peptide pills for brain diseases? reality and future perspectives

Review

Oral peptide therapeutics: the impact of new technologies & clinical development Since the licensing of a vasopressin analog Lypressin® in the 1970s [1], peptides have received interest as treatments for a wide variety of indications. The therapeutic role of peptides is attractive due to their high specificity and potency combined with low toxicity of metabolites and minimal potential for drug–drug interactions. Utilizing nature’s tool kit for designing therapeutics provides an advantage to small molecular drugs as the use of endogenous or structurally similar peptide drug candidates substantially reduces the risk of unforeseen side reactions, which is reflected in over 20% probability of regulatory approval, a rate that is double that of small molecules [2].

Peptides, however, have not always been favored as drug candidates due to their physicochemical characteristics and the necessity for expensive and complicated manufacturing processes. Peptides often have short halflives making chronic administration problematic and costly. However, the most important drawback in their clinical use is the lack of adequate oral bioavailability. Undoubtedly, as the preferred route of administration for pharmaceutical products has been the oral route [3], and given the lack of patient compliance with therapeutics that require chronic self intravenous administration (with the exception of lifethreatening diseases), the pharmaceutical industry had directed its efforts to the development of oral alternatives

opting against peptide/proteinbased drug candidates.

However, due to the increasing costs for R&D and the decreasing number of drugs approved by the regulatory agencies, new alternative approaches are demanded to enhance the productivity of the pharmaceutical industry [4], reviving the focus on peptide therapeutics. Two major technological advancements are responsible for increasing the acceptability of peptide drug candidates. The first involved the advancements of polymer technologies allowing controlled longacting release formulations of peptides encapsulated in biodegradable polymers to be prepared (such as GnRH [2]). Advances in genetic engineering and recombinant technology have radically changed the mindset of the pharmaceutical industry and caused an increase of interest in the chemical manufacture of peptides and the genetic engineering of proteins, offering a window to the therapy of previously untreatable conditions. Recent technological advances in the peptide development process, such as solidphase peptide synthesis, has led to a significant improvement in their physicochemical properties, which has overcome many of their drawbacks such as high cost of manufacture and lack of stability, resulting in a large number of marketed peptidebased drugs [5], making peptide therapeutics one of the fastest growing areas of the pharmaceutical industry. The average number of new candidates entering clinical study every year has steadily increased from 1.2 per year in the 1970s to 4.6 per year

Peptide pills for brain diseases? Reality and future perspectives

The peptide therapeutic market is one of the fastest growth areas of the pharmaceutical industry. Although few orally administered peptides are marketed and many are in different phases of clinical development, there is no marketed oral peptide therapeutic used for CNS disorders. The major challenges involved in orally delivering peptides to the brain relate to their enzymatic instability and inability to permeate across physiological barriers. The paucity of therapies for the treatment of brain diseases and the presence of the blood–brain barrier excluding 98% of therapeutic molecules necessitates parenteral administration. Various approaches have been applied to enhance oral peptide bioavailability, but only nanoparticulate strategies were able to deliver orally therapeutic peptides to the brain. Although industry may be reluctant to invest in developing oral peptide nanomedicines, the increasingly unmet clinical need and economic burden associated with brain diseases will fuel the development of the first marketed oral-to-brain peptide therapy.

Dolores Remedios Serrano Lopez1 & Aikaterini Lalatsa*2

1Department of Pharmacy & Pharmaceutical Technology, School of Pharmacy, Complutense University of Madrid, Plaza Ramon y Cajal S/N, Madrid, 28040, Spain 2Department of Pharmaceutics, School of Pharmacy, University of Hertfordshire, College Lane Campus, Hatfield, Hertfordshire, AL10 9AB, UK *Author for correspondence: Tel.: +44 1707 28 4997 Fax: +44 1707 28 4506 E-mail: [email protected]

479ISSN 2041-599010.4155/TDE.13.5 © 2013 Future Science Ltd Ther. Deliv. (2013) 4(4), 479–501

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in the 1980s to 9.7 per year in the 1990s and 16.8 per year in the 2000s [6]. Over 90% of the peptides undergoing clinical investigation in the last decade possess agonistic activity, and are most frequently used as treatments for cancer (18%) and metabolic disorders (17%; diabetes, obesity and osteoporosis), targeting extracellular targets and particularly Gprotein coupled receptors (GPCRs) [6]. From targeting GPCR type A receptors (rhodopsinlike, accounting for 85% of GPCR genes) almost exclusively in the 1980s, there has been a shift in the last decade to equal proportions of type A and B (secretinlike) receptors. The latter are regulated by peptide hormones such as those of the glucagon hormone family, including vasoactive intestinal peptide or calcitonin and parathyroid hormone (PTH) [6]. More than 50 therapeutic peptides are on the market and over 130 are in clinical development [6]. In 2004, more than 20% of medicines belonging to the top 200 sales were proteins, antibodies or peptidebased drugs, representing 10% of the overall sales for the pharmaceutical industry [7]. Examples of some commercialized peptides are GNRH/LHRH agonists (leuprorelin, goserelin), a somatostatin analog (sandostatin) and an immunomodulator peptide (glatiramer), reaching collective global sales of over US$1 billion in 2010 [6].

As peptide therapeutics are increasingly able to address a growing range of medical challenges, the pharmaceutical industry is today more in need of technologies able to stabilize and effectively deliver therapeutic peptides across physiological barriers. Neuropeptides have been indicated as primary molecules in the therapy of several neurological disorders including pain, depression, neurodegenerative disorders, cancer and infection. Bearing in mind that the WHO has indicated that CNS disorders are the major medical challenge of the 21st century [8] with conventional therapies being either inadequate or absent, peptide therapeutics can provide a solution to treatment as long as they are permeable across the blood–brain barrier (BBB). The BBB naturally limits compounds with a molecular weight greater than 500 Da, eliminating potentially active biologicals from entering the CNS and leading to a higher failure rate for CNS drug development, even though the need of therapies is increasing in parallel with the increase in the incidence of CNS diseases due to aging of the population.

Although the pharmaceutical industry has accepted the need to develop drugs that cannot

be orally administered, technologies that can enable delivery of biologicals across mucosal barriers such as the GI tract and the BBB can offer an unmatchable potential for the development of effective and safe biologicals, and enhance the commercial success of peptide therapeutics. Today, only two peptides are marketed as oral products: desmopressin acetate (DDAVP®), approved for the treatment of diabetes insipidus ;and cyclosporin (Neoral®) as an immunosuppressant [9]. Both are cyclic peptides whose structural features protect them from intestinal enzymatic degradation. Thus, the commercial success of peptide delivery via the oral route is still very limited, and to date there is no oral peptide on the market to treat brain diseases, in spite of being a therapeutic field with unmet needs where the potential of therapeutic peptides is immense. This review gives an overview of the current challenges of oral peptide delivery for brain diseases and the new strategies that are in development, aiming to bring therapeutic peptides to the market.

Current challenges in oral peptide delivery�� Limitations of oral peptide therapeutics

Peptides are justif iably considered by the pharma ceutical industry as poor oral drug candidates as they possess low oral bioavailability (generally bellow <1%; Box 1) [10]. The low oral bioavailability of peptides is a direct result of their physical instability (in the acidic environment of the stomach) and metabolic instability (pepsin, intestinal enzymatic degradation), and their very poor permeation across biological barriers in the absence of a specific transport system due to their hydrophilicity, charge and high molecular weight (>500 Da). Peptides routinely violate the majority or all of Lipinski’s predictors for good absorption and bioavailability [11]. Even if a peptide is able to escape physical and gastrointestinal enzymatic degradation and is absorbed in the blood in adequate amounts, it is still subject to firstpass metabolism by liver enzymes and degradation by plasma enzymes, while due to its small size and relatively small molecular weight (<50 kDa) is subject to clearance via the kidneys (the cutoff molecular weight for glomerular filtration is thought to be 30–50 kDa [12]) [5]. In addition, linear peptides possess high conformation flexibility that can result in peptide denaturation and poor targeting to the tissue of interest, and can further result in poor shelf stability. Although the enhanced potency of the peptides necessitates

Key Terms

Peptide: Biochemical polymer that consists of two or more amino acids (more than 50 amino acids are considered as proteins) linked together by amide bonds between the carboxyl and amino groups of adjacent amino acid residues.

Blood–brain barrier: Unique membranous barrier mainly consisting of tightly bound endothelial cells and perivascular astrocytes imposing a combination of enzymatic and physical barriers. The blood–brain barrier tightly segregates the brain from the systemic blood circulation, regulating the constancy of the internal environment of the brain and ensuring CNS homeostasis in order for neuronal functions to optimally take place.

Oral delivery: Combination of strategies and technologies used in enabling the effective and safe oral administration of therapeutic molecules, overcoming the physical and enzymatic hurdles of the GI tract.

Review | Serrano Lopez & Lalatsa

Ther. Deliv. (2013) 4(4)480 future science group

that very small amounts bind to the receptor of interest to result in a pharmacological response or cascade, the very low oral bioavailabilities require larger doses to be administered, increasing industrial development costs and, essentially, the costs of therapies. This places more constraints on healthcare providers to balance costs with therapeutic efficacy, thereby limiting their use in lifethreatening and unmet diseases. Fortunately, largescale chemical synthesis of peptides up to 50 residues is a viable option for the pharmaceutical industry as solidphase peptide synthesis has considerably reduced their manufacturing costs.

�� Gastrointestinal physical & enzymatic barriers to oral peptide deliveryThe GI tract is designed to digest dietary proteins into smaller peptides and amino acids that can be absorbed across the intestinal epithelium into the blood, as well as the protective role of the GI tract against ingested toxins effectively abolishing intact peptide oral delivery. Although

intact oral absorption of peptides is restricted, it is possible if several barriers encountered after oral administration are overcome (FiguRe 1).

The first hurdle that a peptide faces upon oral delivery is the high salt and acidic environ ment

Box 1. Advantages and limitations of oral peptide therapeutics.

AdvantagesLarge-scale chemical synthesisGood patient compliance Enhanced potencyHigh specificity Minimization of drug–drug interactionLow accumulation in non-targeted tissues Lower risk of immunogenic effects

LimitationsLow oral bioavailability (<1%)Poor physical and metabolic stabilityPoor physiological barrier permeationShort half-life (several minutes)Denaturation – loss of 3D structurePoor shelf stabilityComplex and expensive manufacturing

Lumen

Lamina propria M cell Lymphoid tissue Lymphatic vessels Blood vessels

Epithelial cell

Tight junction

Mucus layer

Paracellular transportTranscellular transport(passive diffusion)

Carrier-mediated uptake

Pino

cyto

sis

+ ++

+++ ---- - --

Ads

orpt

ive

upta

ke

Lymphatic transport

Efflux transporters

Lysosomal peptidases

Cytosolicpeptidases

Brush-border peptidases

Receptor-m

ediated

uptake (a

ctive

)

Pancreaticproteases

COOHH2N

COOH

100–200 µm

+

-

+

-+

-+

-

Figure 1. Enzymatic and physical barriers to oral absorption of peptides.

Peptide pills for brain diseases? Reality & future perspectives | Review

www.future-science.com 481future science group

of the stomach that can denature the therapeutic peptide (e.g., insulin) [13,14]. Pepsin, an aspartic proteinase optimally active at a pH of approximately 3, is the main proteolytic gastric enzyme, hydrolyzing peptide bonds but sparing nonpeptide amide bonds and ester linkages. As digestion progresses, the gastric juices are emptied within the intestinal lumen where the administered peptides are faced with luminally secreted pancreatic proteases (trypsin, chymotrypsin, elastase and carboxypeptidase A and B) [15,16] able to hydrolyze a peptide to amino acids or small di or tripeptides. Complete peptide degradation to amino acids, if not imparted already, can be accomplished by the action of brushborder peptidases (endo as well as amino and carboxypeptidases), or even after if absorption has taken place by intracellular enzymes (cytosolic and lysosomal peptidases), further limiting the amount of the peptide reaching blood circulation [16]. Extracellular peptide metabolism (degluronidation, decarboxylation and reduction of double bonds, ester and amide hydrolysis and dehydroxylation reactions) may occur in the colon by enzymes originating from local microflora and is dependent on the dissolution of the peptide in the intestinal contents and the transit time [17]. However, colon metabolism is less significant if oral delivery is desired unless the major absorptive site for a particular peptide resides in the large intestine and not the ileum, or if a local effect is desired.

Even if the integrity of the peptide to enzymatic degradation is assured, the physical barrier imposed by the presence of the mucous layer that coats the epithelial surfaces of the intestine hinders oral absorption. The mucus barrier is a constantly changing mix of many secretions and consists mostly of mucins and high molecular weight glycoproteins crosslinked via disulfide bonds. The thickness of this 3D network varies throughout the human GI tract from 40–450 µm, being narrower (100–200 µm) in the duodenum and jejunum. Due to the sialic and sulfonic acid substructures, the mucus layer is negatively charged, which means that it can interact with positively charged peptides or functional groups, slowing their diffusion across this layer and thus slowing or reducing their absorption, while possibly allowing for increased brushborder enzymatic metabolism [18–20].

The architecture of the GI tract mucosa further restricts peptide absorption. Peptides can permeate into the intestinal epithelial cells via paracellular or trancellular routes. The paracellular

pathway, an aqueous, extracellular route across the epithelia, allows passive translocation of the peptide across the epithelia with the flux being driven by the electrochemical potential and hydrostatic pressure between the two sides of the epithelium, limited only by the presence of tight junctions [21]. The dimensions of the paracellular space lie between 1 and 5 Å, suggesting that solutes with a molecular radius exceeding 15 Å (~3.5 kDa) will be excluded from this uptake route [22]. Tight junctions are generally accepted as being negatively charged overall, and therefore selective for positively charged permeants [23]. Considering that the intestinal epithelium has a large surface area (more than 2 × 106 cm2) with the paracellular surface area ranging from 200 to 2000 cm2, paracellular permeation of even minute quantities (in the pM to nM range) of a polypeptide drug may be sufficient to exert their required biological effect [23]. However, although paracellular absorption is generally assumed as the route of entry of peptides, it is not clearly established that paracellular transport of peptides is the exclusive route of entry [24]. Bearing in mind that the interaction of tight junction proteins between adjacent cells is the basis of the selective function of tight junctions, paracellular transport across monolayers is governed by the interaction of the extracellular loops of the claudin family members on adjacent cells to create extracellular pores. Confounding factors such as the effect of primary (nonmotile) cilia on tight junction remodeling and the effect of deciliation in increasing transepithelial resistance [25], the absence of primary cilia in the Caco2 monolayers [25], as well as the effect of Ca2+ levels [26] and function of Ca2+/Mg+2ATPase pump [26] also need to be carefully considered. Examples of peptide drugs that have been shown to permeate the intestinal mucosa to an extent via the paracellular route include octreotide, vasopressin analogs, thyrotropinreleasing hormone and salmon calcitonin.

The transcellular pathway (primarily used for more lipophilic therapeutics) involves transport across the apical cell membrane, through the cytoplasm of the cell and, finally, across the basolateral membrane, with lipophilicity, hydrogen bonding potential and size being the most important controlling factors. For passive peptide absorption to occur, energy is required to break water–peptide hydrogen bonds allowing the peptide to enter the cell membrane. Therefore, due to hydrophilicity, hydrogen bonding and large molecular size, passive diffusion of peptide

Key Term

Tight junctions: Intercellular junctions between adjacent epithelial cells with two main functions: sealing neighboring cells together and acting as an impermeable barrier to the diffusion of macromolecules (proteins and lipids), while allowing the passage of small molecules such as ions (Na+).



therapeutics is minimal unless a more lipophilic active derivative is engineered. Transcellular transport can occur through carriermediated uptake (e.g., using di or tripeptide transporters) or by vesiclemediated processes, which are important for those peptides that are too large to be absorbed by the di or tripeptide transporters. The cellular internalization of peptides through a vesiclemediated transport can occur via either pinocytosis (when no interaction between the peptide and the apical membrane is required), receptormediated endocytosis (more efficient than pinocytosis and involves the binding of the peptide to the plasma membrane receptor prior to the incorporation into endocytic vesicles) [13,27,28] or through adsorptive uptake (requiring the electrostatic binding of peptide cationic moieties to negative charges on the membrane constituents such as anionic lipids) [29].

Efflux transporters (such as the Pglycoprotein, multidrug resistanceassociated protein 2 and breast cancer resistance protein) expressed on the apical membrane of intestinal epithelia can reduce intracellular accumulation and, hence, transcellular diffusion of peptides transported across the intestinal epithelium. Examples of peptides that are substrates of efflux pumps are cyclosporine, valinomycin and gramicidin D [30].

Significant transcytosis of polypeptides has been described for microfold cells (M cells) located in the follicleassociated epithelium of the Peyer’s patches that possess the unique ability to sample antigen from the intestinal lumen and deliver it to antigenpresenting cells and lymphocytes located in a unique pocketlike structure on their basolateral side [31]. M cells possess a high transcytotic capacity and are able to transport a broad range of materials, including nanoparticles, via adsorptive endocytosis by way of clathrincoated pits and vesicles, fluidphase endocytosis and phagocytosis representing another potential route for oral peptide delivery as well as for mucosal vaccination [32]. The reduced levels of enzymatic activity compared with epithelial cells makes this route of entry attractive for peptide containing nanoparticles, even if they are less numerous compared with enterocytes (accounting for 1% of total intestinal surface), while the relatively sparse nature of the glycocalyx facilitates the adherence of both microorganisms and inert particles to their surfaces [32].

Peptides can enter the lymphatic circulation utilizing either of the following routes [10,27,33]:

��Paracellularly as lymphatic capillaries have higher porosity compared with blood vessels;

��Transcellularly when coadministrated with lipidbased vehicles utilizing the intestinal lipidtransport pathways incorporating peptides into chylomicrons;

��An endocytic pathway from the M cells situated on the dome of the gutassociated lymphoid tissue, which are able to sample macromolecular antigens from the peptides in the lumen to the lymphatic tissues.

Therefore, designing a successful nanomedicinal strategy for an orally delivered peptide to the brain will hugely depend on enhancing peptide presystemic and systemic stability, as well as enhancing the relative contribution of all of these transport processes to the overall absorption, which in most cases will be a combination of all viable routes in order to ensure that the necessary amounts for eliciting a pharmacological effect are bioavailable.

Oral strategies for marketed peptide therapeutics & nanomedicines in clinical developmentEnhancing the oral bioavailability of peptides from <1% to at least 10–20%, and if possible to 30–50%, is a formidable undertaking for clinical development of oral peptide therapeutics [34]. Strategies that have illustrated the ability to enhance the oral absorption of peptide therapeutics can be divided in two broad groups: chemical modifications of the peptide structure, and formulating the peptide together with other compounds aimed at modifying or masking the unfavorable physicochemical characteristics that limit absorption or are responsible for enhancing enzymatic degradation.

Chemical modifications aim to elicit metabolically stable analogs by minimizing recognition of the analog by peptidase families in the GI tract while retaining the original biological activity and receptor specificity of the native peptide. Similarly, chemical modifications can be imparted to increase the peptide lipophilicity to enhance the transcellular transport of the peptide therapeutic or to minimize immunogenicity [34]. Chemical modifications can involve:

��Substitution of lamino acids with unnatural damino acids in order to enhance stability to proteolytic enzymes (as unnatural amide bonds are not readily recognised) [35];



��Cyclization of a linear peptide, which usually confers stability to peptidases and a decrease in hydrogen bonding; however, this is a major structural and conformational change that can potentially negatively affect the biological activity of the peptide [36];

��Engineering of peptidomimetics able to mimic the biological activity of the peptide, but without the traditional peptide structure replacing labile bonds by enzymatically stable constructs [37];

��Introduction of steric bulk in order to lead to an increase in stability, decrease in hydrogen bonding and/or lipophilicity (e.g., Nalkylation slows the addition of an enzymatic nucleophile to the peptide bond) [38,39];

��Synthesis of prodrugs (chemically modified peptides with improved physicochemical or biopharmaceutical properties) that must undergo spontaneous or enzymatic transformation in vivo to release the active peptide such as tyrosylpalmitoyl leucine5enkephalin, a palmitic ester prodrug of leucine5enkephalin [39].

On the other hand, formulation strategies involve administrating or formulating the peptide with other compounds to enhance the peptide’s oral bioavailability: ��Coadministration of enzyme inhibitors such

as aprotinin (natural inhibitor of trypsin) [40], EDTA [41], sodium glycocoholate, camostat mesylate [40,42] or bacitracin [43];

��Formulation with absorption enhancers (such as low molecular weight surfactants, bile salts, calcium ion chelators, cyclodextrins [44]) modifying the epithelial lining of the GI tract in order to allow for improved trans and paracellular transport by interfering with the mucus layer, modulating tight junctions or affecting membrane components [34];

��Altering the gastrointestinal retention time using mucoadhesive polymers such as chitosans [45,46];

��Loading or conjugating the peptide to a suitable carrier (such as lipidic [27,47] and micro or nanoparticlebased systems [34,45,48]), whereby the in vivo fate of the macromolecule is determined by the properties of the carrier system rather than those of the peptide resulting in controlled and targeted release.

At present there are only a few marketed peptide pharmaceuticals for oral administration (TaBle 1). DDAVP®, a synthetic analog of vasopressin with the first amino acid deaminated and the last larginine replaced by a darginine to enhance its membrane permeation, stability and activity [34], is a viable oral pharmaceutical product, although it only possesses oral bioavailability of 0.08–0.16% [301]. The other marketed oral formulation, Neoral® [302], possesses an unusually high oral bioavailability for cyclosporine of 40% elicited by a microemulsion system that is mostly attributed to cyclosporin’s relative small size, cyclic structure and the high propensity of hydrophobic amino acids within its sequence [9,49]. Recently, oral peptide delivery was effected utilizing the RapidMistTM device for delivery of aerosolized insulin particles (<7 µm) generated using generallyregardedassafe excipients (permeation enhancers, nonchlorofluorocarbon propellant and stabilizers) that are not deposited to the lung, but can be absorbed by the buccal mucosa – a product commercialized in India and Ecuador and currently in Phase III studies in USA [303]. Although there are several peptide delivery technologies for enabling peptides via the oral route, very few have progressed as oral therapeutics beyond the proofofconcept stage to clinical trials. TaBle 1 delineates approaches in various clinical development phases used for the development of orally bioavailable formulations of peptides and identifies the current status of nanomedicinal peptide pharmaceuticals. Oral nanomedicinal approaches for brain delivery rely on stabilization of the peptide within the GI tract, ensuring high oral bioavailability in blood prior to ensuring efficient transport across the BBB, and, thus, they should therefore be considered in light of other successful oral peptide strategies in clinical development.

Designing a successful oral peptide strategy for insulin delivery has been a work in progress for many pharmaceutical industries. The broad existent market entailing 350 million diabetes sufferers worldwide, with this number predicted to double from 2000 to 2030 [50], and an estimated US$376 billion in expenses for treatment and prevention of diabetes, is fuelling research into oral insulin therapeutics [304]. Novo Nordisk has invested in Emisphere’s Eligen® technology [305] for development of oral insulin and GLP1 receptor agonists formulations, which at the moment are being tested in Phase II/III [51,52] and Phase I [306] clinical trials, respectively [53]. This technology is based on low molecular weight

Key Term

Chitosan: Natural linear polysaccharide polymer composed of b-(1–4)-linked 2-amino-b-d-glucopyranose and 2-acetamido-2-deoxy-b-d-glucopyranose units. Chitosan is derived from the exoskeleton of crustaceans by alkaline N-deacetylation of chitin and is increasingly used in the design of delivery systems because of its unique properties (biocompatible, biodegradable and reactive -OH and -NH2 groups allowing functionalization).



Tab

le 1

. Mar

kete

d o

ral p

epti

de

ther

apeu

tics

an

d n

ano

med

icin

es in

clin

ical

dev

elo

pm

ent.

Tech

no

log

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pan

yPe

pti

de

Clin

ical

p

has

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ral s

trat

egie

sO

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om

eR

ef.

Lice

nse

d o

ral s

trat

egie

s

Ana

logu

e p

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de

Ave

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Ph

arm

aceu

tica

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opr

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etat

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Subs

titu

tion

of

the

last

l-ar

gini

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by d

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d d

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ion

of fi

rst

amin

o ac

idO

ral b

ioav

aila

bilit

y b

etw

een

0.0

8 an

d 0.

16%

[34,

301]

Lipi

d-b

ased

sy

stem

Nov

artis

and

Ro

che

Phar

mac

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Cyc

losp

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for

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of a

mic

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aq

ueo

us e

nviro

nmen

tsO

ral b

ioav

aila

bilit

y of

ap

prox

imat

ely

40

%[9

,49,

302]

Ora

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Rap

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Gen

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Bi

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log

y C

orp

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Insu

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Del

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aer

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par

ticl

es (<

7 µ

m)

cons

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pep

tid

e (r

H In

sulin

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GR

AS

exci

pien

ts (

per

mea

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enh

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rs, p

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ella

nt

and

stab

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rs)

Pept

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perm

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and

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syst

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a

dose

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0.8

± 2

6.0

mU

/l an

d w

ith

No

bex

®-i

nsul

in a

t 10

mg

: 10

0.3

± 0

020

66

.7; a

t 15

mg

: 177

.69

± 1

50.

3; a

t 20

mg

: 24

6.2

± 2

45.2

; and

at

30

mg

: 35

2.5

± 2

79.3

mU

/l)

[58–

60,3

09–3

11]

Pept

ellig

ence

TMU

nig

ene

Lab

orat

orie

s an

d ot

her

part

ner

ship

s

Cal

cito

nin,

PTH

,an

orex

igen

ic

pep

tid

e

III,

II, pre-

clin

ical

Ente

ric-

coat

ed t

able

t co

nsis

ting

of u

nmo

difi

ed

drug

, a p

rote

ase

inhi

bito

r (o

rgan

ic a

cid

) and

ab

sorp

tion

enh

ance

r (c

arni

tine)

Effe

ctiv

e fo

rmul

atio

ns w

ith

go

od

safe

ty p

rofil

e fo

r o

steo

por

osi

s (c

alci

toni

n, P

TH) a

nd o

bes

ity

(ano

rexi

gen

ic p

epti

de)

[61,

312–

315]

† Pro

duct

lice

nsed

as

Rec

osul

inTM

.C

max

: Max

imum

pla

sma

con

cent

rati

on

; GR

AS:

Gen

eral

ly r

eco

gni

zed

as s

afe;

PK

: Pha

rmac

oki

neti

cs; P

TH: P

arat

hyro

id h

orm

one

; rH

: Rec

om

bin

ant

hum

an; s

c.: S

ubcu

tane

ous

; STZ

: Str

epto

zoto

cin

; Tm

ax: T

ime

to m

axim

um

pla

sma

con

cent

rati

on.



Tab

le 1

. Mar

kete

d o

ral p

epti

de

ther

apeu

tics

an

d n

ano

med

icin

es in

clin

ical

dev

elo

pm

ent

(co

nt.

).

Tech

no

log

yC

om

pan

yPe

pti

de

Clin

ical

p

has

eO

ral s

trat

egie

sO

utc

om

eR

ef.

Co

nve

nti

on

al o

ral s

trat

egie

s

Pept

ellig

ence

TMU

nig

ene

Lab

orat

orie

s an

d C

ara

Ther

apeu

tics

Kap

pa o

pio

id

rece

ptor

ag

onis

t

IEn

teri

c-co

ated

tab

let

cons

istin

g of

unm

odi

fied

dr

ug, p

rote

ase

inhi

bito

r an

d ab

sorp

tion

en

hanc

er

Ora

l bio

avai

labi

lity

of 1

6% a

nd h

igh

sele

ctiv

ity

for

kap

pa o

pio

id r

ecep

tors

[316

,317

]

GIP

ET

Tech

nolo

gy

Mer

rion

Ph

arm

aceu

tica

lsIn

sulin

,d

esm

opr

essi

n,G

nR

H

anta

gon

ist

IEn

teri

c-co

ated

tab

let

or c

apsu

les

wit

h un

mo

difi

ed d

rug

and

bran

ched

, cyc

lic a

nd

stra

ight

cha

in f

atty

aci

ds

2.4%

ora

l bio

avai

labi

lity

for

des

mo

pres

sin

(~15

-fo

ld

incr

ease

in o

ral b

ioav

aila

bilit

y co

mpa

red

to m

arke

ted

tabl

et).

Hyp

og

onad

al le

vels

of

test

ost

eron

e fo

r G

nR

H

anta

gon

ist

[63,

64,3

18]

Axc

essTM

(Cap

sulin

)D

iab

eto

log

y Lt

dIn

sulin

IIEn

teri

c-co

ated

cap

sule

wit

h G

RA

S ex

cipi

ents

as

abs

orpt

ion

enha

ncer

s an

d so

lubi

lizer

sH

ypo

glyc

emic

act

ivit

y ov

er 6

h. H

ypo

glyc

emic

eff

ect

of o

ral i

nsul

in (3

00

U) s

imila

r to

50

% o

f th

e sc

. ins

ulin

ef

fect

(12

U)

[61,

62,3

19]

Ora

med

TMO

ram

ed

Phar

mac

euti

cals

Insu

linII

Ente

ric-

coat

ed c

apsu

le w

ith

GR

AS

exci

pien

ts

as a

bsor

ptio

n en

hanc

ers

and

solu

biliz

ers

Go

od

safe

ty p

rofil

e in

all

pati

ents

ove

r 6

-wee

k st

udy

and

effic

acy

in t

wo

-thi

rds

of p

atie

nts.

Abl

e to

red

uce

gluc

ose

as

wel

l as

C-p

epti

de

leve

ls in

tw

o-t

hird

s of

all

enro

lled

pati

ents

[320

,321

]

Nan

om

edic

inal

ora

l str

ateg

ies

Ora

del

TM

tech

nolo

gy

Ap

ollo

Life

Sc

ienc

esIn

sulin

,TN

F bl

ock

ers

IaC

arb

ohy

drat

e-ba

sed

po

lym

eric

nan

opa

rtic

les

coat

ed w

ith

vita

min

B12

Prec

linic

al s

tudi

es; 8

0%

of

anim

als

resp

ond

ed a

nd

cont

rol l

aste

d ov

er 6

h. B

etw

een

75 a

nd 8

0%

re

duct

ion

in p

lasm

a gl

uco

se a

fter

5 h

in S

TZ-i

nduc

ed

diab

etic

rat

s

[65,

66,3

22,3

23]

NO

D T

ech

NO

D

Phar

mac

euti

cals

Insu

lin

(No

dlin

),G

LP-1

ag

onis

t (N

od

exen

)

I Pre-

clin

ical

Bio

adhe

sive

ent

eric

-co

ated

nan

opa

rtic

les

Mo

dula

tion

of

PK p

rofil

e fo

r o

ptim

al e

ffica

cy. P

hase

I st

udie

s co

mpa

red

wit

h sc

. ins

ulin

in f

our-

way

cr

oss

-ove

r ra

ndom

ized

tria

l of

heal

thy

volu

ntee

rs

[324

]

Osh

adi O

ral

Insu

linO

shad

i Dru

g A

dmin

istr

atio

nIn

sulin

IEn

teri

c-co

ated

cap

sule

s co

ntai

ning

a m

ixtu

re

of s

ilica

nan

opa

rtic

les,

bra

nche

d p

oly

sacc

hari

des

and

insu

lin s

usp

end

ed in

an

oil

core

Do

se-d

epen

den

t re

duct

ion

in b

loo

d gl

uco

se le

vels

for

9

–12

h

[201

,325

]

HD

V In

sulin

Te

chno

log

yD

iaso

me

Phar

mac

euti

cals

Insu

linII/

IIIEn

caps

ulat

ion

in li

po

som

es w

ith

hepa

tocy

te-

targ

etin

g m

ole

cule

s (b

iotin

) con

juga

ted

to t

he

lipid

s fo

rmin

g th

e bi

lipid

laye

r

Ora

l adm

inis

trat

ion

wit

hin

15–3

0 m

in b

efor

e m

eals

si

gnifi

cant

ly r

educ

es p

lasm

a gl

uco

se le

vels

. Met

abo

lic

cont

rol w

as p

oor

er t

han

wit

h SC

insu

lin

[67,

326,

327]

† Pro

duct

lice

nsed

as

Rec

osul

inTM

.C

max

: Max

imum

pla

sma

con

cent

rati

on

; GR

AS:

Gen

eral

ly r

eco

gni

zed

as s

afe;

PK

: Pha

rmac

oki

neti

cs; P

TH: P

arat

hyro

id h

orm

one

; rH

: Rec

om

bin

ant

hum

an; s

c.: S

ubcu

tane

ous

; STZ

: Str

epto

zoto

cin

; Tm

ax: T

ime

to m

axim

um

pla

sma

con

cent

rati

on.



carriers or delivery agents that create a noncovalent conformational complex with the peptide, increasing its lipophilicity and, thus, promoting the absorption of the macromolecule utilizing passive trans cellular pathways [54,55]. A library with more than 4000 delivery agents has been generated, and sodium N[8(2hydroxybenzoyl) amino] caprylate and N(4chlorosalicyloyl)4aminobutyrate have been identified as the most effective in promoting transcellular absorption. This technology can be applied to a variety of peptides, proteins and poorly soluble drugs, up to approximately 150 kDa in molecular weight [56], without altering the molecule, and, thus, minimizing toxicological data necessary for approval. The technology can be applied in various formulation types (e.g., solutions, tablets, capsules and controlledrelease formulations) by blending the carrier with the active peptide. Another advantage of this technology is enhanced room temperature stability eliminating the need for cold chain, however, applicability to all proteins and peptides may be presumptuous. Faster onset of activity and higher insulin concentrations in a dosedependent manner were achieved with Eligen®insulin compared with injected insulin in Phase II clinical trials [57]. The application of this technology can be beneficial for drugs that require a fast onset of absorption and high liver concentration or delivery to the liver via the portal vein, mimicking the physiological route of a peptide such as insulin, which is not necessarily the case for neuroactive peptides. Emisphere’s Eligen technology was able to orally deliver the incretin hormones GLP1 and PYY 3–36 and elicit a rapid and dosedependent, statistically significant increase in plasma drug concentrations (p < 0.05) at all doses tested versus placebo. They are the first studies illustrating that GLP1 can stimulate insulin releases in humans after oral administration [307]. Applications of Eligen technology in oral delivery of salmon calcitonin have entered Phase III studies, and Emisphere is codeveloping the formulation in combination with Novartis/Nordisc an oral HGH and an oral form of PTH fragment 1–34 [308]. Despite the promising results obtained up to date, the major drawback of this technology is the large amount of delivery agent required to be formulated with the active polypeptide (various orders of magnitude higher than the peptide), yielding formulations that challenge patient compliance [56] and exhibit a high interindividual variability, which can be detrimental in designing an oraltobrain delivery strategy (TaBle 1) [53].

Nobex techonology, under Phase II trials in India and the USA [309], is based on a covalent conjugation with low molecular weight amphiphilic oligomers consisting of an alkyl chain (C6–C16) and a PEG unit (1–10 units) to specific and preselected sites of the molecule [58,59]. This results in improved enzymatic stability, improved solubility for optimal formulation design and increased permeation across the gastrointestinal epithelium, while possessing a prolonged plasma circulation halflife (TaBle 1) [56,58–60]. The rapid absorption profile and the glucoselowering effects appear to be a result of hepatic activation, as oral hexyl insulin (TaBle 1) is delivered to the liver via the portal circulation mimicking the physiological route of insulin secretion in nondiabetic individuals [59], leading to improved control of daily glucose levels and avoidance of comorbidities associated with diabetes such as glaucoma [59]. However, in Phase III clinical trials conducted in India, it failed to reach the necessary control in latestage Type II diabetes patients [310,311]. This technology has been proposed to enable other molecules, such as enkephalins, calcitonin and PTH, but neither this nor their potential for oral delivery to the brain of peptides have yet been demonstrated. Delivery via the portal route to the liver can lead to enzymatic degradation for small labile peptides, thereby reducing the overall increase in circulation halflife offered by this technology.

Unigene Laboratories have three product pipelines under development using PeptelligenceTM technology [312]. It is based on an entericcoated tablet or capsule that allows the drug to pass through the stomach intact and to release its cargo in the intestine, where the pH is above 5.5. Two different groups of excipients are included in the tablet together with the unmodified drug: ��Protease inhibitors such as specific enzyme

inhibitors or organic acids (citric acid), which will inhibit the intestinal enzymes;

��Absorption enhancers such as lauroyl or acylcarnitine, which increase paracellular transport and decrease interaction with intestinal mucus.

To date, this technology is applied to: ��An anorexigenic peptide that is still under

preclinical development for obesity treatment;

��PTH in Phase II trial [313] for the treatment of osteoporosis;



��Salmon calcitonin (with a positive outcome in the Phase III trial [314.315]) for the treatment of osteoporosis in postmenopausal women [61].

Using Unigene’s peptide technology, Cara Therapeutics (CT, USA) has developed an oral kappa peripheral opioid receptor agonist with increased kappareceptor selectivity and enhanced oral bioavailability of 16% (Phase I trials) in fasted individuals [316,317].

Other oral insulin delivery technologies include the Gastrointestinal Permeation Enhancement Technology (GIPETTM), CapsulinTM and OramedTM as described in TaBle 1 [58,62–64,318–321]. It is evident that the main focus of the pharmaceutical industry has been developing oral therapies for the treatment of diabetes, fuelled by the increasing numbers of patients suffering from the disease and the clear clinical need for developing an oral formulation – followed by other metabolic disorders and other conditions where there is a clear market for the orally developed product. However, this limits the applicability of the developed peptide strategies as the peptide therapeutic is left unprotected in the plasma, while their ability to permeate cellular membranes and to bind to intra cellular targets or be transported across a second physiological barrier such as the BBB is limited.

Four nanomedicinal oral peptide delivery approaches were also identified in the quest for an oral insulin pill (TaBle 1). OradelTM technology is based on the entrapment of insulin within a nanoparticulate matrix [322]. These nano particles (100–200 nm) are made up of a carbohydratebased protective polymer, which is coated with vitamin B12 (the delivery agent in this case). Vitamin B12 binds to haptocorrin (R factor) in the stomach, and when the complex enters the duodenum, pancreatic enzymes digest hapto corrin. This allows B12 in the more alkaline environment of the intestine to bind to the intrinsic factor and to be endocytosed by endothelial cells, within which it binds to transcobalamin II and is packaged into chylomicrons that are transported via the lymphatic circulation into the systemic circulation and liver. This delivery strategy thus exploits this natural transport system for delivering insulin, while the carbohydrate nanoparticulate matrix protects the peptide from enzymatic digestion and acid denaturation in the stomach. 5 h after oral administration of Oradelenabled insulin formulation in streptozotocininduced diabetic mice, blood glucose levels reach a nadir of 75–80% of baseline values [65]. An advantage of

this technology is the near 100% entrapment efficiency of insulin within the nanoparticles, while the ratio of insulin to excipient (including Vitamin B12) required is also favorable (1:36,000). This plays a key role as the insulin absorption depends on the B12 uptake, which is limited to 1–2 µg per dose received by humans (1 µg of attached B12 can deliver 160 mg of insulin) [66]. Another advantage involves the transport of the peptideloaded nanoparticles via the lymphatic circulation, which can allow for protection of the peptide when it reaches the systemic circulation. Apollo has reported positive outcomes for preclinical studies of the technology able to entrap, protect and deliver molecules up to 130 kDa and has successfully completed a Phase Ib human study for the oral delivery of a TNFblocker for the treatment of rheumatoid arthritis [323]. However, this technology has not been tested for the ability of the particles or their cargo to permeate across the BBB, and success with other peptide molecules has not been tested.

Another example of a nanoparticulate system for oral insulin delivery is the use of bioadhesiveentericcoated nanoparticles (NOD technology, Nodlin©) [324]. Macromolecules up to 34 kDa can be encapsulated into bioadhesive nanoparticles, creating a unidirectional release system able to enhance permeation across physiological barriers and protection against enzymatic degradation [324]. Oshadi Oral Insulin, also based on a nanoparticulate strategy, is composed of a noncovalent mixture of silica nanoparticles, branched polysaccharides and insulin suspended in an oil phase within entericcoated capsules [201,325].

Lipidbased strategies, and particularly liposome technologies, have also been employed for oral delivery of insulin. Diasome Pharmaceuticals (PA, USA) is undergoing Phase II/III clinical trials [326] with a developed oral insulin formulation based on a hepaticdirected vesicle technology, consisting of insulin entrapped in liposomes (with a diameter of 20–50 nm, stable at low pH and in blood) that contain biotin (hepatocytetargeting molecule) attached to phosphatidylethanolamine within the bilipid layer [327]. A Phase II doseranging study [328] revealed that oral administration of hepaticdirected vesicleinsulin within 15–30 min prior to meals significantly lowers plasma glucose levels by approximately 20% of baseline values [67]. However, some liposomal technologies (OrasomeTM, MacrulinTM) designed to orally deliver insulin failed to meet therapeutic targets set, and some of these development programs have been discontinued [329,330].



It is obvious that novel and emerging technologies have focused on a variety of strategies to develop orally bioavailable peptide formulations, the majority of which are currently under clinical development with few successful marketed formulations. The main reason for their failure lies either in lack of efficacy, illustrated by their inability to meet specified clinical targets, or the lack of adequate toxicological data of excipients or carriers used, delaying progress to the market. However, only a handful of peptides have been explored for their potential as oral therapeutics, limiting applicability of technologies to peptides with specified characteristics. This may increasingly change as the pharmaceutical industry is investing in delivery technologies as a way of stabilizing and ensuring success of peptide and protein therapeutics.

Interestingly, among all of these oral peptide technologies, there is currently no oral peptide therapeutic available for the treatment of neurological diseases. In general, only a small portion of peripherally delivered peptide reaches the brain parenchyma; however, this small amount is often sufficient to affect physiological functions and modulate neuroendocrine and behavioral responses [68]. If on the other hand the example of an oraltobrain active molecule, such as morphine, is considered even though it is not a peptide, it is assumed that it enters the brain freely. However, morphine possesses a poor oral bioavailability (20–30%) and suffers from extensive firstpass metabolism to various metabolites including morphine6glucoronide, which is a major reason for the pain relief achieved. However, even small amounts of morphine reaching the brain can result in analgesia, as only 0.02% of the intravenous dose of morphine is recovered from the brain after intravenous administration [69,70]. Diamorphine has an oral dosedependent bioavailability and is hepatically metabolized to morphine, achieving a maximum plasma concentration of morphine following oral administration of heroin, twice as much as that of oral morphine [71]. Thus, diamorphine, the more lipophilic derivative of morphine, illustrates greater permeability across the gastrointestinal barrier, increased stability to firstpass and plasma metabolism, and is able to permeate across the BBB 100times more easily than its parent drug morphine. Extrapolating from morphine’s example, ensuring that a peptide molecule would be more permeable across biological barriers, possess increased enzymatic stability and the ability to permeate across the

BBB will almost certainly lead to an overall adequate bioavailability to warrant oral use clinically.

Challenges in oral peptide delivery across the BBB & oral-to-brain peptide nanomedicines�� The BBB & brain peptide delivery

The relative clinical success of pharmaceuticals aimed at treating CNS diseases (8% from first inman to registration [72]) is partially explained by the paucity of treatments currently available for brain diseases, which at present affect more than 1.5 billion individuals [5] and lead to a worldwide economic burden of over $2 trillion [73]. The major obstacle to delivery of drugs or macromolecules in the brain is the BBB, a structure comprising two plasma membranes in series formed at the level of the endothelial cells of the cerebral capillaries (FiguRe 2), acting as a physical and metabolic barrier aiming to maintain brain homeostasis [74,75]. The endothelial cells of the BBB are distinguished from those in the periphery by minimal pinocytic activity, lack of fenestrations and increased mitochondrial content, but most importantly, by the presence of tight junctions that severely limit paracellular transport to allow only the flux of small, lipidsoluble molecules requiring invasive therapies [74]. Peptide therapeutics are modulators of CNS functions and can be translated into effective therapies for brain diseases such as psychiatric disorders (e.g., schizophrenia, depression, anxiety, autism, epilepsy), neurodegenerative diseases (e.g., Alzheimer’s, Parkinson’s), pain relief and antiinflammatory therapies, and infective and neoplastic diseases (only if they are deemed permeable across the BBB). Therefore, technologies that can enable their delivery, ideally via nonparenteral routes, hold great promise for developing into successful clinical products.

Despite the impenetrability of the BBB, there are a number of highly controlled routes available for macromolecules and nanomedicines. The majority of successful CNS active agents utilize passive diffusion as the main route for gaining entry to the brain parenchyma (FiguRe 3). The rate of entry across the BBB for permeable peptides depends upon weak hydrogenbonding potential, high lipophilicity (as indicated by the octanol/buffer partition coefficient, log D) and small molecular weight (<500 Da) [76–78], which are characteristics that are rare in endogenous CNS active peptides and necessary for successful oral delivery. The endothelium is characterized



by exhibiting a high transepithelial electrical resistance in the region of 1500–2000 W.cm2, allowing only the active transport of hydrophilic molecules such as peptides [79]. Even essential polar molecules such as glucose and amino acids rely on carriermediated processes for their

uptake. However, some peptides, even when possessing high lipid solubility, may not enter the brain as readily due to the presence of active efflux transporters (generally ATPbinding cassette transporters), which remove substances from the brain and the cerebral endothelium

BBB structure

Luminal and abluminal plasma membraneLack of fenestrationsMinimal pinocytotic activityIncreased mitochondrial contentTight junctions

Hydrogenbond

Endothelial cell

Tightjunction

Pericyte

MitochondriaNeuron

Astrocyte

Braincapillary

Transport through the BBB

Transcellual lipophilic pathwayCarrier-mediated influxCarrier-mediated effluxParacellular aqueous pathwayReceptor-mediated transcytosisAdsortive-mediated transcytosis

Desirable peptide physicochemicalattributes

Weak hydrogen bonding potential (<6 H-bond)High lipophilicityPolar surface area < 80 Å2

MW < 500 Da

Brain delivery strategies

Invasive procedures: transient osmotic opening of the BBB, shunts and biodegradable implantsPharmacological approach: peptide modifications to improve the physicochemicalproperties

CNS diseases

Psychiatric disordersNeurodegenerative diseasesInfective and neoplasic diseasesAnti-inflammatory therapiesPain relief

+

++

++

H

O

H

H

O

H

H

O

H

H

O HH

OH

NH2

N

H

N

H

N

H

N

H

OH

O

O

O O

O

OH

Figure 2. Peptide pill design for oral delivery to the brain. The combination of various strategies is necessary to elicit a clinically successful peptide pill able to deliver the therapeutic peptide to the brain parenchyma. A coated oral dosage form (usually in the form of enteric-coated capsule or tablet) can ensure that the peptide will permeate intact across the intestine, avoiding degradation by the harsh conditions encountered in the stomach. Including generally regarded as safe excipients such as fatty acids, absorption enhancers and protease inhibitors, and stabilizers in minimal amounts to avoid toxicities, can improve the oral absorption of the peptide by decreasing its intestinal enzymatic degradation, enhancing its permeation across the unstirred and mucus layer, enhancing its passive diffusion across the intestinal epithelial cells and/or enhancing its lymphatic transport. In addition, the peptide needs to be loaded or associated with a carrier/nanoparticulate system able to protect the peptide from chemical and enzymatic degradation and further enhance its permeation across the unstirred layer and through the intestinal epithelial cell membrane. The carrier system, if absorbed, needs to ensure stability of the peptide to liver and plasma metabolism and have a sufficiently long circulation half-life to allow the therapeutic peptide to reach the BBB and permeate in adequate amounts. In this case, the safety of the carrier system needs to be clearly demonstrated. If the carrier system is not absorbed, a stable analog of the peptide therapeutic is necessary, which will possess the necessary stability, plasma circulation half-life and ability to permeate efficiently across the BBB. BBB: Blood–brain barrier.



[79]. In addition, the plasma bilayer is high in cholesterol, which allows for a high packing density of membrane components. Due to this the resistance to the passive diffusion of large lipophilic molecules is greater when compared with resistance to the passive diffusion into noncerebral capillaries [80]. Highly lipophilic peptides may be extensively bound to plasma proteins, which could reduce the free peptide available in plasma, and therefore compromise brain uptake [81]. In addition, there is an inverse linear correlation between the polar surface area and brain penetration. The presence of hydroxyl groups on peptides tends to promote hydrogen bonding with water, which increases the free energy required for the peptide to move from the aqueous environment to the lipid of the cell membrane. A polar surface area above 80 Å2 (or 60 Å2 [82,83] in the case of orally administered CNSpermeable molecules) and a strong hydrogen

bonding potential (more than six hydrogen bonds) are factors that restrict passive transcellular diffusion across the BBB [78,84]. Therefore, large peptides (with a molecular weight higher than 500 Da) and the presence of rotatable bonds are unfavorable to BBB penetration [85]. However, the majority of CNS active peptides, as well as the majority of nanomedicinal strategies, rely on receptormediated or adsorptivemediated transcytosis (nonspecific transcytosis necessitating cationic charge that is facilitated by the interaction with the negatively charged glycocalyx and phospholipids groups of the cell membrane) for their transport across the BBB [79,86,87]. Thus, the capacity of a peptide to enter the brain parenchyma is dependent upon several compositional factors, including size, flexibility, conformation, biochemical properties of amino acid, and the amino acid arrangement [88]. As the majority of peptides do not possess desirable

Stomach Epithelialcells

Portalblood

Lymph Brain

Blood–brainbarrier

Blood

Enterohepaticcircualtion

Intestinallumen

Peptide-loaded nanoparticles

M cells First-pass metabolism

Modi�ed peptide(tyrosyl palmitoyl

leucine5-enkephalin)

Unmodi�ed peptide(leucine5-enkephalin)

NH2NH

NH

NH

NH

OH

O

O

O O

O

OH

NH2NH

NH

NH

NH

OH

O

O

O O

O

O

O

Enteric-coating

Peptide pill

Unmodi�edpeptide

Modi�edpeptide

Peptide-loadednanoparticles

Absorptionenhancers

Proteaseinhibitors

Fatttyacids

Figure 3. Peptide delivery across the blood–brain barrier.



physicochemical properties, there is a clear clinical need for the development of effective delivery strategies able to elicit transport across the BBB.

Transient osmotic opening of the BBB by intracarotid administration of hypertonic mannitol solutions [89,90], shunts [91] and biodegradable implants [92,93] is still used clinically for delivery of therapeutic molecules to the brain parenchyma. Similarly, transient opening of the BBB affected by ultrasound disruption or intravenous administration of bradykinin has been employed to create a window for the delivery of therapeutic molecules and peptides [94]. However, all of these strategies have been linked to low therapeutic efficiency as they rely on diffusion for drug penetration, which is low and decreases with the square of the diffusion distance, and thus is only effective for 1–2 mm from the surface of the ventricles [94]. In addition, substantial side effects (tumor dissemination after successful disruption of the BBB, high incidence of seizures and chronic neuropathologic changes in the brain) have been documented with their use, while they also have the potential of being highly traumatic [79,81,94–97]. Increased cost of therapy combined with the need for anesthesia and hospitalization further reduce the risk–benefit profile of these strategies for delivery of peptide therapeutics.

The dynamic regulatory functions of the BBB have become increasingly apparent in relation to transport of peptides. Peptide delivery across the BBB has been affected in certain cases by the exploitation of BBB localized transport carrier systems for nutrients, small peptides and proteins [97]. To date, eight different nutrient transport systems have been identified: hexose, monocarboxylic, choline, adenine, nucleoside, basic, acidic and neutral amino acid transport systems [79,81]. Engineered peptidomimetics with a molecular structure mimicking endogenous nutrients are able to be transported by one or more of the inwardly directed nutrient carriers. However, transport carrier systems have a low capacity for transport. The hexose and large neutral amino acid carriers have the highest capacity and are the best candidates for the transport of peptide, peptidomimetic, prodrug or active targeting of a delivery system decorated with a vector that is a substrate for the carrier system, and loaded with the peptide therapeutic. From an industrial point of view, targeting peptides to a specific nutrient transporter requires good knowledge of both the peptide and the transporter, and establishment of efficacy and

safety of the modified peptide or vectormediated delivery approach [98]. In addition, as these transporters are not specific for the BBB, the developed strategies are subject to uptake by target organs in the periphery.

Interactions of peptides with receptors on the surface of endothelial cells comprising the BBB could lead to endocytosis and, eventually, transcytosis of peptides (FiguRe 3) [79]. Conjugating peptides or nanoparticulate carriers loaded with peptides to these transport ligands is a strategy that has been successfully used to deliver biomacromolecules across the BBB. Earlier research concentrated on receptors that transport large endogenous molecules to the brain, such as transferrin or human insulin [79]. Recent research, however, suggests that even a signaling receptor such as the nicotinic acetylcholine receptor may be used as a portal for BBB delivery, as long as the vector used has sufficient high affinity for the receptor, but allows the cargo to be released in the brain parenchyma. Ideally, the endogenous ligand should not compete with the delivery vector for receptor occupancy at the BBB; thus, careful consideration of the relative binding affinities and the physiological levels of the endogenous ligand is required [79]. This explains the lack of clinical translation of technologies utilizing vectors such as transferrin that have a plasma concentration 1000fold higher than their Kd value (5.6 nM). The vector should also not be pharmacologically active (e.g., insulin is not suitable). The type of linker or spacer used to conjugate the vector to the peptide or carrier may influence this receptor affinity or its ability to release the peptide cargo in the brain parenchyma in order to elicit its pharmacological response. Brain uptake of the vector conjugate should be high enough to achieve clinical efficacy and an uptake of 2% of the intravenously administered dose per gram of brain has been suggested as a reasonable target when corrected for the amount of drug contained in the brain blood capillaries [99]. Finally, as with strategies employing endogenous transporters, these receptors are not specific for the BBB, leading to enhanced losses of doses in the periphery and unwanted peripheral side effects.

Amongst the most successful strategies able to enhance passage across the BBB are pharmacologicalbased strategies, which are aimed at increasing specific biochemical attributes of the therapeutic peptide. Strategies with successful BBB penetration include lipidization (blockage of polar functional moieties),

Key Term

Lipidization: Technology aimed at in increasing the lipophilicity of a therapeutic agent in order to enhance its permeability and then its transport across biological membranes.



structural modifications to enhance stability, glycosylation (addition of carbohydrate moieties), cationization and polymer conjugation/encapsulation of peptide therapeutics [81]. Similar to oral peptide delivery, enhancement of lipid solubility and enzymatic stability of the peptide can lead to a significant increase in its permeability [39,100]. However, enhancement of lipophilicity will not necessarily improve BBB transport. Increased lipophilicity has been linked to recognition by efflux transporters, limiting the amount of the peptide accumulating within the brain parenchyma [101]. Lipidization of therapeutic peptides, if used alone, is likely to increase unspecific uptake in peripheral tissues [100], thus lowering the amount of available peptide for transport across the BBB, and may diminish receptorbinding affinity leading to a reduced biological activity [88,100]. Apart from enhanced permeability, enhanced stability is required not only for effective oral delivery, but also for successful permeation across the BBB. The therapeutic peptide has to be able to withstand enzymatic degradation from blood and brain capillary endothelium enzymes [79]. Lipidization by conjugation of lipophilic or amphiphilic blocks [39], glycosylation [102] and cationization [103] modifications can enhance parent peptide stability. The latter strategy may be of limited value, however, as the concentration of cationic peptides in the brain may be limited by the fact that cationic agents are more readily taken up by the liver and kidney, so that the actual mass taken into the brain is minimal – less than 0.1% of the intravenously injected dose [79,104].

Although there are various different routes for peptide entry to the brain parenchyma, only delivery via the vascular route will result in widespread diffusion of the administered peptide therapeutic due to the large surface area of the BBB (12 m2 of capillaries in human brain [105]). Intravenous delivery of stable peptide analogs, conjugates or nanoparticulate formulations of therapeutic peptides is necessitated in almost all cases in order to render the peptide therapeutically effective, even when adequate brain permeation is present via passive or active transport pathways for achieving clinical efficacy. Oral peptide delivery to the brain is affected by strategies able to enhance peptide stability in both the GI tract and in the blood, and few developed technologies are currently under development. At the heart of oral brain delivery technologies lie nano particulate carriers able to encapsulate the peptide therapeutic or chemically modified peptide therapeutic

(e.g., by lipidization or conjugation) and protect it from enzymatic degradation, as well as enable its permeability across physiologial barriers and the BBB, allowing pharmacologically active concentrations of the peptide therapeutic to reach the brain parenchyma [81].

�� Oral-to-brain delivery of peptidesTranslation of developed oral brain delivery strategies into the clinic still remains an unresolved challenge for industry, considering that development of a CNS drug takes 12–16 years, almost 4 years longer than other drugs [72]. However, the simplicity and convenience of this route for patients necessitates delivery and development approaches to address this challenge. Oral delivery systems targeting the CNS have been investigated in academia for over a decade. In a few cases this research was successful enough to allow for intellectual property protection, which is necessary for the commercial exploitation of these technologies, and at present the most advanced technologies are entering Phase I studies. The pharmaceutical industry is increasingly accepting more complex brain and peptide drugdelivery systems in order to enter niche treatment markets and address the growing demand for brain therapeutics, driven by the medical and clinical success of intravenously administered biologics. However, in order to further improve the transfer from academically discovered oral CNStargeting pharmaceuticals, industrialists would need to be convinced on enhancement of oral bioavailability and stability, versatility of the employed technology for a variety of therapeutic peptides/proteins, receptorspecific binding of the therapeutic biologic, safety of the formulation and possibly modified peptide, and finally the ability to develop the therapeutic peptide formulations from the laboratory to the clinic (manufacturing scalability and costs, activity in all models and intellectual property protection). Using nanoparticulate carriers with a safe toxicological profile, ideally from biocompatible and biodegradable materials, will allow for easier transfer of the developed technologies.

Exploring the strategies that are able to effectively combine oral delivery with brain penetration for a peptide therapeutic resulted in identification of only a few successful preclinical brain delivery strategies after oral administration (TaBle 2). The most developed approaches possessing proofofconcept studies in a preclinical setting have two factors in common; first, they are based on nanoparticulate carriers; second,



both are targeting opioid receptors in order to elicit an analgesic central effect in the brain.

The first reported strategy able to orally deliver a small linear peptide to the brain was dalargin (TyrdAlaGlyPheLeuArg), a leucineenkephalin synthetic analog with enhanced enzymatic stability to aminopeptidases [106–108] achieved by substitution of the second natural amino acid (alanine, lAla) with dAla. Dalargin is a potent agonist of µopioid receptors and exhibits potent analgesic activity after intracisternal administration [109,110]. However, dalargin does not readily penetrate the BBB after intravenous administration by itself resulting in lack of efficacy even after intravenous administration [111]. To overcome this problem, dalargin was absorbed onto the surface of polybutylcyanoacrylate nanoparticles overcoated with polysorbate 85 (Tween 85™) and PEG [202]. The rationale behind this coating was based on two factors. First, polysorbate 80 reversibly opens tight junctions, allowing a temporary enhancement in permeation while it may also cause a temporary fluidization of the mucus present in the unstirred layer of the GI tract, allowing the nanoparticles to be more exposed to the absorptive enterocytes and possibly M cells, leading to increased oral absorption [110]. Second, if the Tween 85 overcoated nanoparticles escape the firstpass metabolism and opsonization, they are able to enter the bloodstream where adsorption of apolipoprotein E onto the surface of the nanoparticles takes place, leading to uptake across the BBB utilizing the LDLreceptors present [112,113]. Tween 85 was necessary for dalargin entry to the CNS (dalarginloaded polybutylcyanoacrylate nanoparticles without Tween 85 were ineffective after intravenous administration [114]) and was responsible for the highest induction of

analgesia [115]. After oral administration of the loaded nanoparticles in mice, the maximum centrally induced analgesic effect was observed at time points of 30 or 45 min [106]. This strategy was improved several years later by Das and Lin as the original work did not provide adequate characterization of the formulation nor the dose of the peptide administered [110]. Similarly, dalargin was adsorbed onto the surface of poly(butyl)cyanoacrylate nanoparticles; however, in this case they were doublecoated with Tween™ 80 and PEG (20,000 Da) [110]. PEG enhanced the protection of the peptide in the harsh gastrointestinal milieu [116] and the long PEG chains reduced reticuloendothelial system opsonization, leading to an enhancement in the halflife of the nanoparticles in the bloodstream [117,118]. A near maximum antinociception was observed in mice in the hot plate bioassay (maximum possible effect of 93.8%) after 60 min post oral administration of nanoparticles composed of 2% Tween and 2% PEG with a duration of antinociception of 2 h. However, a toxic effect at the BBB has been postulated for polysorbate 80 by increasing the permeability of tight junctions or destructions of endothelial cells [114]. No PK studies have been reported quantifying the extent of enhancement of the oral bioavailability or brain levels achieved. Finally, although the intellectual property of this technology was protected [202] almost 15 years ago, the technology has not yet progressed into Phase I studies.

Recently, another promising proprietary technology for oraltobrain delivery involves the molecular envelope technology developed by Nanomerics Ltd [331]. The technology is based on an engineered amphiphilic chitosan polymer (quaternary ammonium glycol

Table 2. Delivery strategies for successful oral delivery of peptides to the brain.

Peptide Strategy Outcome Ref.

Dalargin (Leucine5-enkephalin analog)

Peptide absorbed onto the surface of poly(butyl)cyanoacrylate nanoparticles coated with Tween™ 85

Maximum analgesic effect at 30–45 min post-oral administration

[106,202]

Dalargin (Leucine5-enkephalin analog)

Peptide absorbed onto the surface of poly(butyl)cyanoacrylate nanoparticles double coated with Tween™ 80 and PEG (20,000 Da)

Duration of antinociception: 1 h after oral administration

[110]

Leucine5-enkephalin MET technology: engineered amphiphilic carbo-hydrate polymer (quaternary ammonium palmitoyl glycol chitosan) able to self-assemble into stable nanoparticles encapsulating or associating with the therapeutic agentLipidization of Leucine5-enkephalin and synthesis of plasma-stable and BBB-permeable prodrug (tyrosyl palmitoyl leucine5-enkephalin)

Enhancement in brain AUC after oral administration (67%).Enhanced and prolonged analgesia reaching a maximum 4 h after oral administration (duration of antinociception: 8 h)

[39,45,203,331,332]

AUC: Area under the curve; BBB: Blood–brain barrier.

Key Term

Antinociception: Reduction in pain perception produced when a drug interacts with opioid receptors localized in the periphery or the CNS.



chitosan [GCPQ]) that is carefully tailored to form nanoscale polymer aggregates to package or specifically interact (covalently and noncovalently) with drugs or biologics. The technology has been used successfully in a preclinical setting via both parenteral and oral routes to deliver peptides across the BBB. Leucine5enkephalin has been used as a model peptide for this technology, an endogenous dopioid receptor agonist with a human plasma halflife of 3 min [119]. Leucine5enkephalin was not able to elicit any significant antinociception after oral administration (tailflick bioassay) in a mouse model [39]. However, when formulated with GCPQ (with a molecular weight of 14 kDa), an enhancement was observed in brain bioavailability (67% increase in AUC

0–24

after oral administration and 57% increase in the brain C

max) [39]. The increase in oral bioavailabil

ity is reflected in the elicited antinociception, which was enhanced and prolonged achieving maximum antinociception 4 h after oral administration. The mechanism responsible for absorption was further elucidated [45]. GCPQ polymers with a molecular weight greater than 6 kDa are necessitated for the success of the oraltobrain delivery strategy [45]. GCPQ particles facilitate oral peptide absorption by protecting the peptide from gastrointestinal degradation, adhering to the mucus increasing the gut residence time of the peptide and transporting the GCPQassociated peptide across the enterocytes to the systemic circulation, enabling the GCPQstabilized peptide to be transported intact to the brain [45]. GCPQ particles do not inhibit the efflux pumps or open epithelial tight junctions [120], and the predominant mechanism enabling peptide transport across the BBB does not rely on GCPQ transport across the BBB. However, GCPQ nanoparticles were detected using coherent antiStokes Raman spectroscopy in close association with the luminal surface of brain capillaries or within perivascular spaces, indicative of an interaction between the polymer and the endothelial cell glycocalyx resulting in localization of the nanoparticles at the BBB facilitating peptide delivery [121]. Combining this technology with a lipidization strategy involving GCPQ particles associated with a synthetic lipidized prodrug of leucine5enkephalin (tyrosyl palmitoyl leucine5enkephalin, tyrosylpalmitoyl leucine5enkephalin) can potentiate and enhance the oral antinociceptive effect produced, and lead to analgesia lasting over 8 h after oral administration and significant enhancements in brain bioavailability [39,203]. Peptide lipidization resulted in a peptide analog with enhanced

plasma stability and enhanced brain permeation, and this strategy can be applied to other endogenous neuroactive peptides. Nanomerics Ltd is developing a nanotechnologyenabled peptide pill (METDoloron [332]) for severe chronic pain [122] and Phase I studies are planned within the next 2 years. Developing nanomedicines of endogenous peptides does not require validation of the mechanism of action for the active therapeutic, which is critical for avoiding high attrition rates in late clinical development since proofofconcept in latestage clinical trials is already available [72].

It has to be noted that both successful strategies have targeted opioid cell surface receptors, which are widely distributed in the brain and periphery; thus, analgesia can be elicited by binding to both types of opioid receptors [123]. It is therefore critical to quantify the amount of therapeutic peptide in the brain in order to quantify the brain bioavailability of the therapeutic peptide. Nanoparticulate strategies are particularly advantageous due to their versatility regarding the peptide cargo that can be delivered orally to brain.

Aside from opioid receptor agonists, there are many technologies under preclinical testing aimed at achieving therapeutic levels in the brain after oral administration. Nobex Corporation (NC, USA) is attempting to apply its amphiphilic peptide conjugates technology to create enkephalin, antineoplastic and antimicrobial conjugates that are able to cross the GI tract and the BBB (TaBle 1) [204].

Recently, a fusion peptide between snowdrop lectin (mannosespecific lectin; Galanthus nivalis agglutinin) and insecticidal whexatoxinHv1a peptide was shown in a Mamestra brassicae (cabbage moth) larvae model to have ability to allow oraltobrain delivery of the insticticidal peptide [124]. Snowdrop lectin was used as a carrier for allowing oral absorption of whexatoxinHv1a peptide, a spider venom peptide that targets insect voltagegated calcium channels [125] and acts directly at sites within the CNS, as snowdrop lectin is resistant to proteolytic activity in the insect gut and can mediate delivery to the insect hemolymph [124]. The instecticidal peptide has limited oral toxicity when administered alone, indicated by the mortality of insects receiving either the peptide alone or the fused peptide. However, proofofconcept in a mammalian model is required.

Many different approaches have been used for enhancing the oral bioavailability of therapeutic peptides, but not their brain bioavailability.



Nanomedicines for oraltobrain delivery can provide a unique solution for the unmet clinical need posed by brain diseases that are affecting 1.5 billion individuals worldwide. The increasingly rising economic burden of neurological diseases can justify the high reward expected for industry’s highrisk investment in the translation of nanoparticulate delivery solutions for oraltobrain delivery of therapeutic peptides, from proofofconcept into the market.

Future perspectiveDespite the great advances in oral peptide delivery to the brain, peptide therapeutics currently rely on parenteral routes of administration. Oral peptide therapeutics for CNS indications can only reach the market if they are able to deliver sufficient quantities of the peptide to the brain parenchyma where they would be able to interact with the receptor of interest to elicit a response. Among all the peptide delivery systems developed

Executive summary

Oral peptide therapeutics: impact of new technologies & clinical development

�� The peptide therapeutic market is one of the fastest growing areas of the pharmaceutical industry representing approximately 10% of global sales. Although numerous peptides have been undergoing clinical development in the last few decades, there is no oral marketed peptide therapeutic for brain diseases, despite being a field with unmet clinical needs.

�� The pharmaceutical industry has recognized the immense potential of therapeutic peptides, a direct result of their enhanced potency, high receptor specificity and low toxicity. Apart from a few marketed peptides, parenteral administration is still needed as they are subject to physical and metabolic instability and poor permeation across physiological barriers, which is imparted by their unfavorable physicochemical characteristics. Hence, increasing the oral bioavailability of peptides remains a major challenge for the pharmaceutical industry.

Current challenges in oral peptide delivery

�� An oral peptide therapeutic has to overcome several hurdles, such as the harsh gastrointestinal milieu, proteolytic enzymes, mucin and biological barriers, efflux mechanisms and first-pass metabolism, prior to reaching the bloodstream where it is subject to further metabolism by plasma enzymes.

Oral strategies for marketed peptide therapeutics & nanomedicines in clinical development

�� To enhance the oral bioavailability of peptides, different strategies have been developed, mainly based on: chemical modifications of the peptide (such as amino acid modification, cyclization or synthesis of a prodrug); and different delivery approaches involving enzyme inhibitors, absorption enhancers and novel delivery systems such as mucoadhesive polymers, micro-and nano-particulate carriers and lipid-based systems.

�� The majority of oral delivery systems have focused on the development of oral insulin formulations due to the clinical need for nonparenteral administration and the broad existent market. The most promising oral peptide delivery technologies under clinical development are Nobex®, Eligen®, OradelTM, hepatic-directed vesicle insulin technologies and NOD Tech, which follow a variety of approaches (such as conjugation with amphiphilic oligomers, liposomes, carbohydrate-based polymeric nanoparticles coated with vitamin B12 or enteric-coated bioadhesive nanoparticles).

�� However, none of the technologies developed to enable oral peptide delivery were able to deliver a peptide across the gastrointestinal and the blood–brain barrier (BBB). Oral peptide delivery for brain diseases still remains an unresolved challenge for the industry. Apart from requiring good oral peptide bioavailability, it is necessary to be able to overcome the BBB in adequate amounts to ensure extra- or intra-cellularly receptor binding depending on the location of the targeted receptor.

Challenges in oral peptide delivery across the BBB & oral-to-brain peptide nanomedicines

�� Permeation across the BBB is restricted to unmodified peptides oral delivery. Different technologies are required to enhance the permeation and stability of the peptides across the BBB.

�� Lipophilicity plays a crucial role in BBB penetration, with the majority of active brain therapeutics possessing a lipophilic or amphiphilic character. It can be a useful strategy for enhancing the brain permeation of therapeutic peptides.

�� To overcome limitations imparted by lipid solubility, this strategy can be used in combination with an encapsulation or conjugation strategy (conjugation with different polymers, encapsulation in liposomes and particulate carriers), which are other promising approaches for brain targeting.

�� To date, the only two strategies that have successfully delivered peptides to the brain after oral administration are poly(butyl)cyanoacrylate nanoparticles double-coated with Tween™ 80 and PEG 20,000 Da for dalargin, and MET technology developed by Nanomerics Ltd (Hertfordshire, UK) involving quaternary ammonium glycol chitosan nanoparticles or a combination of this technology with a lipidized prodrug strategy of endogenous peptides. The MET technology is entering Phase I clinical studies.

Future perspective

�� Although commercialization of peptide therapies for brain diseases is still deemed risky by the biopharmaceutical industry, the reward promised by the unmet clinical need and paucity of effective brain therapies will fuel the development of nanomedicines able to enhance oral bioavailbility of peptides to the brain.



over the last 15 years, nanoparticulate strategies were only able to reach the brain parenchyma and obtain preclinical proofofconcept in rodent models after oral administration. The unique features of the nanoparticulate strategies are based on their ability to protect peptides from enzymatic degradation in the GI tract, enhance permeation across physiological membranes such as gastrointestinal mucosa, protect peptides from enhanced firstpass metabolism and plasma enzymatic degradation and opsonization, and enable the peptide therapeutic to cross the BBB. Therefore, they address the critical factors that hamper oral peptide delivery to the brain. However, it is easy to imagine that a single approach based solely on nanoparticulate systems may not be enough to face the multiple obstacles that a peptide encounters in its pathway to the brain. An increase in bioavailability may be potentiated by using a combination of strategies, one of which involves a nanoparticulate carrier, possibly with the use of coatings, absorption enhancers, protease inhibitors and stabilizers, while the second

relies on peptide modification (lipidization, conjugation, fusion), where the advantages of one approach can overcome the deficiencies of the other (FiguRe 2). As the biologics market is flourishing and the clinical needs imposed by CNS diseases are not met, it is not so far from reality to speculate the marketing of an oral peptide nanomedicine able to treat brain diseases via a patientfriendly route.

Financial & competing interests disclosureDR Serrano Lopez was supported financially by the Spanish Ministry of Education Research Fellowship (FPU/AP 2008–00235). A Lalatsa is a co-inventor in a patent com-mercialized by University College London, School of Phar-macy/Nanomerics Ltd (PCT/GB2010/050355. 2010). The authors have no other relevant affiliations or financial involvement with any organization or entity with a finan-cial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.

No writing assistance was utilized in the production of this manuscript.

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peptide pills for brain diseases? reality and future perspectives

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