coarse emulsion
upon dispersion in GI fluid [143]. Labrasol® is a mixture of caprylocaproyl
polyoxylglycerides used for self-emulsification of poorly soluble small molecules.
Labrasol® is embodied in recent Novo-Nordisk patents demonstrating increased
intestinal permeability of BCS Class III drugs. It improved bioavailability of gentamicin
in rats from less than 1% to 54% in colonic instillations [144], increased absorption of
LMWH by four-fold following intra-duodenal administration [145] and increased FD4
bioavailability from 4.4% to 30 % in an in situ closed loop in rats [146]. It is possible that
lipolysis of Labrasol® by intestinal lipases releases medium chain fatty acids that
ultimately may be responsible for permeation enhancement. These dispersions can also
facilitate encapsulated proteins. For example, bioavailability of erythropoietin (EPO) in
rodent instillations was improved from 0.6% to 1.9% when formulated with Labrasol®,
but when EPO-loaded micro/nano particles were dispersed in Labrasol®, bioavailability
increased to 5.3% in microparticulate porous silicon dioxide, 4% in liquid filled carbon
nanohorns, 5.7 % in fullerene and 11.5% in liquid filled carbon nanotubes [147]. The use
34
of permeation enhancers to facilitate oral peptide delivery in nanoparticle compositions is
an important patentable research area [148].
Gelucire® 44/14 is another well defined acyl glycerol admixture that is listed in various
National and International compendia as a SEDDS comprising saturated poly-glycolized
C8-C18 glycerides. It is an effective solubiliser and gelling agent that is used to improve
drug solubility (e.g. Fenofibrate (Lipofen®, Kowa Pharma, USA), ibuprofen (Solufen®;
Galephar, Venezuela), and food supplements (e.g. extracts of Tribulus terrestris). In
rodent instillations, Gelucire® improved bioavailability of EPO from 0.6% to 3.9% when
formulated in liquid filled carbon nanotubes [147]. Furthermore, formulation of the
polypeptide, calcein, in a nano-sized water-in-oil-in-water (w/o/w) emulsion comprised
of Gelucire 44/14 and polyvinyl alcohol (PVA) improved bioavailability following rat
duodenal instillations from 1.8 to 8% [149].
4.5 Oral nanoparticle delivery systems
Oral delivery of peptides in nanoparticles is the focus of many academic research
programs. An optimised nanoparticle carrier (with and without permeation enhancers)
offers the prospect of protecting the peptide from degradation in the gastric and intestinal
milieu as well as improving its permeation across the intestinal epithelium [150]. A
number of different nanoparticle delivery vehicles have been pursued with initial promise
in pre-clinical animal models, although to date none have been optimized for proper
clinical assessment. The current challenges to progression of oral peptide nanoparticles
include insufficient enhancement of bioavailability [151], instability in simulated
35
intestinal fluids and low loading capacity [152]. Outlined below are efforts that have
been made to overcome these issues.
Solid lipid nanoparticles (SLNs) are a suitable vehicle to encapsulate complexed peptides
with poor aqueous solubility. Ionic complexation of a charged peptide with an oppositely
charged surfactant reduces the aqueous solubility of both the target peptide and the
surfactant. At the same time, the complex becomes more soluble in lipophilic solvents
that are used to prepare SLNs. For example, complexation of leuprolide to stearic acid
permitted encapsulation in SLNs based on mineral oil [153]. SLNs based on cetyl
palmitate improved oral bioavailablity of insulin in rats to 5% versus 1.6% for insulin
solution [154]. More recently oral delivery of sCT in SLNs prepared with stearic acid and
tripalmitin improved oral bioavailability in rats to 13% [155]. A patent filing for several
lectin coated polymerised lipid nanoparticles prepared from stearic/palmitic acid, lecithin,
PVA and wheat germ agglutinin demonstrated improved insulin release kinetics in
diabetic rats, although further optimisation is required for these SLNs to be effective
peptide delivery vectors [362].
Tamarisk Technologies (USA) has developed an oral peptide delivery vehicle termed
serum specific nanoencapsulate (SSNe) particles. These are lipoidal nanoparticles with an
aqueous core that are formed by the aggregation of a natural polymer (such carrageen or
alginate), which has been alkylated with C20-C40 chains [363]. The target peptide is
contained within a helical core that is formed by the polymeric backbone (particle
diameter ~35nm). The covalently linked alkyl chains that are exposed on the surface of
36
the particle are thought to protect the peptide from proteolysis and enable better
transcellular permeation across intestinal epithelial cells. Once absorbed, the lipophilic
SSNe particles are thought to be packaged into chylomicrons and absorbed into the
lymphatic system. The ability of SSNe particles to delivery insulin or peptide YY (3-36)
is currently being evaluated in rodent models.
Receptor mediated delivery of a peptide loaded nanostructure is an attractive alternative
to peptide-ligand conjugation as it may protect the therapeutic peptide and improve the
efficiency of permeation to a greater extent. Attempts to overcome the drawbacks of
insulin-vitamin B12 conjugates resulted in pre-clinical evaluation of insulin- and siRNA
loaded, B12 coated nanostructures (Access Pharmaceuticals, USA). Access has
developed CobOralTM (formerly CobalaminTM) a peptide loaded dextran nanoparticle that
is coated with cobalamin, a vitamin B12 analogue.
Binding and endocytosis of cobalamin via endogenous Intrinsic Factor-B12 receptors on
the surface of intestinal enterocytes increased oral peptide bioavailability in animal
models [364-366]. The encapsulation of the peptide within dextran nanoparticles prevents
enzymatic degradation and improves cell uptake efficiency compared with directly
conjugating the peptide to cobalamin. The oral bioavailability of insulin when delivered
in vitamin B12-coated dextran nanoparticles was 29% relative to s.c. injection in rats
[156]. Access reported a reduction of blood glucose levels in rats that was greater than
80% of that achieved by the s.c. route. This delivery platform has not yet advanced to
clinical evaluation. Apollo Life Sciences (Australia) has also developed a vitamin B12-
37
coated carbohydrate based nanoparticle called Oradel™ [36, 131] [367, 368]. This
formulation induced sustained plasma glucose lowering up to 12 hours in diabetic rats
[131]. Others have used FcRn targeted nanoparticles to substantially increase oral
absorption efficiency of insulin in mice using the apical membrane receptor for IgG,
noting that no increases were seen in FcRn knock-out mice [157]. Evidence of
effectiveness of receptor-mediated targeting approaches from conjugates or from ligandcoated nanoparticles is however lacking in man and there are concerns about whether
such technologies can be scaled for manufacturing.
BioSante (USA) has developed a microparticle based insulin delivery platform referred to
as CAPIC (calcium phosphate-PEG-insulin-casein) [369]. This delivery system is
composed of a hydrophobic core containing suspended particles of calcium phosphate,
PEG3350 and insulin with an interfacial layer of casein [158]. Oral delivery of CAPIC to
fasted-diabetic mice reduced serum glucose level to a comparable extent as s.c. injection,
but unlike the injectable format the effect was sustained over 12 h.
Nod Pharmaceuticals (USA/China) developed a bioadhesive nanoparticle delivery
vehicle that is also based on calcium phosphate (Nodlin) [370]. The formulation contains
exenatide mixed with calcium phosphate and a co-precipitating agent selected from either
bile salts (e.g. ursodeoxycholate, taurocholate, chenodeoxycholate) or salts of fatty acids
(e.g. C8 or C10). When delivered orally to diabetic mice, the formulation improved
absorption of exenatide compared to untreated control, but was not as effective as the s.c.
format.
38
NanoMega Medical Corp. (USA) has filed a patent for a chitosan-polyglutamic acid
(PGA) nanoparticle that is prepared by ionic gelation [159] [371]. The chitosan imparts a
positive charge on the particle surface while the core of the nanoparticle is anionic owing
to negatively charged PGA-complexed conjugates, which function to stabilise the
chitosan shell. In addition to encapsulating the peptide, chitosan is mucoadhesive and
some structural forms can facilitate paracellular permeation by reversibly opening TJs
[160]. Oral delivery of chitosan-PGA nanoparticles increased paracellular permeation
thereby improving oral delivery of both exendin-4 and insulin in diabetic rats [161]. The
PGA-complexes also possess enzyme-inhibitory action which could augment the
protection provided by the nanoparticle core upon release [162].
ThioMatrix (Austria) have filed a number of patents that utilise thiolated polymers to
enable oral delivery of peptides and other molecules with poor oral bioavailability.
Thiolated polymers are mucoadhesive [163], have permeation enhancement action [164],
and are thought to be efflux pump inhibitors [165] [372]. Insulin-loaded thiomer
nanoparticles prepared from polyvinylpyrrolidone and poly(acrylic acid)-cysteine or
polyacrylic acid significantly increased plasma insulin concentration to values similar to
that of s.c. injection when delivered orally in an aqueous dispersion or solid dosage form
to rats [166].
Delivery of peptides in liposomal formulations has not yet been successful by the oral
route. This is mainly because of poor stability in gastrointestinal fluids and poor
39
permeation across the intestinal epithelium [167]. Nevertheless, Diasome
Pharmaceuticals Inc. (USA) has developed a liposomal insulin formulation termed
hepatic directed vesicle insulin (HDV-I) to Phase II/III trials (reviewed in [168] [373]).
The phospholipid bilayer of HDV-I vesicles (<150nm) consist of insulin bound to a
proprietary hepatic targeting molecule in order to direct the delivery to the physiological
site of action. HDV-I has been tested in a number of small scale clinical trials, where preprandial oral delivery of HDV-I to type I diabetics was comparable to postprandial
parenteral delivery [168].
Endorex Corp. (USA) previously developed a stable liposome delivery system that was
found to improve oral bioavailability of a number of peptides including insulin and hGH
[131]. The liposomes that constitute OrasomeTM were stabilised by interfacial
polymerisation to prevent deterioration in the upper GI tract. Oral delivery of hGH in low
polymerised OrasomeTM liposomes in mice boosted the plasma concentration of hGH to
levels similar to the s.c. format [374]. To our knowledge, this formulation was not
pursued further for oral peptide delivery due to formulation reproducibility issues and
insufficient oral bioavailability in rodents.
5. Future perspective
In recent years there has been significant investment in the development of oral peptide
dosage forms by specialised drug delivery companies in partnership with major
pharmaceutical companies. There is renewed optimism that a licensed oral peptide
dosage form is an achievable goal. Yet, even the best oral peptide formats will continue
40
to have relatively low bioavailabilities of ≤ 10%. The cost of the delivery system must
also be factored into the development especially those based on complex drug delivery
systems (e.g. drug loaded, ligand coated nanoparticles). Tarsa Therapeutics may file an
NDA in 2014 for their oral sCT (OstoraTM) [212], and it is therefore the most clinically
advanced oral peptide format. They are followed by other delivery platforms associated
with clinical trial data: TPE (Chiasma), ORMD (Oramed), Eligen® (Emisphere), IN-105
(Biocon) and GIPETTM (Merrion). What stands out about these formulations is their
simplicity compared with highly complex delivery constructs. Technologically advanced
formulations incorporating nanoparticles, cell penetrating peptides and peptide basedparacellular permeation enhancers have shown promise in preclinical research, but there
is no commitment to their clinical development to date. The cost associated with
technologically advanced delivery vehicles could restrict their use.
The safety of oral peptide formulations depends on the approach taken to improve
permeation across the intestinal epithelium. Delivery strategies that result in transient
alteration of intestinal barrier function were historically considered unsafe. One of the
perceived drawbacks of formulating a peptide with such a candidate excipient is the
potential adverse effects of repeatedly perturbing the barrier function of the intestinal
epithelium. The selection of an enhancer for this purpose is an important development
consideration because it requires a balance between transcellular enhancement action and
mild mucosal perturbation. For surfactant permeation enhancers, there is a
concentration-dependent relationship between the two effects. Early sentiment on
enhancer safety took the view that there was a too narrow a safety margin for them to be
41
developed [169]. Yet, multiple clinical trials of a range of enhancers suggest that the most
advanced candidates have good safety profiles. In vitro toxicity assays do not accurately
represent the capacity of the small intestine to routinely endure challenge in vivo nor do
they model repair from mild superficial mucosal injury. If the toxicological data from
incubation of surfactants in closed compartment cell cultures or isolated tissue models
were a reflection of an in vivo environment, then even post-prandial bile salts in the
intestinal lumen at concentrations of 8-16mM [170] could be considered harmful.
Similarly, high concentrations of long chain fatty acids achieved in lipid digestion
indicate that mucosal injury and repair are routine [171]. The most advanced permeation
enhancers have similar physicochemical properties to many surfactants and cosurfactants that are already in many approved pharmaceutical formulations. Increased
epithelial permeability caused by superficial mucosal injury occurs in the presence of
alcoholic beverages, spicy and fatty foods and some drugs; it goes unnoticed because the
gut undergoes efficient repair [172]. Mediators such as prostaglandins and nitric oxide
have been implicated in the repair process, and modulation of their action is one of the
reasons why non-steroidal anti-inflammatories (NSAIDs) cause more sustained mucosal
injury compared with permeation enhancers [173].
Selection of additives that have GRAS status or are excipients listed in the Center for
Drug Evaluation and Research’s (CDER) inactive ingredients can alleviate some of the
uncertainty about systemic toxicity when dosed at the same level as those allowed in food
or marketed drug formulations [174]. Specialised drug delivery companies have selected
such excipients where possible and others have funded studies designed to achieve GRAS
42
status for their delivery vehicles [175]. This is important because in order to effectively
control chronic diseases, the dosage form should be amenable to daily administration.
Finally, there is the concern about the consequences of bystander absorption in the
context of repeatedly inducing increased intestinal permeation. The likelihood of
bystander absorption is minimal with enhancers that elicit only a temporary increase in
permeability high up in the GI tract, where there are far lower levels of bacteria compared
to the colon. There have been no significant adverse immunological or pathogen-related
responses reported in clinical studies with any of the candidate delivery systems to date.
The molecular weight and radii of LPS, bacterial toxins and even whole bacteria are
orders of magnitude greater than target payloads. Furthermore, the necessity of intimate
contact of enhancer and payload at the mucosal surface provides the additional argument
that bystanders present will not have their permeation assisted, especially since they are
dilute and not in direct intimate association with the formulation.
6. Conclusions
The market share of therapeutic peptides has grown considerably in the last twenty years
and with it, the incentive to deliver them by the oral route. The time taken for an oral sCT
formulation to progress from an experimental concept to submission of the first NDA has
taken nearly 20 years, but there remain hurdles to the clinical success of these
formulations. The most advanced delivery strategies in clinical development are
relatively simple additives in solid dose or liquid dispersion forms that have a safety
record in food or pharmaceutical formulations. Several new strategies to deliver peptides
by the oral route have shown promise in pre-clinical drug delivery model. Of note, the
43
potential of nanostructures and associated complexation strategies have not yet been fully
realised. A number of permeation enhancers found in more advanced technologies have
been shown to cause superficial mucosal injury, but no more than endogenous secretions,
dietary xenobiotics or currently allowed excipients. To date, safety concerns that
originated from reductionist cell culture models have not be confirmed in several
thousand administrations of oral peptide formulations containing permeation enhancers
that have reached clinical trials.
Acknowledgments
This work was supported by Science Foundation Ireland Cluster grant, 07SRC B1154,
and by the Irish Department of Agriculture FIRM grant, 11/F/042. Research for this
review has also received funding from the European Union Seventh Framework
Programme (FP7 / 2007-2013) under grant agreement n° 281035.
Disclosure
David Brayden has acted as consultant to Merrion Pharmaceuticals and has had previous
grant support from the company for oral delivery research.
44
Table I: Examples of agents in preclinical research which open intestinal epithelial tight
junctions
Non-proprietary
name
CPE (and derivatives)
Mode of action
Reference
Disruption of TJ by binding to an extracellular
domain of claudin
[176]
Synthetic claudin peptides
Disruption of TJ by binding to an extracellular
domain of claudin
[375]
zot (and derivatives)
Activation of PKC
[116]
DerP1
Cleavage of an extracellular region of occluden
[177]
HIV gp120
TNF-α dependent disruption of TJ structure
[178]
VP-8
Possible interaction with extracellular moieties of
claudin and occluden
[179]
Synthetic occluden
peptides
Disruption of TJ by binding to an extracellular
domain of occluden
[114]
Patulin
Occluden proteolysis
[180]
45
Transcellular route
Paracellular route
Microvilli
Tight Junction
Intestinal
Epithelium
Basement
membrane
Capillary
Endothelium
Figure 1: Barriers to the intestinal epithelial flux of peptides. Protection of a peptide
from luminal proteases and capacity to permeate mucus will enable the peptide to reach
the intestinal epithelium. If the peptide has sub-optimal lipophilicity it will not passively
diffuse across the cell membrane and small peptides may divert to carriers. Furthermore,
the high molecular weight of most therapeutic peptides prevents them from passing via
the TJs between epithelial cells. While the basement membranes of the epithelium and
endothelium do not restrict peptide permeation they can reduce the rate of peptide
diffusion. Upon entry into the capillary lumen the peptide drains into the hepatic portal
vein delivering the peptide to the liver where first pass metabolism may further reduce
oral bioavailability circulation [Figure prepared using images courtesy of Servier Medical
Art].
46
Executive summary
 This article provides an outline of the current clinical status of oral peptide delivery,
and details the advantages and disadvantages for patients, healthcare professionals,
and manufactures, should selected low molecular weight potent injectable peptides
be reformulated for delivery by the oral route.
 The impediments to oral peptide delivery are summarised along with the patented
approaches that drug delivery scientists have made to progress past each barrier.
 We outline the patent and scientific literature behind many of the drug delivery
systems that have matured into proprietary oral peptide formulations currently being
evaluated in clinical trials.
 Also, included is an outline of advanced drug delivery systems that have not yet
matured to clinical evaluation, but nonetheless are of commercial interest if they can
be suitably optimised to overcome the barriers to progression.
 We concludes with a perspective on the safety and efficacy of oral peptide delivery
and a discussion of the final hurdles that need to be surmounted for selected
candidates
47
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