Lipophilic peptide character – What oral barriers fear the most

Accepted Manuscript Lipophilic peptide character – What oral barriers fear the most

Ožbej Zupančič, Andreas Bernkop-Schnürch PII: DOI: Reference:

S0168-3659(17)30553-9 doi: 10.1016/j.jconrel.2017.04.038 COREL 8783

To appear in:

Journal of Controlled Release

Received date: Revised date: Accepted date:

19 February 2017 21 April 2017 25 April 2017

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ACCEPTED MANUSCRIPT Lipophilic peptide character – what oral barriers fear the most Ožbej Zupančič, Andreas Bernkop-Schnürch* Review article Department of Pharmaceutical Technology, Institute of Pharmacy, Leopold-Franzens-

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University Innsbruck, Innrain 80/82, Center for Chemistry and Biomedicine, 6020 Innsbruck,

Corresponding author:

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*

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Austria / Europe

Department of Pharmaceutical Technology

Leopold-Franzens-University Innsbruck

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Institute of Pharmacy

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Innrain 80/82, Center for Chemistry and Biomedicine

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Tel.: +43-512- 507-58601

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6020 Innsbruck, Austria / Europe

Fax: +43-512-507- 58699

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E-Mail: [email protected]

ACCEPTED MANUSCRIPT Keywords: lipophilicity, hydrophobic ion pairing, REAL, cyclization, esterification, presystemic metabolism, intestinal permeation. Abstract Peptide therapeutics is currently one of the fastest growing markets worldwide and

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consequently convenient ways of administration for these drugs are highly on demand. In particular, oral dosage forms would be preferred. A relative large molecular weight and high

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hydrophilicity, however, result in comparatively very low oral bioavailability being in most

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cases below 1%. Lipid based formulations (LBF), in particular self-emulsifying drug delivery systems (SEDDS) and solid lipid nanoparticles (SLN) as well as liposomes are among the

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most promising tools for oral peptide delivery. Key to success in orally delivering peptides via

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LBF seems to be a sufficiently high lipophilic character of those therapeutic agents. Hence, different non-covalent and covalent peptide lipidization methods from drug delivery point of

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view are presented. On the one hand, among non-covalent lipidization methods hydrophobic

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ion pairing seems to be a promising way to sufficiently increase peptide lipophilicity providing high drug payloads in the lipid phase, a protective effect against presystemic metabolism via thiol-disulphide exchange reactions and proteolysis as well as an improved

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intestinal membrane permeability. On the other hand, covalent methods like conjugating fatty

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acids via amidation, esterification, reversible aqueous lipidization (REAL) and cyclization also show potential. The present review therefore describes those lipidization methods in detail and critically evaluates their contribution in successfully overcoming the oral barriers.

ACCEPTED MANUSCRIPT 1. Introduction Recent advances in biotechnology have led to rapid increase in synthesis and commercialization of numerous peptide drugs [1]. Currently more than 600 peptide drugs are in preclinical or clinical trials, giving the global peptide market a high growth potential [2,3]. On the one hand, peptides have advantages over conventional small molecule drugs including

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high potency, selectivity, excellent safety profile, tolerability and fewer side effects [1,4]. On

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the other hand, the majority of them has to be administered via injections being related with

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pain, discomfort and consequently low patient compliance [1]. Oral peptide delivery is indeed an ongoing challenge, which is evident from numerous published strategies over the past

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decades [3,5]. Comparing the data of so far available in vivo studies and clinical trials, delivering peptides via LBF such as (micro)emulsions including SEDDS [6–12], SLN [13–

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20] and liposomes [21–33] is likely one of the most promising strategies. Increasing intestinal membrane permeability, modulation of tight junctions and reducing proteolytic degradation

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are just some of the benefits when utilizing lipid excipients in oral peptide delivery [3].

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However, only few oral peptide formulations containing lipophilic auxiliary agents have up to date reached clinical trials or commercialization [34,35]. The only commercialized oral

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peptide LBF Sandimune Neoral® contains cyclosporine dissolved in lipid preconcentrate,

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forming an o/w emulsion under in vivo conditions, where the peptide remains incorporated in the lipid matrix of LBF. Following this example, this review will focus therefore just on one important aspect having a substantial impact on the success of oral peptide delivery strategies – namely the lipophilic character of the therapeutic peptide. 2. Lipidization 2.1 Hydrophilic character of peptides and its consequences

ACCEPTED MANUSCRIPT Peptide chemical structure has two fundamental disadvantages when it comes to oral administration – relative high molecular weight and high hydrophilicity [35–40]. The former restricts peptides to paracellular absorption, which comprises of a very small percentage of the total epithelial surface area [37,41–43]. The latter, on the other hand, prevents the therapeutic peptides from permeating the phospholipid bilayer of epithelial cell membranes as

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they are forced to break its hydrogen bonds with solvating water in order to interact with the

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lipid bilayer [37,43–45]. As a consequence of this low membrane permeability systemic

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uptake via the passive transcellular route is strongly limited. Thus, the key factor in increasing peptide lipophilicity is the reduction of its hydrogen bonding potential [44]. Furthermore,

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orally administered peptides containing disulphide bonds may interact in the intestine with endogenous glutathione and thiol substructures of food via thiol-disulphide exchange

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reactions leading to peptide conformational changes and subsequently their inactivation [46,47]. Moreover, an insufficient lipophilic character exposes peptides to harsh presystemic

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metabolism by intestinally secreted trypsin, α-chymotrypsin, elastase, carboxypeptidases A

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and B as well as membrane bound peptidases leading to their inactivation [48–51]. Additionally, polar functional groups of peptides lead to ionic interactions and hydrogen

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bonding with intestinal content and mucus contributing to a low oral bioavailability and high variability in drug uptake [44]. Indeed, the mucus layer covers the luminal surface of the

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gastrointestinal tract and is composed up to 95% of water as well as cross-linked and entangled mucin fibres, carbohydrates, DNA, lipids, salts, antibodies, bacteria and cellular debris [52]. As such, mucus acts as a filter for charged hydrophilic peptides either by size exclusion mechanism or ionic peptide-mucin interactions, resulting in poor peptide mucus permeation [53]. However, there are two opposing strategies to improve peptide diffusion through mucus – the mucoadhesive and the mucodiffusion principle [52]. Firstly, incorporating peptides in mucoadhesive polymers and their thiolated counterparts was shown

ACCEPTED MANUSCRIPT to facilitate peptide diffusion through mucus layer due to close contact of polymer matrix with absorption membrane, providing steep concentration gradient. Moreover, polymer matrix can protect peptides against proteolysis in GIT as well as offers controlled drug release [3,54]. On the contrary, mucodiffusive LBF with neutral or negatively charged surface show even greater potential [3]. The hydrophilic PEG surface corona of LBF enhances their mucus diffusion and

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also prevents their interaction with biological enzymes and mucin [3]. From oral peptide

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delivery point of view, it would be highly beneficial if LBF contain the peptide drug in its

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lipid matrix. Accordingly, a high lipophilic character of orally given peptides seems to be essential.

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2.2 How to measure lipophilicity

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Lipophilicity is defined as the affinity of a molecule or moiety for a lipophilic environment. Indeed, as from the drug delivery point of view it is advantageous to incorporate therapeutic

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peptides in lipophilic carrier systems such as SEDDS, SLN and liposomes, the determination

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of their solubility in lipophilic excipients used in these formulations is essential. Lipophilicity is commonly measured by peptide distribution behaviour in a biphasic system, which can be liquid-liquid or solid-liquid. On the one hand, the gold standard for direct liquid-liquid phase

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lipophilicity characterization of neutral substances is the octanol/water partitioning coefficient

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(log P) [55,56]. However, since most of peptides are ionized, distribution coefficient (log D) expressing the contribution of all neutral and ionized species at given pH, is utilized. For simplicity reasons, in the scope of this review, log P values of peptides are compared. Octanol is used due to its structural similarity with phospholipids in the cell membrane. However, octanol is also a known hydrogen bond donor/acceptor and therefore alternative solvents with different hydrogen bond properties such as heptane, chloroform, cyclohexane and propylene glycol dipelargonate have been proposed [57,58]. One the other hand, since chromatographic retention of substances in a reversed phase column and n-octanol/water partitioning are

ACCEPTED MANUSCRIPT energetically analogous, reversed phase chromatography is becoming the preferential indirect solid-liquid phase method for determining log P [56,59]. Octadecyl-bonded silica (C-18 RPHPLC) and immobilized artificial membranes mimicking the phospoholipid bilyer (IAM HPLC) are normally used as stationary phases. Here, a prolonged retention time of lipidized peptide is observed with respect to the native peptide [60–63]. From difference in retention

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times the representative lipophilicity factor, log kw, which is directly related to log P, can be

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calculated [56]. Noteworthy, peptide lipophilicity can be also determined by potentiometric

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titration, where the peptides are partitioned between liposomal membrane and water and the dissociation constants (pKa) are determined by adding high precision titrators [56]. In

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addition, fast and simple indirect methods to measure lipophilicity include thin layer chromatography (TLC) [56] and various computational methods [64].

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2.3 Covalent vs. non-covalent lipidization

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Lipidization not only alters peptides lipophilicity, but it also may have a substantial impact on

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its secondary structures, self-assembling properties and its ability to bind to target receptors. Moreover, a significant change in ADME properties including metabolic and plasma stability, membrane permeability and bioavailability can be expected [55]. In some cases, these

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changes may cause partial or total loss of the peptides biological activity [61,65,66]. From the

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commercial point of view, non-covalent lipidiziation is preferred. On the one hand, noncovalent lipidization offers a straightforward lipophilicity increase excluding intensive labour with multiple reactions and purification steps. Additionally, non-covalent interactions enable native peptide regeneration in vivo, restoring its secondary and tertiary structures and thus preserving its affinity to target receptors. Moreover, the peptide might not be regarded as a new active pharmaceutical ingredient (API) as it would definitely be the case for all covalently lipidized peptides. On the other hand, the shortcoming of such labile non-covalent bonds is the dissociation of peptide-complex in vivo before it reaches the desired target [67–

ACCEPTED MANUSCRIPT 69]. In such cases, reversible covalent lipidization methods, where the parent peptide is regenerated in vivo, is recommended. In the following chapters, different strategies to increase the lipophilic character of peptide drugs including non-covalent and covalent lipidization

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approaches as illustrated in Figure 1 are described.

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Figure 1: Schematic presentation of non-covalent and covalent peptide lipidization methods. On the one hand, lipophilicity can be increased via ionic interactions between charged amino acids and oppositely charged surfactants. On the other hand, covalent methods utilize peptide primary amino groups, hydroxyl groups and disulphide bonds.

ACCEPTED MANUSCRIPT 3. Non-covalent lipidization Peptides can interact non-covalently with lipidization agents through hydrogen bonding, hydrophobic interactions, complexation with divalent metal ions and ionic interactions, forming water insoluble complexes. A typical example of hydrogen bonding and hydrophobic interactions are tannines. For instance, it was shown in the previous studies that protein-tannin

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complexes are stable at gastrointestinal pH and in the presence of proteolytic enzymes such as

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pepsin, trypsin, α- chymotrypsin, elastase and carboxypeptidase A and B as well as bile salts

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[70,71]. Furthermore, metal ions such as Zn2+, Cu2+ or Ni2+ are able to form coordinative complexes with peptides, which cause conformational changes and stabilize the peptide

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structure [72–74]. Oxytocin, for example, undergoes a conformational change when coordinated with Cu2+ or Zn2+. This causes the carbonyl oxygens of the ring to be directed

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towards the core reducing the peptides hydrogen bonding potential and rendering its surface more hydrophobic [73,75]. Also, Zn2+ is well known to coordinate with peptides, forming

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water insoluble complexes. In previous studies, various water insoluble zinc-peptide

gonadotropin-releasing

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complexes such as with insulin [76–78], hirudin [79,80], dalarein [81,82], buserelin [83], hormone

[81],

thyrotropin-releasing

hormone

[81],

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adenocorticothropic hormone [81] and thyroliberine [82] have been prepared. However, the

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most efficient approach for non-covalent peptide lipidization seems to be via ionic interactions, where peptide lipophilicity is increased by peptide net charge neutralization by anionic, amphoteric or cationic surfactants. 3.1 Interactions between peptides and surfactants Peptide-surfactant interactions are a complex process, dependent on peptides primary structure, pH, ionic strength, temperature, surfactant type (ionic, non-ionic) as well as on the surfactant to peptide molar ratio (S/P) [84]. Various well established analytical techniques to measure such interactions as presented in Table 1.

ACCEPTED MANUSCRIPT Table 1: Methods to measure peptide-surfactant interactions Technique

Description

Ref.

Morphology

Scanning Electron Microscopy X-ray Powder Diffraction

The morphological changes of unprocessed peptide, surfactants, physical peptide and surfactant mixture and PSC are compared

[85] [85] [86]

Peptide backbone and secondary structure

UV-circular dichroism Fourier transform infrared spectroscopy (FT-IR)

Near UV and far UV circular dichroism spectra are recorded in order to determine possible peptide backbone and secondary structure changes due to ionic interactions with surfactants. Spectra of peptide and PSC are compared to determine secondary structure of peptide in the PSC.

[84,85,87] [88]

Ionic interactions between peptide and surfactant

Fourier transform infrared spectroscopy (FT-IR)

DSC and FT-IR spectra of peptide, surfactant, physical mixture of peptide and surfactant and peptide-surfactant ion pair are compared.

Lipophilicity (noncovalent peptidesurfactant interactions Lipophilicity (covalent peptide-surfactant interactions)

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Stoichiometry of binding

Differential scanning calorimetry (DSC) Isothermal titration calorimetry

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Parameter

[85–87,89] [14,85]

[84,87]

Partitioning/distribution coefficient (log P/log D) (“shake flask method”)

Extraction of tested substance fromwater/1-octanol. Determination of concentation of the drug in aqueous and organic phase

[56,90,91]

Reverse phase highperformance liquid chromatography (RP-HPLC)

Partitioning of subsatances between aqueous mobile phase and C-18 RP-HPLC or IAM HPLC stationary phase. Lipophilicity is calculated from the retention time shift between the substance and its analogues

[56,92,93]

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Electrostatic binding of surfactants to peptides is an exothermic process whereas peptide unfolding is an endothermic process. By measuring the heat-flow the stoichiometry of binding could be determined

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Indeed, this review primarily is focused on ionic interactions between peptides and surfactants as they result in significant lipophilicity increase of the peptide-surfactant complex (PSC).

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There are different concentration dependent mechanisms of surfactant binding on the peptide surface. Initially, below the critical micelle concentration (CMC), ionic surfactants bind non-

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cooperatively as monomers to the peptide native state [94]. Polar head groups of individual surfactants bind on oppositely charged side chains via electrostatic interactions, whereas alkyl chains interact through hydrophobic interactions with nearby hydrophobic patches [95,96]. The alkyl chain length has an impact on the degree of binding and stabilization of the complex [97]. With increasing surfactant amounts, the initial surfactant binding sites are saturated and the excess of surfactant molecules such as sodium dodecyl sulphate (SDS) may promote the peptide to unfold and aggregate. This leads to formation of clusters, which are stabilized by the association of peptide aggregates, leading to formation of shared micelles

ACCEPTED MANUSCRIPT [84,98]. Further addition of surfactant provides sufficient number of molecules for each peptide to form its own cluster. Above CMC, typically at 100 surfactant molecules per each peptide molecule, cooperative surfactant binding takes place, where interactions between peptides and micelles are predominant [84]. Such interactions are more common between peptides and non-ionic surfactants, where the micelles interact with the peptide hydrophobic

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surface [99–101]. Recently, non-ionic surfactants are being extensively investigated as the

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main component of niosomes, a cheap and more stable alternative for liposomes, where

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phospholipids are substituted with non-ionic surfactants [102]. For instance, physically stable niosomes with droplet size 95.7 nm and negative zeta potential containing peptides have been

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designed [103]. In addition, orally administered niosomes containing insulin-Tat complex showed significant increase in hypoglycemic activity in mice [104].

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3.2 Hydrophobic ion pairing

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Many peptides contain at least one ionisable amino acid. At physiological pH, lysine, arginine

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and histidine are positively charged, whereas glutamic and aspartic acid are negatively charged. By adjusting the pH value above their isoelectric point, peptides pose a negative net charge and vice versa, by decreasing pH below isoelectric point yields peptides of a positive

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net charge. Lipophilicity of such ionized peptides can be effectively increased by hydrophobic

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ion pairing [105–107]. Here, ionic interactions between charged peptides and oppositely charged surfactants lead to the formation of water insoluble PSC. In the scope of this review, surfactants are classified according to their charge in anionic, cationic and amphoteric. In Table 2 structures of representative surfactants, which successfully formed water-insoluble PSC with indicated peptides are presented. Table 2: Structures of representative surfactants, which successfully formed lipophilic complexes with indicated peptides. Type

Class

Structural formula

Name

Peptide

Ref.

ACCEPTED MANUSCRIPT Anionic

Fatty acids Sodium decanoate

Octreotide

[108]

Vancomycin

[88]

Desmopressin, leuprolide

[14,90]

[108–112]

Sodium oleate

Leuprolide, octreotide, salmon calcitonin, desmopressin

Sodium dodecylsulp hate

Desmopressin, octreotide, insulin, bivalirudin

[87,90,108,1 13–115,132]

Sodium laurate Sodium stearate

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Organo sulphates

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Sodium octadecylsul phate

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Bile acids

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D

Salicylatefatty acid conjugates

Alkyl phosphates

Phospholi pids

Amphote ric

Phospholi pids

Cationic

Fatty amines

Sodium docusate

Desmopressin,lanre otide, insulin, bivalirudin

[90,116,117, 132]

Sodium deoxycholat e

Octreotide, lanreotide

[108,116,118 ]

Lanreotide

[116]

Salmon calcitonin

[119,120]

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Organo sulphonate s

[90,116]

Desmopressin, lanreotide

Sodium taurocholate

Sodium N[8-(2hydroxybenz oyl)amino]c aprylate (SNAC)

[121,122]

8-(N-2hydroxy-5chlorobenzyl)aminocapryli c acid (5CNAC)

Sodium hexadecylph osphate

Thymopentin

[123]

DMPG

Salmon calcitonin Insulin bivalirudin

[112] [89] [132]

Phosphatidyl choline

Insulin

[85,86,124– 127]

Dodecylami ne chloride

Daptomycin, bivalirudin

[91,132]

ACCEPTED MANUSCRIPT Quarterly amines Distearyldim ethylammoni um bromide

Insulin

[85]

Most therapeutic peptides contain at least one cationic centre and could be therefore ion paired with anionic counter ions. Anionic surfactants such as fatty acids are preferred due to

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their ability to open tight junctions, binding to fatty acid binding proteins as well as solubilizing membranes and thus enhance drug permeation. In addition, fatty acids may also

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mask peptides from the gastrointestinal environment and eluding first pass effect by

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disguising the drug as a triglyceride as well as carry them across mucus as part of their

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absorption [128,129]. Moreover, fatty acids are generally considered as safe [130]. In contrast, ion pairing of anionic peptides with lipophilic cationic counter ions is more rarely

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used due to their potential toxicity [35]. Nonetheless, there are numerous reports about the use of fatty amines such as dodecylamine chloride [91,131,132], hexylamine [133] and quaternary

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amines such as distearyldimethylammonium bromide [85], dodecyltrimethylamonium

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chloride, cetylprydinium chloride, stearalkonium chloride and cetrimide [133]. In addition, cationization of bile salts via diethylamine spacer like for example Nα-deoxycholyl-L-lysyl-

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methylester [134] or deoxycholylethylamine [135] might be used as a safer alternative. Also, a likely promising alternative to cationic surfactants for the ionic complexation of peptides

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exhibiting an anionic net charge are amphoteric surfactants. On the one hand, they are still capable of ion pairing with anionic peptides and on the other hand most of them can be regarded as safe.

Hydrophobic ion pairing efficiency is a function of pH, which determines peptides net charge and ionization state of peptide and surfactant functional groups. However, it seems that the S/P also plays a prominent role in peptide complexation efficiency and the lipophilicity of the PSC. So far, SDS has been most commonly used as ion pairing agent. It was observed that an

ACCEPTED MANUSCRIPT equimolar ratio of SDS led to complexes of comparatively highest lipophilicity [113,136,137]. Similarly, practically quantitative complex formation of antibiotic peptide daptomycin was achieved at equimolar amounts of dodecylamine [91]. In contrast, as illustrated in Figure 2, the most efficient complex formation of desmopressin, which contains

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one cationic centre was at S/P of 1.8 [90].

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Figure 2: Hydrophobic ion pairing of desmopressin acetate with sodium octadecyl sulphate

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(○), sodium stearate (▲), sodium dodecyl sulphate (□) sodium oleate (●) and sodium docusate (■). After the addition of surfactants, the precipitated desmopressin complex was

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centrifuged and the amount of remaining peptide in supernatant determined by HPLC. Data are shown as mean ± SD (n =3).

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Furthermore, the optimum S/P ratio of 3.0 was observed when peptides with two cationic centres – lanreotide and leuprolide – were ion paired with sodium oleate and sodium deoxycholate [109,116]. An explanation for this phenomenon might be given by peptidesurfactant aggregation and mixed micelles formation as described above. In addition, when large excess of surfactant is added to PSC solution, its aqueous solubility is regained. Indeed, with increasing amounts of surfactant beyond the critical micellar concentration (CMC) micelles are formed, which most probably resolubilize the PSC. This assumption was

ACCEPTED MANUSCRIPT confirmed by performing the inverse experiment – adding increasing amounts of insulin to an excess of SDS. At the beginning of the experiment, where the amount of SDS was in great excess, no PSC was observed, indicating its immediate solubilization via micellization [87]. As mentioned earlier, PSC lipophilicity is also a function of S/P as illustrated in Figure 3. For instance, daptomycin log P was increased with dodecylamine chloride from - 5.0 at S/P 0:1 to

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+4.8 at S/P 4:1[91], whereas ion paring of lanreotide with sodium deoxyclolate increased its

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log P from -10.0 at S/P 0:1 to remarkable 9.0 at S/P 3:1[116]. In addition, the surfactant itself

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also has an impact on PSC lipophilicity. Desmopressin log P of -6.13, for instance, was increased to a positive value of 0.33 when using SDS and sodium docusate, but remained

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negative (log P = -1.80) when sodium oleate was used at S/P 1.8:1[90].

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Figure 3: (A) Log P (octanol/water) increase of daptomycin dodecylamine complex (●), lanreotide deoxycholate complex (■) and lanreotide docusat complex (□) as a function of surfactant : peptide molar ratio. (B) Log P (Capmul 907 P/water) increase of desmopressin oleate complex (●), desmopressin dodecyl sulphate complex (■) and desmopressin docusat complex (□) as a function of S/P. In relation to that, increased lipophilic nature of PSC enables its incorporation in the lipid matrix of LBF. Table 3 summarizes most representative examples, where hydrophobic ion

ACCEPTED MANUSCRIPT pairing resulted in successful peptide incorporation in LBF. For instance, 8.0% and 10.5% ion pair complexes, corresponding to 5.5% and 6.4% of daptomycin and lanreotide were dissolved in SEDDS formulations [91,116]. Moreover, ion pairing of thymopentin with hexadecylphosphate increased the apparent log P of the complex 280-fold with respect to bare peptide and subsequently the drug payload in SLN, which was increased from 1.7% to 5.2%,

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respectively [123]. In connection to that, ion pairing of vancomycin with lauric acid increased

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peptides log P from -1.41 to 1.37, yielding SLN with a 0.1% drug payload [88]. Also, PSC

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drug payloads of about 10% (w/w) can be achieved with high amounts of hydrophilic

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surfactants and co-solvents in LBF [138].

Table 3: LBS containing peptide-surfactant ion pair and their characteristics. Counter ion

LBF

Formulation

Desmopressin

Sodium docusate

SEDDS

40% Cremophor RH40 50% Capmul 907 P 10% Transcutol

Insulin

Dimyristoyl phosphatidylglycerol (DMPG)

SEDDS

Soyphosphatidylcholine

SEDDS

Leuprolide

Result

Ref.

Increased desmopressin docusate complex log P (Capmul 907 P/ water) from -6.30 to 0.33 SEDDS protected desmopressin against α– chymotrypsin and glutathione degradation

[90]

33% Labrafil 1944 47% Cremophor EL 20% Transcutol

1.13%

SEDDS protected insulin against in vitro trypsin and α – chymotrypsin degradation Sustained insulin release form SEDDS Mucus permeating SEDDS

[89]

35% Cremophor EL 32.5% ethyl oleate 32.5% ethanol

0.30%

The relative insulin oral bioavailability of SEDDS congaing insulin was 7-fold increased compared to oral insulin PBS solution

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Lanreotide

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β - Lactamase

Drug Payload 0.08%

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Peptide

Soyphosphatidylcholine

SEDDS

42% Lauroglycol FCC, 33% Cremophor EL 25% Transcutol

0.17%

The protein-phospholipid complex in SEDDS resulted in a relative bioavailability of 6.34%, which was 2.6-fold higher compared to protein in aqueous solution

Sodium deoxycholate

SEDDS

25% Capmul MCM 45% Mygliol 840 30% Cremophor EL

10.5%

25% Captex 8000 30% Capmul MCM 30% Kolliphor EL 15% Propylene glycol

9.7%

Increased lanreotide deoxycholate log P (octanol/ water) to 9.0 Sustained lanreotide release form SEDDS SEDDS protected lanreotide against in vitro glutathione and thiol-enriched casein,peptones thiol-disulphide exchange reactions

30% Ethyl oleate 30% Capmul MCM

0.4%

Sodium oleate

SEDDS

SEDDS protected leuprolide against in vitro trypsin and α –

[139]

[140] [141] [142]

[116]

[109].[110]

ACCEPTED MANUSCRIPT EP 30% Cremophor RH40 10% Ethanol 30% Captex355 30% Capmul MCM EP 30% Cremophor EL 10% Propylene glycol 33.3% Brij 97 33.3% Oleic acid 33.3% Ethanol

Dodecylamine hydrochloride

SEDDS

Vancomycin

Lauric acid

SLN

Thymopentin

Sodium hexadecyl phosphate

SLN

Leuprolide

Sodium stearate

Lipase stable SEDDS

57% olive oil 38%Tween 80 3% propylene glycol

2.0%

75.5% paraffin 25%, Brij 35 8.0% propylene glycol 1.5%, ethanol

2.0%

73.1 ± 8.1% of octreotidedeoxycholate complex was found in the octanol phase. Sustained release of octreotide from SEDDS for at least 24 h, Pancreatic lipase stable SEDDS

30% Dermofeel MCT 30% Capmul MCM 30% Cremophor EL 10% propylene glycol

[110]

[108]

Increased daptomycindodecylamine complex log P (octanol/ water) from -5.0 to 4.8 SEDDS protected daptomycin against in vitro α – chymotrypsin degradation Mucus permeating SEDDS

[91]

0.1%

Increased vancomycin log P from -1.41 to 1.37 and higher encapsulation of the drug in the lipid core of SLN

[88]

3.9% egg lecithin 9.7% sodium turodeoxycholate 4.0% butanol 5.9% stearic acid 1.5% sodium hexadecylphosphate

5.2%

Apparent log P increase by 280fold with respect to bare peptide and subsequently the drug payload in SLN

[123]

Stearic acid Liquid paraffin Span-80

-

Sustained drug release from SLN

[14]

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5% Compritol 888 ATO 3% Lutrol F68

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SLN

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0.4%

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SEDDS

[109] in vivo study in rats showed a 17.2-fold improved oral bioavailability of SEDDS containing leuprolide oleate compared to leuprolide acetate solution

8.0%

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Daptomycin

Sodium deoxycholate

0.4%

SC

Octreotide

chymotrypsin degradation

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4. Covalent lipidization

Most of peptides contain at least one primary amino, hydroxyl or carboxyl functional group, which can be converted to amides and esters. Additionally, disulphide bonds can also be manipulated to attach lipid moieties [143–147]. “The classical approach” to lipidize peptides is via amide bond formation between the primary amino group of the N-terminus or lysine subunits and a fatty acid [148–155]. Interestingly, coupling a fatty acid to tyrosine hydroxyl group via O-etherification was also reported [156]. Moreover, stabile ethers can also be

ACCEPTED MANUSCRIPT synthesized through S-ether formation. Such method was utilized in lipidizing salmon calcitonin with ε-maleimido lysine derivative of palmitic acid [62]. Worth mentioning is also a powerful iodoacetamide derivatization method, used mainly in analytical field [63,157]. Some lipophilic peptide derivatives have already been synthesized by this pathway and according to the increased lipophilicity of the synthesized derivatives as illustrated in Table 4,

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the iodoacetamide method might also find potential in oral peptide delivery.

Peptide sequence

Log kb

Log P calc.a

-12.75

0

-8.86

0.45

-8.28

0.55

-6.50

0.78

a

Log kb

-15.83

0

-8.45

0

-13.88

0.69

-4.56

1.30

-13.59

0.77

-3.98

1.30

-12.70

1.02

-2.20

1.30

0.92

-12.50

1.09

-1.80

1.32

1.22

-10.72

1.17

1.75

1.53

MA

D

CE

-2.54

Log P calc.a

PT E

-6.10

Log kb

NU

Log P calc.a

SC

substituent R

RI

Table 4: lipophilicity increase of the model peptides with iodoacetamide derivatization [63].

Calculated log P values with chemAxon software (www.chemicalize.org) Retention factor calculated from retention times of derivatives with respect to iodoacetamide derivatization

b

AC

4.1 Reversible aqueous lipidization (REAL) To keep the advantages of lipidization without taking the risk to lose the conjugates biological activity, various studies have utilized the REAL technology by forming in vivo labile disulphide bonds between model peptides and amphiphilic PEG-stearic acid conjugate 2-(2(2-(hexadecyloxy)ethoxy)ethoxy)ethyl 3-(2-(pyridin-2-yl)disulfanyl) propanoate or cysteinepalmitic acid conjugate N-palmitoyl-S-(pyridin-2-ylthio)cysteine. Such conjugates were shown to quantitatively regenerate the parent peptide in vivo. The common REAL synthetic pathway, where the peptide is lipidized in aqueous medium is presented in Figure 4. REAL

ACCEPTED MANUSCRIPT technology has already been successfully used on some model peptides and proteins containing disulphide bonds [61,62,93,158–160]. For instance, REAL lipidization of desmopressin (log P = -6.13) with various fatty acids yielded in increased RP-HPLC retention times and capacity factors of the lipidized derivatives. The most lipophilic palmitic acid derivatives with log P values of 5.65 and 5.32 were too lipophilic to be eluted from the In addition, in vivo results showed that REAL

PT

column with the chosen method [161].

RI

lipidization might be a promising tool in orally delivering peptide and protein drugs. Orally

SC

administered REAL lipidized calcitonin, for example, showed a 19-fold increased area under curve compared to unmodified peptide [92]. Nevertheless, one limitation of REAL technology

NU

is its restriction to peptide drugs containing disulphide bonds. However, most of therapeutically significant peptide drugs contain at least one primary amino group. Thus, an

MA

amine-reacting lipidizing reagent based on REAL concept, 3,4-bis(decylthiomethyl)-2,5furandione was used to reversibly modify Leu5-enkephalin. The lipidized conjugate showed a

D

significantly increased antinociception effect using a rodent inflammatory pain model. After

PT E

oral administration, the peak concentration and area under the curve of the conjugate were 4.4

AC

CE

and 21-fold higher compared to Leu5-enkephalin, respectively [162].

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

PT E

Figure 4: Common synthetic pathway in REAL lipidization. The water soluble lipidized derivatives are water soluble and converted to the parent drug under in vitro and in vivo

4.2 Cyclization

CE

conditions.

AC

Conventional linear peptides are charged under physiological conditions due to C- or Nterminal ends. As such, linear peptides show poor membrane permeability and high proteolytic instability compared to their cyclic counterparts [163,164]. In contrast, peptide cyclization was shown to alter peptides physicochemical properties such as charge, conformation, hydrophobicity and subsequently led to improved oral absorption [165–167]. Lipophilicity of such cyclic derivatives, commonly referred to as macrocycles [168], is most probably increased due to restricted conformational freedom, leading to formation of

ACCEPTED MANUSCRIPT intramolecular hydrogen bonds. Additionally, more lipophilic non-charged cyclic peptides can traverse the cell membrane by passive diffusion via transcellular pathway [169]. The poster child of cyclization technology is cyclosporine [163,168,170]. Although cyclization can be performed in different ways [171–173], the use of cyclization linkers as illustrated in Table 5 has shown to increase lipophilicity and Caco-2 monolayer permeation of cyclic peptides

RI

PT

compared to their linear counterparts.

SC

Table 5: Partitioning coefficient and Caco-2 monolayer permeation increase between linear

Linker

Structure

NU

encephalins and their cyclic counterparts.

Leu5-enkephalin

Papp x10-6 [cm/s] < 0.0031

log k'IAMa 0.17

Leu5-enkephalin cyclic prodrug

2.66 ± 0.12 ____

2.70 -

DADLE

0.086 ± 0.005

0.43

-1.30

3.16 ± 0.26

2.91

2.98

H-Try-Ala-GlyGly-Asp-AlaOH

< 0.17

-1.10

-5.20

H-Try-Ala-GlyGly-Asp-AlaOH cyclic prodrug

12.97 ± 1.48

-0.32

-2.24

Leu5-enkephalin

< 0.31

0.17

-1.86

Leu5-enkephalin cyclic prodrug

1.80

1.36

0.92

DADLE

0.78

0.43

-1.30

1.86

1.63

1.49

Leu5-enkephalin

< 0.0031

0.17

-1.86

Leu5-enkephalin cyclic prodrug

13.3 ± 0.426

3.32

3.94

DADLE

0.086 ± 0.005

0.43

-1.30

8.27 ± 0.51

3.32

4.51

Peptide

MA

Coumarinic acid

cyclic

D

DADLE prodrug

AC

CE

PT E

(Acyloxy)alkoxy

Phenylpropionic acid

DADLE prodrug

DADLE prodrug a

cyclic

cyclic

---

Log Pcalcb

Ref.

-1.86

[169]

2.41-- ---------------

[174,175]

[176]

Partitioning coefficient between 0.01 M phosphate buffer pH 7.4 and an immobilized artificial membrane column, composed of phosphatidylcholine analogues (IAM.PD.DD) b Partitioning coeficent calculated withChemAxon software (ww.chemicalize.org)

ACCEPTED MANUSCRIPT 4.3 Esterification with fatty acids A great advantage of forming peptide O- or S- esters with fatty acids is their weak enzymatic or chemical stability, regenerating the parent drug upon hydrolysis in vivo [177]. In previous studies, several prodrugs of desmopressin have been prepared by forming O-esters with tyrosine hydroxyl group using various fatty acids. The lipophilicity and the transport of ester

PT

prodrugs across Caco-2 monolayer was remarkably higher compared to desmopressin alone as

RI

shown in Table 6 [178]. Furthermore, O-esterification of tyrosine hydroxyl group with

NU

dalargin and dermorphin derivative [179–181].

SC

palmitic acid was shown to increase the oral bioavailability of leu5-enkephalin analogue,

MA

Table 6: Increased log P and Caco-2 permeation of desmopressin and its corresponding ester

D

prodrugs [178].

log Pa

log Pcalcb

Papp x 10-7 [cm/s]

Enhancement ratio

< -3.5

-6.20

5.15 ± 1.67

-

< -3

-5.43

9.44 ± 1.54

1.8

-2.25

-4.33

15.1 ± 1.1

2.9

-1.25

-4.10

9.77 ± 1.19

1.9

0.41

-3.21

8.50 ± 1.60

1.7

O-2-Ethylhexanoyl desmopressin

0.09

-3.11

ND

ND

O-isobutyloxycarbonyl desmopressin

-1.77

-4.27

5.99 + 0.57

PT E

Peptide Desmopressin O-propionyl desmopressin O-pivaloyl desmopressin

a

AC

O-octanoyl desmopressin

CE

O-hexanoyl desmopressin

1.2

0

experimental partitioning coefficient between octanol and aqueous buffer pH 7.40 and 21 C Partitioning coeficent calculated with ChemAxon software (ww.chemicalize.org)

b

5. Impact of lipidization on thiol-disulphide reactions Intestinal thiol compounds such as glutathione, cysteine, N-acetyl cysteine and homocysteine usually originate from food [182]. In addition, endogenous glutathione is present in most mucosal tissues as a part of antioxidant defence system [183,184]. Thus, peptides containing thiol or disulphide bonds can undergo chemical degradation via thiol/disulphide exchange

ACCEPTED MANUSCRIPT reactions with intestinal thiol compounds. To preserve the integrity of disulphide bonds, peptides containing disulphide bonds can be incorporated in LBF. Since the concept of so called “sulfhydryl barrier” is relatively fresh, only a handful of studies demonstrating protective effect of LBF against thiol/disulphide exchange reactions are available. For instance, two disulphide bond containing peptides, lanreotide and desmopressin were

PT

incorporated in different SEDDS via ion pairing and emulsified in reduced glutathione

RI

solution [90,116]. Figure 5 illustrates the protective effect of SEDDS containing PSC against

SC

glutathione degradation. The decelerated peptide degradation was observed by all SEDDS formulations except one, which contained 80% hydrophilic surfactants. It seemed that the

NU

increased amount of surfactants enabled diffusion of hydrophilic glutathione in the droplets, inactivating desmopressin [90]. Furthermore, an impact of potential thiol containing

MA

substances from protein dietary source on lanreotide inactivation was tested, where lanreotide was incubated in thiol enriched casein hydrolysed peptones. Similarly, the stability of

D

lanreotide in SEDDS was significantly higher compared to lanreotide in aqueous solution

PT E

[116]. On the other hand, the disulphide bonds may be protected by “preactivation” via REAL lipidization, where the peptide would remain protected against thiol compounds in

CE

gastrointestinal tract. The parent peptide would be regenerated under in vitro and in vivo

AC

conditions by reduction of labile disulphide bonds [160].

ACCEPTED MANUSCRIPT Figure 5: (A) Protective effect of two different SEDDS formulations containing lanreotide deoxycholate against thiol-disulphide exchange reactions with reduced glutathione (□; composed of 25% Capmul MCM, 30% Cremophor EL, 40% Myglyol 840 and ○, composed of 25% Capmul MCM, 30% Cremophor EL, 25% Captex 8000 and 15% propylene glycol) and thiol-enriched peptones (■,●) compared with blank lanreotide (▲). (B) Protective effect

PT

of SEDDS (○, 40% Cremophor RH40, 50% Capmul 907 P, 10% Transcutol) containing

RI

desmopressin docusate complex (○) against thiol-disulphide exchange reactions with reduced

SC

glutathione compared with blank desmopressin (▲).

NU

6. Impact of lipidization on peptide proteolytic stability

MA

The major barrier for orally administered peptides to overcome is the enzymatic barrier. The luminally secreted and brush border membrane enzymes, as well as the cytosolic enzymes of

D

the absorption membrane play prominent role in the peptide presystemic metabolism [48,49].

PT E

More specifically, trypsin, which cleaves basic amino acids arginine and lysine and αchymotrypsin, cleaving aromatic amino acids phenylalanine and tyrosine are among the most

CE

important pancreatic proteases responsible for rapid peptide degradation. Various in vitro studies have demonstrated that LBF can decrease the rate of proteolysis when the peptide is

AC

incorporated in LBF. Figure 6 depicts a typical degradation profile of leuprolide incorporated in SEDDS by trypsin and α-chymotrypsin [110]. Additionally, prolonged proteolytic stability of peptide drugs in SEDDS was also observed among desmopressin [90], daptomycin [91], dalargin [185], insulin [89]. Furthermore, modified SLN [17,186–191] and liposomes [32,192–194] containing peptide drugs also demonstrated protective effects against enzymatic degradation in the gastrointestinal tract. In addition, covalently lipidized peptides showed prolonged proteolytic stability when administered orally. For instance, a 12-fold increased half-time of REAL lipidized Leu5-enkephalin in mouse small intestinal mucosal homogenate

ACCEPTED MANUSCRIPT was observed with respect to native Leu5-enkephalin[162]. REAL, thioether and PEGylated thioether salmon calcitonin derivatives were shown to be significantly more stable in intestinal fluid with respect to native peptide [62,195]. Furthermore, fatty acid acylation of thyrotropin-releasing hormone, tetragastrin and insulin on the amino terminus improved the stability of N-terminus lipidized derivatives against intestinal enzymatic degradation

D

MA

NU

SC

RI

PT

[196,197].

PT E

Figure 6: Protective effect of SEDDS containing leuprolide oleate in three different formulations (□; 33% Brij 97, 33% oleic acid, 34% ethanol ■; 30% ethyl oleate, 30% Capmul

CE

MCM, 30% Cremophor RH40, 10% ethanol ●; 30% Captex 355, 30% Capmul MCM, 30% Cremophor EL, 10% propylene glycol) with respect to blank leuprolide (▲) against α-

AC

chymotrypsin (A) and trypsin (B). 7. Impact of lipidization on peptide intestinal permeation One of the crucial rate limiting factors in oral peptide delivery is the intestinal epithelium, which is comprised of a single monolayer of cells. A generally accepted in vitro model to assess intestinal drug absorption is the Caco-2 cell monolayer model [198]. Using the Caco-2 model, insulin and rhPTH1-34 in SEDDS showed up to 12-fold and 7-fold increased permeation with respect to peptide solutions, respectively [199–201]. Furthermore, SEDDS

ACCEPTED MANUSCRIPT containing β-lactamase showed a 24.9-fold increased diffusion of β-lactamase through MDCK monolayer compared with β-lactamase solution, respectively [142]. In relation to that, SLN decorated with cationic cell penetrating peptide octaarginine containing insulin increased the Caco-2 permeability by up to 18.44 times [16]. The use of such cationic cell penetrating peptides is indeed a remarkable way to boost LBF cellular uptake. For instance, SEDDS

PT

loaded with covalently lipidized HIV-1 Tat-protein 49-57 with oleoyl chloride showed 1.7-

RI

fold improved transfection efficiency for pDNA compared to Lipofectin [202]. However,

SC

covalent peptide lipidization without using cell penetrating peptides also significantly improved peptide Caco-2 permeability. For instance, Caco-2 permeability of REAL lipidized

NU

Bowmann-Birk protease inhibitor and Leu5-enkephalin were increased 140-fold and 131-fold compared to native peptides [66,162]. In addition, fatty acid acylation of tetragastrin on the

MA

amino termini improved the amount of acetyl, butyl and hexyl tetragatrin transported for 13fold, 19-fold and 18-fold, respectively [203]. Also, S-ether of salmon calcitonin with ε-

D

maleimido lysine derivative of palmitic acid showed a 2-fold higher cellular uptake than

PT E

native peptide in Caco-2 model. Additionally, oral administration of conjugate suppressed plasma calcium levels (60% of baseline) for up to 10 h [60,62].

CE

8. Oral delivery lipid-based formulations for peptides

AC

The most commonly investigated LBF used in oral peptide delivery are SEDDS, SLN and liposomes. All these LBF show common characteristics favourable for oral peptide delivery such as controlled drug release properties, biocompatibility, protective effect against proteolysis and enhanced absorption though intestinal epithelium [3,34]. However, they differ in some significant features as illustrated in Table 7. In order to understand the parameters influencing the choice of LBF for effective oral delivery, their physico-chemical features and limitations should be taken into consideration.

ACCEPTED MANUSCRIPT Table 7: LBF used in oral peptide delivery and their properties [3,34].

Description

Composition

SEDDS

Isotropic mixture of lipids, surfactants and co-solvents, spontaneously forming o/w emulsion upon mixing with water.

Middle chain mono-, di- and triglycerides

Advantages

Disadvantages

Simple preparation procedure

Poor in vitro - in vivo correlation

W/O/W double emulsion

Spontaneous selfemulsification

Limited peptide drug payload

Melt dispersion

Thermodynamically stable

Dilution effect in vivo may cause phase inversion and drug leakage

Hydrophobic surfactants (HLB < 12)

RI

Mucus permeability

Hydrophilic surfactants (HLB > 12)

Could be filled in soft/hard gelatine capsules

SC

Peptides dissolved in the oily preconcentrate

Long chain and medium chain fatty acids

Peptide entrapment methods Hydrophobic ion pairing

Co-solvents Long-chain triglycerides

W/O/W double emulsion

Partial glycerides

Hydrophobic ion pairing

Fatty acids

Reverse micelles

Waxes

Melt dispersion

Phospholipids

Limited peptide payload in the lipid core

Ease of large scale production – facile and liable scale up

Unpredictable gelation tendency of SLN dispersions

Low toxicity due to omitting organic solvents

Aqueous core with one or more amphiphilic bilayers made of phospholipids and cholesterol.

Phospholipids (dipalmytoyl phosphatidylcholine, distearoyl phosphatidylcholine ,dicetyl phosphate)

Hydrophilic peptides are incorporated in the inner aqueous core, lipophilic peptides in the lipid bilyer, amphiphilic peptides are partitioned between both phased

Cholesterol

AC

Liposomes

CE

PT E

Steroids

Reduced drug leakage due to solid core

D

Peptides are dissolved or physically dispersed in the solid core.

NU

Solid state at room and body temperature, lipid core with emulsifier layer

MA

SLN

PT

LBF

Saturated and unsaturated fatty acids

Film hydration or reverse-phase evaporation, followed by particle size reduction via sonication, extrusion, high pressure homogenization Detergent dialysis Solvent injection method

Restructuring process can lead to potential drug expulsion and the inability to achieve a prolonged release of the encapsulated drug

High biocompatibility

Low stability in the GIT

Weak immunogenicity and low intrinsic toxicity (natural phospholipids)

High production costs

High specificity for localized delivery

Limited drug payload capacity of the aqueous core and bilayer Leakage of the incorporated peptide drug Sterilization difficult due to phospholipid sensitivity to radiation and heat Difficult batch-tobatch

ACCEPTED MANUSCRIPT

9. Lipid-based formulations as oral vaccine delivery systems

PT

reproducibility and large scale manufacturing

RI

As many antigens are peptides, they are exposed to same barriers as »classic peptides« as

SC

presented above. To illicit an immune response and achieve mucosal immunization, orally administered antigens must reach M-cells, which represent only about 1% of intestinal

NU

epithelial cells and lie below the level of villous tips [204]. Thus, repeated high doses of

MA

antigens must be applied to achieve an immune response. However, such methods usually do not induce systemic immunity, but may lead only to a short-lived secretion of antigen-specific

D

IgA and the development of systemic tolerance. In addition, high doses of antigens may cause

PT E

severe allergic reactions and toxic effects [204,205]. However, effective immunization with low doses of antigens is feasible by co-administration of adjuvants, which enhance the immune response. Certain LBF have shown to be versatile adjuvants with an excellent safety

CE

profile. In addition, there are already some examples of marketed vaccines containing LBF as

AC

adjuvants such as emulsions and liposomes as well as immune-stimulating complexes (ISCOMs) [206,207]. Emulsions usually contain squalene oil as adjuvants, whereas ISCOMs resemble liposomes, being composed of cholesterol, phospholipids and saponine adjuvants, in particular Quil-A or QS-21 [206]. Although limited data on oral delivery of peptide vaccine are available [208,209], peptide lipidization might be a promising approach in achieving mucosal and systemic immunity. For instance, covalent lipidization of a peptide antigen derived from the E7 oncoprotein of human papillomavirus type 16 on with palmitic acid resulted in significantly increased antigen entrapment efficiency in liposomes, from initial

ACCEPTED MANUSCRIPT 27.1% to 94.1%, respectively [210]. Hence, incorporation of peptides in such LBF would

PT

provide the added value of immune response.

10. Concluding remarks

RI

Oral peptide delivery is still an ongoing challenge due to several physiological barriers

SC

including presystemic metabolism by thiol-disulphide exchange reactions and proteolysis as well as poor intestinal membrane permeability. However, it seems the key to successfully

NU

overcome these barriers lies in sufficient peptide lipophilicity. In this review, systematically

MA

structured ways to increase peptide lipophilicity were presented. From drug delivery point of view, the lipidization via non-covalent hydrophobic ion pairing and incorporation in LBF

D

seems to bare the highest potential due to its reversibility, simplicity and efficiency. In

PT E

addition, covalent methods also offer efficient ways in protecting peptides against presystemic metabolism and increasing intestinal permeability. Moreover, a simultaneous combination of

AC

the lipid core.

CE

two or more different lipidization strategies may lead to a higher peptide or protein payload in

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Graphical abstract