Advances in oral macromolecular drug delivery.

Review

Advances in oral macromolecular drug delivery Seung Rim Hwang & Youngro Byun† Introduction

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Strategies to improve oral bioavailability of macromolecules

Expert Opin. Drug Deliv. Downloaded from informahealthcare.com by Mcgill University on 11/19/14 For personal use only.

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Conclusion

4.

Expert opinion

Seoul National University, College of Pharmacy and Graduate School of Convergence Science and Technology, Department of Molecular Medicine and Biopharmaceutical Sciences, Seoul, Republic of Korea

Introduction: Various macromolecules including polypeptides, proteins, genes and polysaccharides have been drawing attention for their therapeutic potential. The passage through intestinal epithelium is the major barrier for the oral delivery of macromolecules, by either paracellular or transcellular pathways. However, most macromolecules are poorly absorbed in oral route due to their high molecular weight and low stability in the gastrointestinal (GI) tract. Nonetheless, advancing in oral macromolecular drug delivery will be significant in expanding the clinical use of therapeutic macromolecules. Areas covered: Technologies using chemical conjugation, absorption enhancers and nano-/micro-particulate systems have been developed to improve oral bioavailability of macromolecules, and some of them are in the process of clinical trials. In this review, they are discussed in the context of their progression states, hurdles and modes of action. Expert opinion: According to the better understanding of receptor or transporter structure and transport mechanisms in the GI tract, the progress ineffective oral delivery systems for therapeutic macromolecules is anticipated over the next decades. In addition, the advent of numerous particulate systems will also speed up the development of novel drug delivery technologies. This offers an optimistic perspective on the potential clinical usage of oral macromolecular drugs. Keywords: absorption enhancer, chemical conjugation, macromolecular drug, oral delivery, particle Expert Opin. Drug Deliv. (2014) 11(12):1955-1967

1.

Introduction

Drugs in the intestinal lumen enter the cell through the brush-border membrane, diffuse into epithelial cells, permeate the basolateral membrane and then migrate into the adjacent capillary. Among passive paracellular diffusion, transportermediated active transport, facilitated transcellular diffusion and passive transcellular diffusion, the primary route of drug absorption through biological membranes is determined according to the physicochemical properties of drug molecules. Molecular weight (MW) of drugs is one of the determining factors for permeability in the pH-partition-hypothesis, affecting the gastrointestinal (GI) absorption [1,2]. Hydrophilic drugs with MW below 200 Da are mainly absorbed via the paracellular route across the small intestinal mucosa, partly via transcellular route. Substances with a MW of 500 -- 700 Da can be marginally transported by passive diffusion through small intestinal epithelial cells [3,4]. Macromolecules with greater MW than 700 permeate into the membrane via active transport rather than passive transport. The membrane permeability of drug is also dependent upon its lipophilicity or partition coefficient in tissue to be absorbed through passive diffusion [5]. Drugs with appropriate partition coefficient between the water and oil phase can easily pass through the biological membrane that is composed of the phospholipid bilayer. 10.1517/17425247.2014.945420 © 2014 Informa UK, Ltd. ISSN 1742-5247, e-ISSN 1744-7593 All rights reserved: reproduction in whole or in part not permitted

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S. R. Hwang & Y. Byun

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Most of macromolecules are poorly absorbed by oral route because of their high molecular weight and low stability in the GI tract. Advances in oral macromolecular drug delivery using chemical conjugation, absorption enhancers and particulate systems have significance for expanding the clinical use of therapeutic macromolecules. Chemical conjugation of a macromolecular drug to the small molecule can target intestinal transporters or receptor-mediated transport pathways, and alter the lipophilicity or permeability of macromolecules. Coadministration with absorption enhancers can increase lipophilicity and permeability of drugs mainly by modifying the tight junction barrier. Nano-/micro-particulate systems are usually transported to the intracellular site via M cells as well as enterocytes.

This box summarizes key points contained in the article.

Hydrophilic compounds with low partition coefficient pass through the membrane mainly via aqueous pores. However, since the mean radius of aqueous pores in the intestinal epithelium is about 4 A˚, macromolecules can hardly permeate the membrane compared to small molecules such as water, urea, methanol and formamide. In addition, peptidases in the GI tract act as enzymatic barriers to peptides and proteins [6]. The lumen of the small intestine and the brushborder membrane of the epithelial cells contain a large quantity of peptidases that can degrade peptides and proteins. Thus, the oral absorption of macromolecules such as polypeptides, proteins, genes and polysaccharides is limited by their poor permeability across physiological barriers [7,8]. However, since the administration of therapeutic macromolecules via the oral route is convenient for long-term treatment for patients and significant for expanding the clinical use, there have been studies on elucidating transport mechanisms of macromolecules in the GI tract in order to create orally active preparations [9]. The passage through intestinal epithelium is the major barrier for the oral absorption of macromolecules, which occurs by either paracellular or transcellular pathways [10]. Paracellular transport of ions and larger solutes is restricted by the presence of narrow channels and tight junctions [11]. Tight junctions comprise a complex array of integral transmembrane proteins, such as claudins, occludin and junctional adhesion molecules along with several regulatory proteins anchoring the transmembrane proteins to cytoskeletal actin [12,13]. The resulting cellular sheets function as a barrier for the diffusion of solutes and recruit the protein kinase C required for claudin assembly. Paracellular transport electrophysiologically elucidated by measuring the degree and selectivity for transport of ions and solutes shows enormous differences in electrical resistance among epithelia and small differences in ionic selectivity [14,15]. Transcellular transport is started by the internalization of drugs into epithelial cells, 1956

followed by release of drugs out of the basolateral membrane [16]. The intestinal epithelial cell layer comprises a variety of cell types including enterocytes, goblet cells and M cells [17]. The epithelial enterocytes play their roles in uptake of ion, water, sugar, amino acid or vitamin B12, and goblet cells can secrete the major component of mucus. M cells are contained in follicle-associated epithelium of Peyer’s patch, one of gut-associated lymphoid tissues, and transport soluble macromolecules or small particles as well as antigens from the lumen to cells of the immune system [18]. Meanwhile, peptide-type drugs such as b-lactam antibiotics and angiotensin-converting enzyme inhibitors can be transferred by the peptide transporter or carrier-mediated transport system for di-/tri-peptides [19]. Receptor-mediated transport is also useful for oral delivery of peptide or protein drugs with a modification in receptor-specific ligands [20]. It facilitates the internalization of substrates binding to specific cell-surface receptors through clustering of the substrate-receptor complexes in endocytotic vesicles [21]. It was observed that transport of insulin-transferrin conjugate across enterocyte-like Caco-2 cell monolayers increased by 5- to 15-fold compared to free insulin in both apical-to-basal and basal-to-apical directions [22]. Based on the better understanding of transport mechanisms for macromolecules to cross the intestinal membrane, various attempts were made to improve oral bioavailability of macromolecular drugs. Oral delivery technologies using chemical conjugation, absorption enhancers and nano-/micro-particulate systems have been reported, and some are in the process of clinical trials. They are discussed below in the context of their progression states, hurdles and modes of action.

Strategies to improve oral bioavailability of macromolecules

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Chemical conjugation Chemical modifications such as cross-linking, acylation, PEGylation and glycosylation have been applied to therapeutic macromolecules to improve oral bioavailability (Table 1). One type of the strategies involves chemical conjugation of a drug to the small molecule that targets intestinal transporters or receptor-mediated transport pathways [21,23]. Cellular transport systems such as the intestinal bile acid transporter, glucose transporter, di-/tri-peptide transporters, Fc transporters, and vitamin B12 receptor have been explored for oral delivery of macromolecules [24-26]. It was reported that low molecular weight heparin (LMWH) conjugated with deoxycholic acid (DOCA) derivative demonstrated the enhanced oral bioavailability in rodents (7.8% at the 20 mg/kg dosage) and monkeys (6.8% after administrating 200 mg/kg of free DOCA) as compared to the unmodified LMWH (0.8%) [27,28]. A series of LMWH-DOCA conjugates were synthesized according to the degree of substitution, and their physicochemical properties and oral bioavailabilities were assessed [29]. They are assumed to target apical sodium-dependent 2.1

Expert Opin. Drug Deliv. (2014) 11(12)

Advances in oral macromolecular drug delivery

Table 1. Chemical conjugation strategies to improve oral bioavailability of macromolecular drugs. Systems

Applied drugs

Progression states

Features

Ref.

DOCA conjugate Protein s1-polylysine Vitamin B12-insulin CPP conjugate

Heparin DNA Insulin Gastrin Insulin GLP-1 Paclitaxel Insulin

Preclinical Preclinical Preclinical Preclinical Preclinical Preclinical Preclinical Discontinued after Phase III Preclinical Preclinical Preclinical

Targeting ASBT Targeting M cells of intestinal Peyer’s patches Vitamin B12 uptake pathway Transcellular transport

[27-33] [34] [35] [36-39]

Enhancing solubility and mucoadhesive property Enhancing stability against intestinal degradation and direct absorption into portal circulation

[40-44] [45-49]

Enhancing stability Enhancing lipophilicity and reducing degradation

[50] [51-53]

PEG conjugate Fatty acid conjugate

hBNP Antibody Insulin

ASBT: Apical sodium-dependent bile salt transporter; CPP: Cell-penetrating peptide; DOCA: Deoxycholic acid; GLP-1: Glucagon-like peptide-1; hBNP: Human brain-type natriuretic peptide.

Streptavidin pull down

38 kDa

p-Tyr ASBT ASBT GAPDH

C.

ASBT

- +

+

+

IP: ASBT Whole

Membrane

Cytoplasm

tra

38 kDa

IP: ASBT

Cell lysate

B. D

A. C on LH ete


Chitosan conjugate Amphiphilic oligomer conjugate

LHe-tetraDbiotin

98 kDa 64 kDa 50 kDa 38 kDa

Heavy chain ASBT

22 kDa

* Light chain

Cell lysate

Biotin ASBT Biotin

Cell lysate LHe-tetraD

Merge

Figure 1. Physical interactions of ASBT and LHe-tetraD in cells. A. Immunoprecipitation-immunoblot analysis for p-ASBT (IP-ASBT, IB-pTyr) and the total ASBT (IP-ASBT, IB-ASBT) from the non-treated and LHe-tetraD-treated MDCK cells transfected with ASBT gene (MDCK-ASBT). B. Biotin-labeled LHe-tetraD was incubated with MDCK-ASBT cells and pulled down by streptavidin beads from membrane and cytoplasmic fraction as well as from the whole cell lysates, followed by immunoblotting for bound ASBT protein and biotin. The control included the ASBT band from lysates and the immunoprecipitates of the non-treated MDCK-ASBT cells. Asterisk indicates nonspecific band. C. The co-localization of LHe-tetraD (red) and ASBT (green) was also visualized in MDCK-ASBT cells. Scale bar indicates 20 µm. Reproduced with permission from [33]. ASBT: Apical sodium-dependent bile salt transporter; LHe-tetraD: The conjugate of low molecular weight heparin with tetrameric deoxycolic acids; MDCK: MadinDarby canine kidney.

bile salt transporter (ASBT), leading to the transmembrane transport of LMWH (Figure 1) [30]. ASBT usually maintains enterohepatic circulation of bile salts by moving its endogenous small molecular substrates from the apical site toward

the basolateral site of enterocytes and cholangiocytes [31,32]. When the high-affinity oligomeric bile acid conjugate binds to the extracellular pocket in ASBT, the ‘receptor-like’ functional transformation of ASBT can be induced, and ASBT/


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Control

LHe-tetraD treatment

A.

B.

C.

D.

E.

ASBT


F.

Drug treatment

Figure 2. LHe-tetraD stimulates ASBT internalization in vesicles. The vesicular transport of ASBT (A -- E) as illustrated by the schematic representation (F) was observed by transmission electron microscopy. ASBT was detected (arrow) in the membrane of MDCK-ASBT cells as gold marks (A). Following the LHe-tetraD treatment, the gold marks appeared in distinctively concaveshaped membrane curvatures (B), in vesicles near the membrane (C), and in the multivesicular bodies (asterisks) in the cytoplasm (D and E). Scale bar indicates 200 nm. Reproduced with permission from [33]. ASBT: Apical sodium-dependent bile salt transporter; LHe-tetraD: The conjugate of low molecular weight heparin with tetrameric deoxycolic acids; MDCK: MadinDarby canine kidney.

substrate complexes can be internalized in vesicles to move into the cytoplasm (Figure 2) [33]. Receptor-mediated transport pathways can be used for the improvement of oral macromolecular delivery. Reovirus, one of the enteric pathogens, is attached to M cells of intestinal Peyer’s patches to infect the host, which is mediated by protein s1. The recombinant fusion protein s1, covalently coupled to polylysine complexed with DNA, could successfully transfect cells expressing the receptor for protein s1 in vitro and be bound to lymphoid tissues with M cell structure ex vivo [34]. Meanwhile, the vitamin B12 conjugation was also applied to the oral delivery of macromolecules, and the vitamin B12-insulin conjugate showed a 4.7-fold greater reduction in the area under the blood glucose curve compared to free insulin [35]. Since insulin is susceptible to enzymatic degradation and poorly uptaken by oral-enteric way, the vitamin B12 uptake pathway can be an alternative for noninvasive delivery of insulin. Peptide-based ligands including cellpenetrating peptides (CPPs) are also applicable to noninvasive delivery of macromolecules [36]. Gastrin, insulin or glucagonlike peptide-1 (GLP-1) can bind to D-form arginine octamer, leading to the increased intestinal absorption in the in situ study [37]. When CPPs are covalently bound to oligonucleotides, proteins or nanoparticles, they facilitate the transcellular transport of conjugates across the phospholipid bilayer of the cell membrane [38]. It was reported that the in vitro intestinal absorption of CPP linked to insulin was 6 -- 8 times increased compared to normal insulin without damaging Caco-2 cell monolayer [39]. Importantly, insulin hybridized with CPP kept intact after passing through the cell monolayer. Chemical conjugation also contributes to the improvement of drug solubility in the intestinal media. The conjugation of low molecular weight chitosan (LMWC) to drug enhanced its water solubility and oral bioavailability [40]. Orally administered LMWC-paclitaxel conjugate (5 mg paclitaxel/kg) with 1958

the enhanced water solubility showed 42% of oral bioavailability. Tracking I (125)-labeled conjugate revealed that the conjugate was mainly absorbed in the ileum, reaching the blood circulation as intact form. Orally administered polymer-drug conjugate, especially chitosan-conjugated drug with mucoadhesive property, has been evaluated for improving therapeutic efficacy and reducing side effect [41,42]. Chitosan has positively charged amino groups, and its solubility according to pH values limits its bioactive properties [43]. It can be chemically modified in order to improve its properties for oral delivery, such as trimethyl-chitosans, thiolated chitosans, PEGylated chitosans or chitosan-enzyme inhibitor conjugates [44]. In addition, the chemical conjugation can alter the lipophilicity and permeability of macromolecules. Hexyl-insulin monoconjugate 2 (HIM2; Nobex Corp., NC) was exploited using covalent linkage of an amphiphilic oligomer to the Lys-b29 residue of recombinant human insulin, although currently abandoned after Phase III clinical trials [45]. Oral HIM2 showed the enhanced stability against intestinal degradation and direct absorption into the portal circulation, leading to the fast and effective suppression of glucose production [46]. In another group, human brain-type natriuretic peptide (hBNP) conjugated with several amphiphilic oligomers was designed for the oral treatment of heart failure and hypertension [47,48]. The activity of conjugates was related to the region-selectivity of conjugation, which was affected by the hydrophobic/hydrophilic balance of oligomers. Orally administered two-arm branched PEG-hBNP (350 µg/kg) activates cyclic guanosine monophosphate, the second messenger of BNP, and significantly decreased the mean arterial pressure, contrary to oral native hBNP [49]. Besides, conjugation of antibodies to PEG has been reported to improve stability of orally administered antibodies against proteolytic degradation [50]. There were reported numbers of additional studies using chemical conjugations for the oral delivery of macromolecules.



Non-covalent drug-enhancer complex formation Paracellular route

Transcellular route


Mucus layer

Figure 3. Schematic illustrations of the transport of macromolecular drugs associated with absorption enhancers across the intestinal epithelial cells.

Absorption characteristics of insulin derivatives modified with fatty acids after the large intestinal administration showed the improvement according to the number of fatty acid molecules attached to insulin [51-53]. When administered into in situ large intestinal loops of rats, 125I-labeled palmitoyl insulin exerted a sixfold increase in the maximal plasma radioactivity compared to native insulin. It may be due to the increased lipophilicity and reduced degradation of insulin in palmitoyl insulin. Tetragastrin conjugated with caproic acid that was more stable than the native tetragastrin in homogenates of intestine, liver or plasma slightly inhibited the hepatic first-pass metabolism. Absorption enhancers Coadministration with absorption enhancers such as bile acid/salt derivatives, sodium caprate, chitosan derivatives, labrasol, thiomers and sodium N-[8-(2-hydroxybenzoyl) amino] caprylate (SNAC) has been reported to increase lipophilicity or permeability of hydrophilic drugs by noncovalent complex formation [54-57]. It is mainly due to the enhancement of paracellular drug transport with the tight junction between intestinal epithelial cells (Figure 3) [58]. On the other hand, some enhancers have been reported to alter the intracellular drug transport by inducing the solubilization of membrane lipid and protein or membrane perturbation. Since absorption enhancers lower the barrier function of mucous membrane that can selectively absorb the essential elements and protect from foreign substances, there is a need for 2.2

a development of safe enhancers that cause no or slight temporary damage to the biological membrane. Enhancers also must be fast-acting and continue their effects for a time. However, the small intestinal absorption has difficulty in maintaining the enhancer concentration, because the enhancer is dispersed and diluted in the large volume of intestinal fluid. In addition, the oral bioavailability of drug in human clinical trials might be significantly different from that in animal studies at the same dose of enhancer. Although there have been obstacles including lack of intersubject reproducibility in efficacy and safety concerns, various attempts were made to develop absorption enhancers (Table 2) [59]. Bile acid/salt derivatives have long been in interest as absorption enhancers. Bile salts can mask the hydrophilic surface of drugs or solubilize fatty acids via micelle formation [60,61]. Further, ASBT in the ileum can be targeted for enhancing oral absorption of oral macromolecular drugs that are physically complexed with bile acid/ salt derivatives [62,63]. The physical complexation of insulin with Na-deoxycholyl-L-lysyl-methylester, one of the derivatives of bile acid salts, can protect insulin from the enzymatic degradation and be absorbed in the intestine of streptozotocin-induced diabetic rats [64]. When anionic LMWH and cationic deoxycholylethylamine were physically associated by ion-pair interactions, the complexation was saturated above a molar ratio of 1:10, and improved the lipophilicity and oral absorption of LMWH (oral bioavailability = 3.08% at 50 mg/kg of the complex compared to 0.12% at 50 mg/kg of native LMWH) through the rat intestine without causing tissue damage [65]. Fatty acids such as sodium caprate were also evaluated as an oral absorption enhancer for hydrophilic macromolecules. Sodium caprate showed the enhanced in vitro Caco-2 permeability (1.97-fold at 0.0625% compared to control) and oral bioavailability of ardeparin in rats (27% at 1200 IU/kg ardeparin with 100 mg/kg sodium caprate compared to 11% at ardeparin without enhancer) [66]. Its main mechanism of action lies in the paracellular transport by dilatations of the tight junctions. Although there had been concern about the local toxicity of sodium caprate, histological examination of GI tissues after single oral administration (100 mg/kg of sodium caprate) had no evidence of damage. Chitosan derivatives such as mono-N-carboxymethyl chitosan (MCC) and N-sulfonatoN,O-carboxymethyl chitosan (SNOCC) could increase the paracellular permeation and intestinal absorption of LMWH in rats [67,68]. MCC, a polyampholyte chitosan derivative, significantly decreased the transepithelial electrical resistance on Caco-2 monolayers and increased the permeability of LMWH (enhancement ratio > 25 compared to control). When intraduodenally administered to rats, MCC significantly increased the absorption of LMWH (sevenfold and 5.4-fold increase in area under the curve and serum peak concentration at LMWH with 3% [w/v] of low viscous MCC compared to LMWH without enhancer) to reach the therapeutic window. MCC formed a gel with LMWH, which provided sustained release of LMWH. Water-soluble anionic


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Table 2. Absorption enhancers to improve oral bioavailability of macromolecular drugs. Systems

Applied drugs

Progression states

Bile acid/salt derivatives

Insulin Heparin Heparin

Preclinical Preclinical Preclinical

Heparin Insulin Heparin LacZ gene

Preclinical Preclinical Preclinical Preclinical

Insulin GLP-1 Heparin

Phase II Preclinical Discontinued after Phase III Preclinical Phase III

Sodium caprate Chitosan derivatives Thiomers b-cyclodextrins


Hydrophilic aromatic alcohols SNAC

Combination of fatty acids and chitosans

PYY Octreotide

Features

Ref.

Masking hydrophilic surface and targeting ASBT Paracellular transport by dilatation of tight junctions Increasing paracellular permeation Mucoadhesive properties

[60-65]

Enhancing adenoviral-mediated gene transfer in the intestine Increasing paracellular permeation via opening of tight junctions Non-covalently interacting with the intestinal mucosal lining

[70]

Facilitating a transient paracellular passage of drug

[77-80]

[66] [67,68] [69]

[71-73] [74-76]

ASBT: Apical sodium-dependent bile salt transporter; GLP-1: Glucagon-like peptide-1; lacZ: The Escherichia coli b-galactosidase gene; PYY: Peptide tyrosine tyrosine; SNAC: Sodium N-[8-(2-hydroxybenzoyl) amino] caprylate.

SNOCC derivatives were also assessed for their ability to enhance the intestinal absorption of reviparin. Formulations containing Labrasol was reported to increase the in situ intestinal absorption of LMWH, when administered to jejunum of the fasted rats. Thiolated polymers with mucoadhesive properties also can improve the permeation of macromolecules such as insulin and LMWH [69]. Besides, positively charged b-cyclodextrins were reported to increase adenoviral-mediated transfer of the Escherichia coli b-galactosidase gene in the intestine by enhancing binding and internalization of viral particles [70]. Currently, some absorption enhancers have emerged as key components for oral macromolecular delivery in advanced preclinical or clinical development. Diabetology (Jersey, UK) completed Phase II clinical trials of an enteric-coated capsule of insulin-containing hydrophilic aromatic alcohols as the absorption enhancer [71]. The absorption enhancer used in the formulation has been generally recognized as safe in pharmaceutical practice for many years, and it presented low toxicity profile during 10-day repeated oral dosing in patients with insulin-independent type 2 diabetes [72]. Oral delivery of GLP-1 has been investigated using the same technology [73]. At a relatively high local concentration, aromatic alcohols are postulated to increase paracellular permeability across the intestinal epithelium via opening of the tight junctions between intestinal epithelial cells. Peptide tyrosine tyrosine (PYY), a peptide released from intestinal cells in response to feeding, is also rapidly degraded in the upper GI tract. It can be formulated with SNAC, an oral absorption enhancer developed by Emisphere Technologies, Inc. (Roseland, NJ) [74]. Moreover, coadministration of GLP-1 and PYY mixed with SNAC to mimic endogenous secretion of the peptides had an additive inhibitory effect on appetite in humans [75]. It is thought that hydrophobic 1960

SNAC non-covalently associated with peptides improves their absorption across the GI epithelium. After the absorption, the peptides disassociated from the SNAC carrier freely pass into the circulation and provide their pharmacological profiles that mimic the physiological release of gut hormones [76]. Meanwhile, hypersecretion of growth hormone can be normalized by long-acting analogs of somatostatin such as octreotide [77]. Oral octreotide using transient permeability enhancer technology (Chiasma, Jerusalem, Israel) performed pharmacokinetics and efficacy comparable to subcutaneous octreotide in human [78]. Combining octreotide, fatty acids, chitosans and other excipients was shown to facilitate a transient paracellular passage of octreotide across the GI wall [79]. Except for the impairment of its absorption by food or proton pump inhibitors, oral octreotide has advantage in the longterm treatment of acromegaly and is now proceeding through Phase III clinical trials [80].

Nano-/micro-particulate systems A variety of nano-/micro-particulate vehicles have been investigated to protect the loaded macromolecules from enzymatic or hydrolytic degradation and increase the intestinal absorption (Table 3) [81-83]. Particles are transported to the intracellular site mainly via M cells as well as enterocytes (Figure 4), and drug delivery efficiency across the intestinal epithelium is related to particle size [84-86]. In general, the uptake efficiency of 100 nm-sized particles is much higher than microparticles because of diffusion throughout the submucosal layers. On the other hand, microparticles are localized in the epithelial lining of the tissue, and exclusively uptaken by the Peyer’s patch. The particle uptake also can be affected by the stability of particles in the small intestine, surface hydrophobicity or charge of particles, and specific ligands. 2.3



Table 3. Nano-/micro-particulate systems to improve oral bioavailability of macromolecular drugs. Systems

Applied drugs

Progression states

Features

Ref.

CS/g-PGA nanoparticles

Insulin Exendin-4 Insulin

Preclinical

[87-90]

Preclinical

Insulin

Phase II

Cobalamin-coated vitamin B12 nanoparticles

Insulin Growth hormone

Preclinical Preclinical

Silica nanoparticles Gold nanoparticles BEODAS

Insulin Insulin Heparin

Phase II Phase I Preclinical

Gagomeric microparticles

Insulin

Preclinical

Microparticles coacervated with gelatin

Heparin

Preclinical

Protecting drugs from enzymatic or hydrolytic degradation Adsorptive transcytosis in epithelial cells and Peyer’s patches Targeting hepatocytes and protecting drugs from the degradation Uptake of nanoparticles linked to vitamin B12 through receptor-mediated endocytosis Nanoparticles have hydrophobic surfaces Rapid onset of activity The inert polymer protects drugs from degradation Targeting to the intestinal mucosa using mucoadhesive hyaluronan Complex coacervation compensates the strong negative charge of drugs


Dextran sulfate/chitosan or alginate/chitosan HDV-I

[91,92] [96] [97,98]

[99] [100] [102] [103,104] [105]

BEODAS: Bio-erodible enhanced oral drug absorption system; CS/g-PGA: Chitosan and poly(g-glutamic acid); HDV-I: Hepatic-directed vesicle insulin.

Particulate system

Enterocyte

M cell Enterocyte

Peyer’s patch

Figure 4. Schematic illustrations of M cell-mediated transport of drug-loaded particulate systems in Peyer’s patches.

Chitosan-based nanoparticles have long been considered as promising vehicles for the oral macromolecular drug delivery. An enteric-coated capsule filled with pH-responsive nanoparticles composed of chitosan and poly(g-glutamic acid) (CS/g-PGA) was reported to be capable of increasing the oral bioavailability of insulin (20% compared to native insulin) and providing a prolonged reduction of blood glucose levels in diabetic rats [87]. The in vivo toxicity study indicated that these self-assembled nanoparticles were shown to be safe at an 18-fold higher dose than that for pharmacodynamic/ pharmacokinetic studies [88]. The CS/g-PGA nanoparticles could also be used for oral delivery of insulin aspart, a rapid-

acting insulin analog [89]. Insulin aspart loaded nanoparticles via the oral route exerted the prolonged pharmacodynamic effects contrary to subcutaneous insulin aspart. An oral formulation of exendin-4, a 39-amino acid peptide in the saliva of the Gila monster, was developed using an enteric-coated capsule containing pH-responsive chitosan nanoparticles [90]. Dextran sulfate/chitosan or alginate/chitosan nanoparticles were also evaluated for oral delivery of insulin [91,92]. Confocal microscopic studies using fluorescein-labeled insulin nanoparticles revealed the adhesion and internalization of nanoparticles into the rat intestinal mucosa. Chitosan nanoparticles can undergo transcellular uptake via adsorptive transcytosis,


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and be found in both epithelial cells and Peyer’s patches [93]. Transport of these nanoparticles is influenced by physicochemical properties of nanoparticles, such as their particle size and surface properties [94,95]. Hepatic-directed vesicle insulin (HDV-I; Diasome Pharmaceuticals Inc., Cleveland, OH) was also developed as an oral gelcap nanoformulation, and Phase II clinical trial had been completed. Insulin bound to HDV is selectively targeted to the hepatocytes by biotin-phosphatidylethanolamine in phospholipid bilayer of the vesicle and protected from the degradation in the upper GI tract [96]. Insulin-loaded nanoparticles coated with cobalamin form of vitamin B12 (Access Pharmaceuticals, Inc., Dallas, TX) were also investigated for enhancing the uptake capacity of vitamin B12 transport and oral absorption of insulin [97]. Preclinical data demonstrated that oral administration of cobalamin-coated nanoparticles containing insulin resulted in the lowering of blood glucose levels (70 -- 75%) and prolonged antidiabetic activity (54 h) [98]. The same technology has been adapted to the oral formulation of human growth hormone for the treatment of growth hormone deficiency. The uptake mechanism of vitamin B12 through receptormediated endocytosis is utilized in the transport of nanoparticles linked to vitamin B12, and then the loaded drug is released from the nanoparticles. Besides, there are other insulin-loaded nanoparticles explored in clinical trials. Oshadi drug administration Ltd. (Rehovot, Israel) completed Phase I clinical trials of oral insulin based on nanoparticulate system for the treatment of type 1 diabetes [99]. Its oral insulin is composed of insulin incorporated into the oil phase, silica nanoparticles with hydrophobic surfaces and branched polysaccharides. Currently, it has been in the process of a 4-week, multiple-dose, nonrandomized, open-label Phase II study. Midatech (Oxford, UK) and MonoSol Rx (Warren, NJ) have developed transbuccal insulin stabilized on biocompatible gold nanoparticles and formulated into a thin polyethylene-oxide-based film [100]. With rapid onset of activity and safety profile compared to native insulin, Phase I clinical trials of transbuccal insulin have been successfully completed. Meanwhile, there were several studies using polymeric nanoparticles for the oral delivery of heparin. Nanoparticles prepared with biodegradable poly-"-caprolactone and nondegradable positively charged Eudragit RL facilitated the oral absorption of loaded heparin in rabbits [101]. In addition, oral formulation of LMWH was also investigated by the bio-erodible enhanced oral drug absorption system (BEODAS; Elan Corp PLC, Dublin, Ireland) [102]. BEODAS can be achieved through the entrapment of LMWH in a range of sub-micrometer sizes within a biodegradable polymer matrix. The inert polymer protects the loaded drug from degradation and improves oral absorption through increased surface area and altered chemical properties. Microparticles have also been developed as an oral delivery system for macromolecules. Gagomeric insulin (Ramot At 1962

Tel-Aviv University Ltd., Tel-Aviv, Israel) is the lipidated microparticle formulation that has hyaluronan on its surface, and insoluble fibrils of insulin are encapsulated in the microparticles with high loading efficiency [103]. Fibrillar insulin provides stability in the GI environment compared to the soluble monomeric form, and it should be dissociated into active monomers thereafter. In vivo studies in diabetic mice showed stable glucose reductions following a single oral dose of fibrillar insulin formulation. Insulin-loaded gagomers can be targeted to the intestinal mucosa using mucoadhesive properties of hyaluronan [104]. Meanwhile, LMWH-loaded microparticles coacervated with gelatin were also investigated for cell binding on Caco-2 and in vivo oral absorption [105]. The strong negative charge of LMWH (tinzaparin) could be compensated by complex coacervation. An optimized tinzaparin microparticle showed mono-dispersed size distribution, high encapsulation rates (> 90%) and increased oral bioavailability (4.2 ± 2.9%). Drug release rate depended on pH of the environment triggering the dissociation of tinzaparin/acacia gum mixture and gelatin. 3.

Conclusion

Most of the therapeutic macromolecules including polypeptides, proteins, genes and polysaccharides have low oral bioavailability because of their high MW and low stability in the GI tract. On the other hand, oral delivery technologies using chemical conjugation, absorption enhancers and nano-/ micro-particulate systems have been in the process of preclinical or clinical trials. Based on the elucidated transport mechanisms in the GI tract, the effective and safe oral delivery systems for therapeutic macromolecules have been progressively developed. It has significance for the potential clinical usage of oral macromolecular drugs. 4.

Expert opinion

There has been a recent upsurge in demand for orally active preparations of therapeutic macromolecules. However, macromolecular drugs could be hardly absorbed in the GI tract because of their physicochemical properties and enzymatic degradation in the intestine. A variety of oral delivery systems using chemical conjugation, absorption enhancers and nano-/ micro-particulate systems have been designed and validated for their enhancing capacity of oral macromolecular absorption. Chemical conjugation approaches enable conjugated drugs to be recognized by receptor or transporter, and offer the increased solubility or amphiphilicity to drugs. Chemical modifications such as cross-linking, acylation, PEGylation and glycosylation have been applied to therapeutic macromolecules to improve oral bioavailability. Intestinal transporters or receptor-mediated transport pathways have been targeted by chemical conjugation of a drug to the specific small ligand. LMWH-DOCA conjugates, vitamin B12-insulin conjugates




and peptide-based ligands including CPPs were reported. HIM2 was one of the Phase III pipeline candidates using chemical conjugation technologies. Using covalent linkage of an amphiphilic oligomer to the macromolecular drug enhanced the intestinal absorption and stability against intestinal degradation, leading to the comparable or superior efficacy to the original injectable formulations. The therapeutic potential of various absorption enhancer systems has also been explored in spite of hurdles related to reproducibility and safety concerns. Bile acid/salt derivatives, sodium caprate, chitosan derivatives, labrasol, thiomers and SNAC have been reported to enhance the intestinal absorption of hydrophilic drugs by non-covalent complex formation. The main mechanism of action appears to be the enhancement of paracellular drug transport by modification of the tight junction between intestinal epithelial cells. Currently, the enteric-coated capsule of insulin containing hydrophilic aromatic alcohols as the absorption enhancer and oral octreotide using transient permeability enhancer technology are in the process of clinical trials. In the cases of oral enhancer system, the intestinal lumen volume should be considered. Since the intestinal lumen volume in human is much larger than those of rodents and monkeys, the oral bioavailability of drug in clinical trials might be significantly different from that in animal studies at the same dose of enhancer. Nano-/micro-particulate vehicles can protect loaded macromolecules from degradation and increase the oral absorption of macromolecules through transcytosis into epithelial cells and M cells. The particle uptake into the intestine is dependent on the stability, size, surface hydrophobicity and surface charge of particles. Chitosan-based nanoparticles and cobalamin-coated vitamin B12 nanoparticles have been investigated for the oral macromolecular drug delivery. HDV-I, one of oral gelcap nanoformulations, has completed its Phase II clinical trial. Besides, there are several insulinloaded nanoparticles explored in clinical trials. Oshadi is going on Phase II study of oral insulin, which is composed of insulin incorporated into the oil phase, silica nanoparticles with hydrophobic surfaces and branched polysaccharides. MidaSol Therapeutics, a joint venture between Midatech and MonoSol Rx, has successfully completed Phase I clinical trials of transbuccal insulin, which is stabilized on biocompatible gold nanoparticles and formulated into a thin polyethylene-oxide-based film. In a range of sub-micrometer sizes, BEODAS can be achieved through the entrapment of LMWH within a polymer matrix. Meanwhile, microparticles have also been developed as oral delivery systems for macromolecules. Insoluble fibrils of insulin can also be encapsulated in the lipidated microparticle formulation called as ‘Gagomeric insulin’ that has hyaluronan on its surface. In the cases of nano-/micro-particulate system, it would be important to find a strategy to be effectively endocytosed by the specific binding of drug carriers to the epithelial cell

membrane. The use of nanoparticles for macromolecular delivery is also faced with difficulties due to high dose required to achieve a desired efficacy and uncontrollability of the absorbed drug amount. Besides strategies described in this review, oral insulin and oral GLP-1 are also under development by companies like Extrawell (Changchun, China) and Novo Nordisk (Bagsvaerd, Denmark). Extrawell completed Phase I and Phase II clinical trials of oral insulin in capsule form for the treatment of type 2 diabetes in 2006 and is planning on conducting an extended clinical trial. Its oral insulin comprising insulin dissolved in acidic buffer, surfactant and oil can resist degradation by digestive enzymes in GI tract and be easily absorbed. An acylated GLP-1 analog developed by Novo Nordisk was approved for the treatment of type 2 diabetes in Europe and USA. Exhibiting an extended in vivo half-life and significant weight loss in obese patients, it is also in the process of Phase III clinical trials for antiobesity therapies. The ultimate goal is that potential candidates for the oral delivery of therapeutic macromolecules could be successful on the market and be used as a safe and effective long-term treatment option. To achieve this goal, structural information on membrane receptor or transporter in the intestine and detailed mechanisms for ligand-binding and translocation are needed. For example, the crystal structure of ASBT revealed the size of substrate-binding cavity bigger than taurocholate, and stronger substrates of bile acid transporter could be designed and attached to macromolecules for enhancing oral absorption. The detailed mechanism study suggested that a DOCA-based tetramer conjugated to LMWH induced internalization and redistribution of ASBT. The proposed transport mechanism will be further applied to many other macromolecules for their ASBT-mediated delivery. In addition, the advent of numerous particulate systems also speeds up the development of novel drug delivery technologies. Drug delivery efficiency of particulate systems might be closely related to the accessibility to target cell membranes and clearance by the reticuloendothelial system. For the potential clinical usage, the efficacy and safety of particulate systems should be thoroughly evaluated in preclinical studies. The authors anticipate the progress in effective oral delivery systems for therapeutic macromolecules over the next decades.

Declaration of interest This study was supported by research fund from Chosun University, 2013. The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties.


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Affiliation Seung Rim Hwang1 PhD & Youngro Byun†2 PhD † Author for correspondence 1 Assistant Professor, Chosun University, College of Pharmacy, Gwangju 501-759, Republic of Korea 2 Professor, Seoul National University, College of Pharmacy and Graduate School of Convergence Sciences and Technology, Department of Molecular Medicine and Biopharmaceutical Sciences, Seoul 151-742, Republic of Korea Tel: +82 2 880 7866; Fax: +82 2 872 7864; E-mail: [email protected]

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