Pharmacokinetic, pharmacodynamic and ...

ADR-13019; No of Pages 14 Advanced Drug Delivery Reviews xxx (2016) xxx–xxx

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Pharmacokinetic, pharmacodynamic and biodistribution following oral administration of nanocarriers containing peptide and protein drugs☆ Brendan T. Griffin a, Jianfeng Guo a, Elena Presas a, Maria D. Donovan a, María J. Alonso b, Caitriona M. O'Driscoll a,⁎ a b

School of Pharmacy, University College Cork, Cavanagh Pharmacy Building, Cork, Ireland CIMUS Research Institute, University of Santiago de Compostela, Santiago de Compostela, Spain

a r t i c l e

i n f o

Article history: Received 22 April 2016 Received in revised form 7 June 2016 Accepted 10 June 2016 Available online xxxx Keywords: Pharmacokinetics and pharmacodynamics of peptide and protein drugs Nanoparticles for oral administration Bioavailability Regulatory challenges Physiologically-based pharmacokinetic modelling

a b s t r a c t The influence of nanoparticle (NP) formulations on the pharmacokinetic, pharmacodynamic and biodistribution profiles of peptide- and protein-like drugs following oral administration is critically reviewed. The possible mechanisms of absorption enhancement and the effects of the physicochemical properties of the NP are examined. The potential advantages and challenges of physiologically-based pharmacokinetic (PBPK) modelling to help predict efficacy in man are discussed. The importance of developing and expanding the regulatory framework to help translate the technology into the clinic and accelerate the availability of oral nanoparticulate formulations is emphasized. In conclusion, opportunities for future work to improve the state of the art of oral nanomedicines are identified. © 2016 Elsevier B.V. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Absorption . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. The gastrointestinal mucus barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Mechanistic studies at the membrane/cellular level . . . . . . . . . . . . . . . . . . . . . . . . . . The influence of physicochemical properties of NPs on ADME profiles . . . . . . . . . . . . . . . . . . . . . 3.1. Size . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Charge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Surface polarity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. Bioadhesive properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Functional modification — targeted NPs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pharmacokinetic (PK) and pharmacodynamic (PD) data following oral administration of p/p loaded nanoparticles . 4.1. Nanoparticles formulated using biodegradable materials . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Nanoparticles formulated using inorganic materials . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.1. Silica Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2.2. Gold Nanoparticles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Biodistribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Role of the intestinal lymphatic system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Whole body distribution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Physiologically-based pharmacokinetic modelling (PBPK) as a predictive tool for oral administration of NPs . . . . Regulatory considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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☆ This review is part of the Advanced Drug Delivery Reviews theme issue on “SI: Oral delivery of peptides”. ⁎ Corresponding author. Tel.: +353 21 4901396; fax: +353 21 4901656. E-mail address: [email protected] (C.M. O'Driscoll).

http://dx.doi.org/10.1016/j.addr.2016.06.006 0169-409X/© 2016 Elsevier B.V. All rights reserved.

Please cite this article as: B.T. Griffin, et al., Pharmacokinetic, pharmacodynamic and biodistribution following oral administration of nanocarriers containing peptide and protein drugs, Adv. Drug Deliv. Rev. (2016), http://dx.doi.org/10.1016/j.addr.2016.06.006

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B.T. Griffin et al. / Advanced Drug Delivery Reviews xxx (2016) xxx–xxx

8. Conclusions and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Oral delivery of peptide- and protein-like drugs (p/p) offers many advantages in terms of being non-invasive, relative ease of administration and correspondingly higher patient compliance. Compared to parenterals, oral formulations are less expensive to produce and are amendable to self administration. In addition, in the case of insulin absorption via the intestine would be a more physiologically relevant route versus the routinely utilised subcutaneous (SC) administration. Following oral absorption insulin would be transported via the portal vein to the liver where it can regulate hepatic glucose production thus mimicking the natural endogenous fate of insulin in contrast to SC administration which delivers insulin into the peripheral circulation [1]. However, the oral route is a very hostile environment for p/p, as discussed in detail in a related chapter, and delivery systems including nanoparticles (NPs) can help overcome many of the barriers encountered following oral administration including enzymatic degradation and poor membrane permeability. The impact of nanocarriers on drug pharmacokinetics and pharmacodynamics (PKPD) can be significantly different from that observed

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with conventional formulations. Consequently a thorough understanding of the mechanism of absorption, distribution, metabolism and elimination (ADME) of NP formulations, (illustrated in Fig. 1), is essential in order to develop technologies to enhance oral p/p delivery, facilitate targeting and minimise off-target toxicity. The objectives of this review are: to examine how orally administered NP formulations can alter the PKPD profiles of p/p, and to provide insights into emerging in silico approaches that more may accurately interpret and predict PKPD of NPs. 2. Absorption The impact of nanocarrier delivery on oral absorption can generally be classified into three potential scenarios: (i) nanocarriers may be absorbed intact along with the cargo into the systemic circulation; (ii) nanocarriers fuse or directly interact with the intestinal membrane aiding the absorption of the cargo but without absorption of the nanocarrier; and (iii) the nanocarrier prematurely releases the cargo in the gut lumen prior to interacting with the intestinal membrane thus leading to degradation of the p/p. In the case of the first two

Fig. 1. Mechanisms of absorption, distribution, metabolism and elimination (ADME) of nanoparticle formulations following oral administration.



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scenarios, where the nanocarrier interacts with the intestinal membrane leading to enhanced uptake, the two major barriers to absorption are the mucus layer secreted by the goblet cells and the intestinal epithelial membrane.

in vitro models facilitate cell-based mechanistic studies, it is more challenging to perform these studies in vivo where the complex physiological environment is likely to influence the behaviour of the NPs.

2.1. The gastrointestinal mucus barrier

3. The influence of physicochemical properties of NPs on ADME profiles

The effects of mucus on intestinal absorption of NPs are extensively reviewed in a related chapter. In summary, the major impacts of mucus are related to the highly viscous nature which can reduce NP diffusion and the negative charge which may hinder transport of charged particles. Recently NP surface characteristics have been modified to: optimise mucoadhesion thus prolonging contact; increase gastrointestinal (GI) retention time; enhance mucus penetration thereby promoting cellular uptake in the GI tract [2–5]. 2.2. Mechanistic studies at the membrane/cellular level Following transport across the mucus layer the next significant barrier to absorption of NPs is the intestinal membrane. The extent of transcytosis will be influenced by cell adhesion, cellular uptake and intracellular trafficking and exocytosis. With respect to membrane transport, Jani et al. reported the uptake of latex particles and microspheres following oral administration to rats [6]. Uptake was reported to be related to size and charge. The mechanism of uptake was speculated to be via the M cells and the gut associated lymphatic system. However in the absence of lymphatic sampling studies this mechanism could not be confirmed [7]. In the ensuing studies, the concept of particulate uptake from the gastrointestinal tract (GIT) and the mechanisms by which transport across the membrane occurs have been the subject of much debate in the literature [8]. It is frequently reported that both phagocytic (M cells) and endocytic (enterocytes) pathways are involved in NP uptake from the GIT (Fig. 1). While it is widely acknowledged that NPs can be transported across the enterocyte membrane via endocytic pathways, it is also considered that the extent of transport is limited by the low levels of endocytic activity. In the case of M cell mediated uptake, transport may also be limited given the M cells constitute less than 5% of follicle-associated endothelium (FAE) and ~1% of total intestinal surface area [9]. In spite of these potential limitations, the increased understanding regarding phagocytosis and non-phagocytic transport mechanisms has been successfully exploited to design NP with the capacity to harness these transport mechanisms and enhance drug delivery. Uptake via the phagocytic route (M cells) has been relatively well characterised and numerous studies have described NPs designed to specifically target this pathway [10,11]. In contrast, endocytic uptake via enterocytes is a more complex process involving contributions from a range of mechanisms. In vitro mechanistic studies in cell culture models have been successfully employed to quantify the relative roles of the various uptake processes. The Caco-2 model [12], has been used to comparatively evaluated poly(lactic-co-glycolic acid) (PLGA) NP uptake via endocytosis, intracellular trafficking, exocytosis and transcytosis. Endocytosis of NPs was shown to involve clathrin, lipid raft/caveolae and macropinocytosis and also incorporated different proteins such as actins, protein tyrosine kinase (PTK) and cyclooxygenase (COX). Intracellularly, the NPs were transported to apical early endosome (AEE) and then onto lysosomes via the AEE/late endosome (LE)/lysosome pathway, as well as recycling to the endosome compartment (REC) or endoplasmic reticulum (ER) through the AEE/REC and AEE/ER pathways, respectively. Similar trafficking mechanisms were previously shown by [13] and are reviewed in detail in Chapter 6. Such well-designed mechanistic in vitro studies provide vital information to help elucidate intracellular trafficking and this can then be applied to optimise the transport of ligand targeted NP designed to enhance cell specific receptor mediated uptake (discussed below). While

3.1. Size NP size is known to influence uptake by M cells and enterocytes [14,15]. In addition, depending on size, NP uptake via the paracellular route may play a role, particularly if the NP material itself acts as a permeability enhancer by opening tight junctions (TJ). However, paracellular uptake is limited by the surface area of the TJ and by size restrictions of the TJ [16]. Recently a systematic study [17] using carboxylated chitosan grafted poly(methyl metacrylate) NPs, with sizes in the range of 300, 600 and 1000 nm and similar zeta potentials, was performed to investigate size-dependent absorption mechanisms. In vitro studies, using a range of techniques including Caco-2 monocultures and co-cultures with M cells, ex vivo and in situ rat ileum, and biodistribution in mice, indicated that these particular NPs were transported via a combination of routes including transcellularly (via both enterocytes and M cells) and paracellularly. The smaller particles were transported to a greater extent, by all routes, compared to the larger NPs due to a number of contributing factors including superior membrane adhesion and lower drug release in the gut lumen. Following oral gavage in mice the in vivo biodistribution confirmed the size dependent absorption predicted by the in vitro studies. Similar sizedependent absorption has previously been reported for biodegradable PLGA NPs in the Caco-2 model [14]. NP size will also influence the biodistribution (Fig. 2). For example, following uptake into the enterocyte and subsequent basolateral secretion into the interstitial space, larger NP may be selectively taken up by the mesenteric lymphatic vessels, leading to altered systemic distribution and potentially avoidance of first pass hepatic metabolism (as discussed in a subsequent section). For NPs that are taken up via the blood, NP size can influence the degree of clearance via the reticuloendothelial system (RES). Alternatively, passive targeting to tumours via the enhanced permeation and retention (EPR) effect can be exploiting by engineered particles within specific size ranges [18,19]. NP size may also impact elimination. Intact NPs may be cleared via either the kidneys or within bile. In the context of renally mediated clearance it is generally considered that filtration of NPs through the glomerular capillary wall—glomerular filtration—is highly dependent on size where it is estimated that b 6 nm are typically filtered, while those N8 nm are not typically capable of glomerular filtration [20]. The hepatobiliary system represents the primary route of excretion for particles that do not undergo renal clearance. A primary function of the liver is to efficiently capture and eliminate particles in the 10–20 nm, and therefore NPs in this scale often undergo rapid liver uptake [20].

3.2. Charge The charge on the NP may influence stability and thus promote aggregation in the gut luminal fluids, and in turn this may influence absorption. It should also be noted that the charge density on NP may be altered in contact with the physiological media. In addition, depending on the nature of the charge, electrostatic interaction with mucus may result in prolonged residence time, or alternatively entanglement with the mucus may hinder uptake [21]. Once absorbed into the systemic circulation charged NPs, especially cationic NPs, are known to interact with plasma proteins resulting in aggregation. In addition, larger charged NPs are likely to undergo enhanced clearance via the RES prior to uptake into target tissues [22].


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Fig. 2. (A) Schematic diagram illustrating the rate constant influencing absorption, distribution, metabolism and excretion following oral administration of peptide/protein loaded nanoparticles. (B) The table summarises the likely impact of nanoparticle characteristics on pharmacokinetic rate constants and the relevance (high, medium, limited) of these effects.

3.3. Surface polarity Pegylation has frequently been used as a means of enhancing NP stability. Pegylated or ‘stealth’ NPs can protect against aggregation in the gut lumen and may also decrease the extent of enzymatic degradation of p/p [23–25]. In addition, although pegylation has been reported to promote mucus penetration [3], the increased surface hydrophilicity may also result in a decrease in intestinal membrane permeability. In contrast, NP with lipophilic or amphiphilic surfaces may promote fusion with the enterocyte membrane, alter the membrane fluidity and thus enhance permeability. The potential of lipid-based nanocarriers are comprehensively reviewed in Section 4. Post-absorption, pegylated NPs have also been widely reported as a strategy for prolonged drug half-life and decreased clearance [26]. Proteins adsorbed on the surface of the NP promote opsonisation, leading to aggregation and rapid clearance via uptake by Kupffer cells in the liver. Typically, the majority of opsonised particles are cleared by a receptor-mediated mechanism in less than a few minutes due to the high concentration of phagocytic cells in the liver. In this regards surface pegylation offers the most widely recognised strategy to reduce protein adsorption on NP surface and therefore have reduced hepatic clearance [18]. 3.4. Bioadhesive properties Mucoadhesive materials have frequently been used to fabricate NPs for oral administration [27,28]. The advantages of bioadhesive NPs include an increased residence time in the intestine and a prolonged contact with intestinal surface membranes, which may collectively act to improve absorption. In contrast, however, mucoadhesive NPs may be compromised by the continuous turnover of mucus in the GIT [29,30]. A recent in vivo study has examined not only the effects of bioadhesion on absorption but also on distribution. In this study, attachment of a bioadhesive coating, poly(butadiene-maleic anhydride-co-Ldopa) (PBMAD), to non-bioadhesive polystyrene (PS) beads increased uptake, in an in vivo isolated rat jejunal loop, from 5.8 ± 1.9 to 66.9 ± 12.9 SEM % [31]. Following uptake the pattern of organ distribution varied markedly between NP formulations. The PS beads were mainly taken up by the liver. In contrast, with the bioadhesive NPs

uptake in the liver was negligible but relatively high in the blood, heart, lungs and spleen. As all NPs were similar in size (500 nm), surface characteristics rather than NP size influenced the biodistribution. This indicates that modification of surface properties may be useful as a method for passive targeting, and it remains to be demonstrated whether this strategy can be extended to enhance drug delivery to target tissue sites employing drug loaded NPs. 3.5. Functional modification — targeted NPs There are many advantages associated with targeted drug delivery. By matching a cell receptor with a suitable ligand, drug delivery to specific cell populations may be possible. Likewise if the disease microenvironment (e.g. tumour) is known, it may be possible to exploit this for disease specific drug delivery. Examples include the acidic microenvironment of a tumour and the application of pH sensitive ligands [32], and the EPR effect in tumours which can be exploited by formulating particles within specific size ranges. With respect to oral delivery of p/p surface modification of NPs with an appropriate targeting ligand, chosen to interact with a receptor in the intestinal membrane, may be a suitable method for enhancing membrane permeability and absorption of the intact NP. The challenges associated with this approach include identifying a suitably specific receptor, confirming the degree of expression, not only in the intestine, but also in other areas of the body. If the receptor is expressed in other tissues (e.g. the brain or lungs) potential exists for a targeted NP to distribute to these other organs following oral absorption. However the possibility also exists that interactions with off target tissue receptors may result in faster clearance. Targeted NPs may also be cleared by the RES. In addition, depending on the nature of the targeting ligand used, generation of an immune response may be an issue. However, in the latter case, such a response may be exploited for therapeutic effect as in the case of oral vaccines [33]. A range of targeting moieties has been investigated for oral delivery of NPs with encouraging improvements in PK and PD results (Table 1). Solid lipid NPs (SLNs) containing insulin (entrapment efficiency N60%) and modified with lectin were shown to protect insulin from enzymatic degradation following in vitro incubation with trypsin and pepsin. After oral administration to rats, blood glucose levels indicated



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Table 1 Targeted nanoparticles for oral peptide/protein drug delivery. NP composition

Targeting ligand

CSKSSDYQC peptide [CSK peptide] NPs of PGA-g-DA coated with CSKSSDYQC peptide TMC modified with CSK targeting goblet cells

Cargo

Pharmacokinetic (PK), pharmacodynamic (PD) and biodistribution (BD) data

Ref

Insulin

PK: Pharmacokinetic analysis of CSK-NPs showed a relative bioavailability (versus SC) of 7.05% which was slightly but significantly higher than 5.95% for the untargeted NP. PD: CSK-NPs produced a hypoglycemic response and decreased serum glucose level by 1.19 fold higher versus the untargeted NP. PK: F % of sCT CSK-SLNs and sCT IRQ-SLNs increased by 2.45-fold and 1.98-fold respectively compared with untargeted SLNs: PD: Total plasma calcium levels decreased 2.4 fold with CSK 1.9 fold IRQ relative to the untargeted SLN.

[34,35]

[36]

Polyoxyethylene (40) stearate solid lipid nanoparticles (SLNs)

CSKSSDYQC peptide (CSK) vs IRQRRRR (IRQ), cell penetrating peptide

Calcitonin (sCT)

RGD peptide Alginate coated chitosan NPs

RGD peptide

Bovine serum PD: Alginate coated chitosan NPs tagged with RGD stimulated higher levels of IgG albumin and IgA relative to untargeted alginate-chitosan NPs.

[37]

Neonatal Fc receptor Pegylated PLGA NPs

IgG Fc

Insulin

PK: A mean absorption efficiency of 13.7% per hour for the targeted NP compared to 1.2% per hour for the non-targeted NP (11.5 fold enhancement) PD: hypoglycemic effect at a clinically relevant dose (1.1 IU/kg) of insulin BD: High levels of C14 NPs in spleen, liver and lungs

[38]

Vitamin B12 receptor Dextran NPs

Vitamin B12

Insulin

PD: Studies in STZ induced diabetic rats, resulted in a 70–75% reduction in plasma glucose at 5 h, reaching basal levels in 8–10 h, with a prolonged second phase lasting up to 54 h. The pharmacological availability (PA) of the optimum formulation was 26.5% compared to NPs without vitamin B12

[39]

Calcitonin

PD: The pharmacological efficacy (blood calcium levels) with the targeted NP was significantly enhanced by more than 20-fold (P b 0.001) and 3-fold (P b 0.05) when compared to non-modified and CP liposomes, respectively. PK: Relative bioavailabilities compared to SC administration, calculated using AUC, of 4.99% and 7.11%, for the untargeted versus targeted NP respectively. PD: Pharmacological bioavailabilities (calculated from the area over the hypoglycemic curve versus time) of 4.46% and 6.08% for the untargeted and the targeted NP respectively, versus SC. PD: The LTA anchored chitosan NPs increased IgA levels in the salivary, intestinal and vaginal secretions, and cytokine (IL-2 and IFN-g) levels in the spleen homogenates. PD: The LTA anchored PLGA nanoparticles have demonstrated approximately four-fold increase in the degree of interaction with the bovine submaxillary mucin (BSM) and were efficient in stimulating mucosal, isotyping response IgG1/IgG2a and cell-mediated (IFN-γ and IL-2 level) immune responses

[40]

Lectins Carbopol liposomes

Soya lecithin-based solid lipid NPs

Chitosan NPs

Wheat germ agglutinin [WGA]

Insulin

[41,42]

[43]

PLGA NPs

Lotus tetragonolobs lectin (LTA) lectin targeting Hepatitis B M-cells antigen

Biotin DSPE liposomes

Biotin receptors

Insulin

PK: Only limited absorption occurred after oral administration of the untargeted [45] liposomes (CLPs). In contrast, the biotinylated liposomes (BLPs) achieved significant INS absorption, with a lower Cmax vs s.c. insulin. The bioavailability of the insulin-loaded CLPs and BLPs, calculated by the trapezoidal method, was 3.30% and 8.32%, respectively. PD: Hypoglycaemic response in diabetic rats seen only with the BLP targeted liposomes. The pharmacokinetic profiles correlated well with the hypoglycemic profiles. The Tmax for the BLPs was approximately 5 h, whereas the pharmacological action lasted up to 12 h.

Folate receptor Pegylated PLGA NPs

Folate

Insulin

PK: The targeted NPs exhibited a twofold increase in oral bioavailability versus SC insulin solution. PD: The NPs maintained decreased blood glucose levels for up to 24 h compared to 8 h in the case of the SC injection.

relative pharmacological activities (calculated from the area over the hypoglycemic curve versus time) of 4.46% for the untargeted NP versus 6.08% for the targeted NP, relative to subcutaneous administration. The pharmacokinetics of insulin in plasma for both formulations was compared to SC administration resulting in relative bioavailabilities of 4.99% and 7.11%, for the untargeted versus targeted NP respectively. The enhanced oral bioavailability may be associated with binding of lectin to receptors on the intestinal membrane resulting in increased contact time. However, in the absence of a mechanistic transport study confirming the transport mechanisms of insulin NPs across the intestine, it is unclear whether the SLNs were absorbed intact or the SLNs release their cargo within the intestine [41,42]. Pegylated PLGA NPs containing insulin and tagged with an antibody fragment (IgG Fc) have been reported to successfully mediate intestinal permeability via the Fc receptor (FcRn) [38]. This receptor is expressed throughout the intestine thus facilitating increased surface area for

[44]

[46]

absorption. Specific intestinal permeability via the Fc receptor occurred following oral administration to wild type mice with a mean absorption efficiency (calculated from the AUC) of 13.7 ± 1.3 SEM% per hour for the targeted NPs compared to only 1.2 ± 0.2 SEM % per hour for the non-targeted NPs. The effect was not evident in FcRn knock-out mice which confirmed the receptor mediated transport mechanism. Biodistribution studies, using 14C labelled NPs, detected high levels of the targeted NPs in the spleen, liver and lungs thus confirming oral absorption. The NPs generated a hypoglycemic effect (pharmacodynamic response) at a clinically relevant dose (1.1 IU/kg) of insulin. Targeted NPs with peptidic ligands have also been used to enhance oral protein delivery [47]. The peptide CSKSSDYQC (CSK) has been shown to specifically recognise goblet cells and thereby increase binding to the gut epithelium. Consequently, conjugation of CSK (peptide ligand) to NPs has been used to enhance peptide delivery [34,35]. A novel self-assembled polyelectrolyte complex NP was prepared by


6


coating insulin loaded doodecylamine-graft-gamma-polyglutamic acid (PGA-g-DA) micelles with trimethyl chitosan (TMC). The TMC was further modified with CSK. The resulting formulation (CSK-NPs) displayed improved stability and slower drug release versus controls. Following administration by oral gavage to diabetic rats, the CSK-NPs produced a hypoglycaemic response and decreased serum glucose level by 1.19 fold higher versus the untargeted NPs. Pharmacokinetic analysis showed a relative bioavailability (versus SC) of 7.05% which was significantly higher than 5.95% for the untargeted NPs. As only fluorescently labelled insulin was monitored in the in vivo work, it should be noted that it is not possible to clearly define the extent, if any, of absorption of the intact NP. Peptide ligands (CSK and IRQ) have also been utilised to enhance oral delivery of calcitonin [36]. Another peptidic ligand, arginine-glycine-aspartic acid (RGD) targets Beta1 integrins at the apical pole of M cells. Alginate coated chitosan NPs tagged with RGD delivering BSA for oral immunisation stimulated higher levels of IgG and IgA relative to the untargeted NPs [37]. Similarly folate tagged pegylated PLGA NPs have been used to enhance oral absorption of insulin [46]. The targeted NPs exhibited a twofold increase in oral bioavailability versus SC insulin solution. The NPs maintained decreased blood glucose levels for up to 24 h compared to 8 h in the case of the SC injection. Dextran NPs have also been labelled with vitamin B12 to promote intestinal uptake. The entrapment of insulin was 45–70% and the NP conjugates were found to protect 65–83% of entrapped insulin against in vitro gut proteases. In vivo PD studies, in streptozotocin (STZ)induced diabetic rats, resulted in a 70–75% reduction in plasma glucose at 5 h, reaching basal levels in 8–10 h, with a prolonged second phase lasting up to 54 h. The pharmacological availability (PA) of the optimum formulation was 26.5% compared to NPs without vitamin B12, consistent with the hypothesis that uptake was mediated by the vitamin B12 transport route [39]. 4. Pharmacokinetic (PK) and pharmacodynamic (PD) data following oral administration of p/p loaded nanoparticles 4.1. Nanoparticles formulated using biodegradable materials PKPD data reported following oral administration of NPs formulated with a range of biomaterials including chitosan, PLGA, lipids are summarised in Table 2. While the majority of studies are focused on insulin, oral delivery of other p/p including cyclosporine, GLP1, exendin-4 has also been investigated. However in the majority of cases a full pharmacokinetic analysis is absent, with PK data generally restricted to Cmax, Tmax and AUC with few studies reporting t1/2, Cl, Vdss or MRT parameters. In general relative bioavailability (BAR) versus absolute bioavailability (BAA) is provided. Pharmacodynamic outcomes (e.g. blood glucose levels in the case of insulin [47] and calcium levels for calcitonin [23,28]) are frequently compared to drug plasma levels which allow comments to be made on the rate, extent and duration of oral absorption and how these factors correlate with the resulting pharmacological/clinical effect. In addition, in the case of insulin PD/PK data for parenteral administration are often included as a benchmark for assessing the comparative clinical efficacy of the oral products. In general, it is difficult to make direct comparisons based on PK/PD/ BD data across the different studies due to the differing nature of the NP formulations, the design of the studies and the extent of the data reported. Select case studies which incorporate more extensive PK/PD/BD data are discussed below. Sonaje et al. investigated the pharmacokinetics, pharmacodynamics (blood glucose) and biodistribution of a rapid-acting monomeric insulin analogue (aspart-insulin) following oral versus SC administration in rats [53]. The formulation was a pH-responsive NP containing chitosan and poly(gamma-glutamic acid). The biodistribution of radiolabelled chitosan and insulin were monitored by single-photon emission computed

tomography (SPECT). Using this technique, it was reported, following oral administration, that while insulin was absorbed, appearing in the kidneys and bladder within 30 min post-administration, the chitosan was mainly retained in the GIT and facilitated insulin absorption by opening of the TJ. BD studies following oral versus SC administration produced very different profiles. Following SC administration insulin was rapidly (peaked at 20 min post injection) absorbed into the peripheral tissue/ plasma. At 3 h post injection 80% of the dose was absorbed and 50% was degraded and excreted into the bladder. In contrast, following oral delivery of the NPs, insulin was retained in the GIT for a relatively long period and 30% of the dose appeared to be absorbed 3 h post-administration. The levels of insulin in the peripheral tissue/plasma were sustained (at 10%) for up to 6 h post administration and 20% of the dose was excreted into the bladder at 3 h post dosing. However, attempts to confirm absorption by monitoring insulin levels in the portal vein failed due to poor levels of sensitivity of the assay method. In addition, the oral NP changed the PD profile of insulin, increasing the half-life of aspart-insulin to 1.79 h, a 2.6 fold increase relative to the SC administration of the solution, producing a relative bioavailability of 15.7% [53]. Comparison of the PK/PD data for oral versus SC showed that while oral aspart-insulin NPs produced a more prolonged hypoglycaemic effect versus SC administration of aspart-insulin, the oral NP profile was similar to that of the SC administered NPH-insulin (intermediate-acting insulin product). These results indicate that the oral chitosan NP could be used for maintaining basal insulin levels in diabetic patients. Sharma et al. co-encapsulated antacid-insulin in PLGA NPs and evaluated the effect of the NPs on PK, BD and PD of insulin following oral administration to healthy and diabetic rats [59]. Relative to an oral insulin solution, the NPs produced a higher Cmax and a longer Tmax (24 h versus 1 h) and increased the AUC six fold. While no direct evidence was available to support absorption of intact NPs, as only insulin levels were monitored, tissue distribution studies indicated the presence of insulin in various organs including liver, spleen, kidneys and pancreas after 72 h post-administration. In contrast, insulin was not detected after 24 h in the case of an oral solution or intravenous (IV) administration. The results show that oral administration of the PLGA NPs changed the pharmacokinetics of insulin relative to an oral solution. This may be related to enhanced protection versus enzymatic degradation or may also indicate that the NPs are absorbed into the blood where they control the slow release of insulin. Pharmacodynamic studies were performed in diabetic rats. However, an oral dose of 120 IU/kg was needed to elicit the equivalent PD response observed following a SC injection of 20 IU/kg. The maximum PD response following oral NP administration which occurred at 24 h (corresponding to the Cmax in plasma) and persisted for 72 h imply a controlled release of insulin. Tissue distribution studies indicated that insulin was present in a variety of tissue up to 72 h in a dose dependent fashion, a similar tissue distribution pattern was observed following SC injection of the NPs. In addition, the authors also examined the ability of the NPs to alleviate clinical complications frequently encountered in diabetic patients, including STZ induced chronic inflammation manifested by increased serum levels of inflammatory biomarkers including IL-6, CRP and TNFalpha. Levels of these biomarkers were monitored following oral administration of the NPs and while a trend toward a reduction in levels was detected, changes relative to the controls were not statistically relevant. In addition, a further clinical complication of diabetes namely an abnormal lipid profile, especially hyper-triglyceridemia and hypercholesterolemia, was also monitored however in this case SC insulin was found to be superior to oral insulin. 4.2. Nanoparticles formulated using inorganic materials 4.2.1. Silica Nanoparticles Recently mesoporous silica has attracted attention as a material with potential for oral drug delivery. The advantages of silica include the high



7

Table 2 Pharmacodynamic (PD) and pharmacokinetic (PK) data in animals following oral administration of peptide/protein loaded nanoparticles formulated with a range of biomaterials. Protein

Chitosan Insulin

PD

50% decrease in blood glucose level at 10 h

PK

BA

Dose

Cmax

Tmax

t1/2

AUC

CL/Vss/MRT

30 IU/kg

46 μlU/ml

5h

1.5 h

177 μlU h/ml

N/A

BAR (15.1%)

Refs

[48] a

Insulin


30 IU/kg

50 μlU/ml

5h

N/A

236 μlU h/ml

N/A

BAR (20%)

[49] a

Insulin


50 IU/kg

63 μlU/ml

2h

N/A

230 mg/Lh

N/A

BAR (20%)

[35] a

Insulin

45% hypoglycaemic effect at 24 h

50 IU/kg

16.6 μlU/ml

8h

N/A

160 μlU h/ml

N/A

BAR (13.2%)

[50] a

Exendin-4


300 μg/kg

300 pg/ml

5h

N/A

2140 pg h/ml

N/A

BAR (14%)

[51] a

TPLENK

60% analgesia at 2–8 h

70 mg/kg

0.17 μg/ml

0.5 h

N/A

3.1 μg h/ml

N/A

N/A

[52] c

Poly(g-glutamic acid) derivatives with chitosan Insulin 40% decrease in blood glucose level at 6 h

30 IU/kg

38.5 μlU/ml

3h

1.79 h

140 μlU h/ml

N/A

BAR (15.7%)

[53] a

Insulin


30 IU/kg

39 μlU/ml

4h

N/A

180 μlU h/ml

N/A

BAR (20%)

[54] a

Insulin


30 IU/kg

45 μlU/ml

5h

N/A

245 μlU h/ml

N/A

BAR (21%)

[55] a

Pluronic F127-lipid, PEO and chitosan Insulin 50% decrease in blood glucose level at 12 h

50 IU/kg

40 μlU/ml

3.3 h

N/A

232 μlU h/ml

N/A

BAR (7.8%)

[56] a

Poly(lactic-co-glycolic acid) Insulin 50% decrease in blood glucose level at 12 h

20 IU/kg

35 μlU/ml

6h

4.55 h

227 μlU h/ml

MRT = 9 h

BAR (7.7%)

[57] a

Insulin


20 IU/kg

45 μU/ml

6h

N/A

N/A

MRT = 9.4 h

BAR (6.3%)

[58] a

Insulin


20 IU/kg

4.8 μlU/ml

24 h

N/A

206 μlU h/ml

N/A

N/A

[59] a,b

Insulin

30% hypoglycemic effect at 10 h

50 IU/kg

N/A

10 h

N/A

N/A

N/A

PA (9.2%)

[60] a

CyA

N/A

10 mg/kg

2.3 μg/ml

1.4 h

N/A

11.4 μg h/ml

N/A

BAR (111%)

[61] d

Poly[MPC-co-BMA] CyA

N/A

10 mg/kg

1.3 μg/ml

4.8 h

N/A

26 μg h/ml

N/A

BAR (21%)

[62] b

Glyceryl monooleate and poloxamer 407 cubic nanoparticles CyA N/A

100 mg

1371 ng/ml

2.42 h

12.14 h

9005 ng h/ml

MRT = 6.56 h

BAR (178%)

[63] d

Silica nanoparticles and pH-sensitive Eudragit® GLP-1 77% hypoglycaemic effect at 4 h

1 mg/kg

2.2 μg/ml

0.25 h

8.58 h

5.23 μg h/ml

MRT = 12 h

BAR (35%)

[64] b

Nanoporous silica and Eudragit® CyA N/A

15 mg/kg

1.4 μg/ml

9h

N/A

23 μg h/ml

N/A

BAR (91%)

[65] b

Lipid nanoparticles Solid lipid nanoparticles Insulin


25 IU/kg

N/A

N/A

N/A

N/A

N/A

PA (13.86%)

[66] a

Self-emulsifying drug delivery systems (SEDDS, SMEDDS, SNEDDS) Epidermal growth factor N/A

4500 mU/kg

1.90 mU/ml

4–6 h

N/A

23.9 mU h/ml

MRT = 12 h

BAR (6.3%)

[67] b

Leuprorelin

N/A

1 mg

51.68 ng/ml

0.5 h

N/A

7385 min ng/ml

N/A

BAA (1.3%)

[68] b

Liposomes Insulin


50 IU/kg

34.1 μIU/ml

6h

N/A

316 μIU h/ml

N/A

BAR (19.3%)

[69] a

CyA

N/A

10 mg/kg

1.37 μg/ml

1.68 h

N/A

11.6 μg h/ml

N/A

N/A

[70] b

a b c d

Diabetic rats. Healthy rats. Healthy mice. Healthy dogs.


8


surface area, large pore volume, tuneable pore architecture and ease of chemical modification. The ADME properties of silica nanoparticles (SNs) have been evaluated following administration (after 24 h and 7 days) by various routes including IV, SC, IM and oral [71] using a range of techniques including TEM, fluorescently labelled particles (SNs-FITC) and silicon content analysis using ICP-OES. Following oral administration of SNs (110 nm) to mice the SNs were absorbed from the intestine and the distribution was visualised (TEM and fluorescence) in the gut wall, spleen but mainly in the liver. Levels of silica were quantified using ICP-OES and while levels following oral exposure were low relative to IV, silica was detected in organs including the liver, spleen, lungs, kidneys and intestine. Following oral administration, up to 80% of the dose was excreted via the faeces within 24 h. Histological examination of the gut tissue including the duodenum, jejunum, ileum, lymph node and Peyer's patches, following oral administration indicated no obvious signs of toxicity. In addition, an acute toxicity study indicated that an oral dose of 5000 mg/kg was tolerated by the mice, in contrast death occurred at 2400 mg/kg following intramuscular (IM) administration. In summary these results indicate that oral delivery of SNs is possible in the absence of toxicity and potential exists to exploit this material for oral delivery of drugs including p/p. Silica particles chemically modified using different functional groups have been proposed for drug delivery including for oral insulin delivery. Andeani et al. prepared pegylated (20,000 and 60,000 Mw) silica NP and reported slow release of insulin due to the association of insulin with the silanol groups on the silica surface [72]. These particles failed to enhance membrane permeability across the everted rat gut and no in vivo data was reported. Mahkam et al. prepared pH sensitive modified silica particles using N-(3-trimethoxysilylpropyl)imidazole [73]. The NPs connected through an ionic liquid-like network entrapping insulin. At physiological pH 7.4 a partial negative surface charge on the modified silica NPs was generated due to the deprotonation of silanol groups, and the strong electrostatic repulsion triggered a sustained release of the insulin. Zhao et al. prepared and characterised insulin loaded silica nanospheres by adsorption in HCl solution [74]. The particles were enteric coated with hydroxypropyl methylcellulose phthalate. Following oral administration to diabetic rats the formulation produced a hypoglycaemic effect which lasted up to 7 h post-administration. 4.2.2. Gold Nanoparticles Due to the easily modified surface chemistry gold NPs (AuNPs) have been investigated as multifunctional carriers for orally transporting p/p. Bhumkar et al. synthesised novel AuNPs using chitosan as the reducing and stabilising agent [75]. The loading of insulin onto the resultant AuNPs was predominantly achieved via hydrogen bonding between insulin and Au, resulting in a loading efficiency of 53%. Following oral administration of insulin-loaded AuNPs (50 IU/kg) at 2 h, blood glucose was significantly reduced (~30%) in diabetic rats compared to insulin solution and blank AuNPs. Intestinal absorption which was most likely due to the interaction of chitosan with the TJ resulting in enhanced transport of insulin through the paracellular pathway. Although AuNPs are still very much in the infancy, they can be functionalised by grafting multifunctional polymers and natural or synthetic biomolecules onto the surface of gold [76], potentially becoming oral p/p carriers in the future. 5. Biodistribution 5.1. Role of the intestinal lymphatic system As discussed previously, the intestinal lymphatic system can play a key role in influencing distribution of drug loaded NPs. Following absorption, NPs may be distributed via either the portal blood or via the lymphatic system (Figs. 1 and 2).

While uptake via the intestinal lymphatics confers the advantage of avoiding first pass metabolism and thereby increasing systemic bioavailability, there are also some additional potential benefits. These include the possibility of a more controlled kinetics of distribution into the systemic blood circulation, and the possibility of treating diseases such as certain cancers or infectious disease (e.g. HIV) that spread via the lymphatics [7,77]. These delivery advantages arise from the physiology and architecture of the lymphatic drainage system [78]. The mesenteric lymph vessels drain lymph from the intestine, which then flows via lymph pools into the cysterna chyili and onwards via the thoracic lymph vessel. The thoracic lymph vessels empty into the blood circulation at the junction with the left subclavian vein, hence avoiding passage through the liver and first pass hepatic metabolism [79]. There are three major routes for accessing the mesenteric lymphatic system from the GIT. Firstly by incorporation into chylomicrons (i.e. lipoproteins formed within, and secreted by, the enterocytes), secondly via a size filtration mechanism whereby larger NPs that are secreted basolaterally are selectively taken up by lymph lacteals which have a more open or ‘leakier’ vasculature relative to blood capillaries, and thirdly via the gut-associated lymphoid tissue (GALT) and the M cells (Fig. 1). Recent studies have described the involvement of the lymphatic in the absorption and biodistribution and pharmacokinetic profile of nanostructured lipid carriers (NLCs, 216 nm in size) loaded with Tamoxifen (Tmx) and formulated using long chain lipids following oral administration to rats [80]. Compared to administration of a Tmx suspension, the pharmacokinetic profile produced by the NLCs was markedly different, with a slower rate of absorption, a 2.71 fold higher Cmax, a longer Tmax of 8.17 h versus 1.15 h, and an overall 2 fold higher AUC. Drug concentrations in the mesenteric lymph nodes following oral administration of the NLCs were consistently higher versus plasma levels up to 8 h post-administration suggesting a slow redistribution of drug from the lymphatics into the systemic circulation. No drug was detected in the lymph nodes following administration of the suspension. Taken together these results suggest that the NLCs are transported via the mesenteric lymphatic system, where it is suggested that the long lipids employed may stimulate chylomicron mediated uptake. In a similar study, the pharmacokinetic and biodistribution profiles for lipid nanoparticles (LNs) (103–110 nm) loaded with edelfosine were studied after IV and oral administration to mice [81]. The LNs resulted in an increase in relative bioavailability of 1500% compared to 10% for a solution of the free drug. Systemic clearance of the LNs was also decreased by approximately 50%. This resulted in a corresponding increase in the volume of distribution which would indicate a greater extent of tissue distribution of the drug following oral administration of the LNs. Mesenteric lymph nodes were harvested from the mice 24 h post-dosing. While drug levels were below the limit of detection for the drug solution, relatively high levels of the drug were detected in the lymph nodes following administration of the LNs. The study also demonstrated improved PD outcomes in a tumour mouse model where oral LNs inhibited metastases to the lymph nodes implying a link between lymphatic transport and clinical efficacy. Nanocapsules (NCs) containing docetaxel, a drug with low oral bioavailability due to efflux by P-gp and metabolism by Cyp3A4, dissolved in the oil core of PLGA NCs and further embedded in microparticles (MPs) made with a blend of polymers (Eudragit plus HPMC) when administered orally to rats and minipigs yielded oral plasma area under the curves (AUCs) 1.77-times higher than that after IV administration of the same dose of the commercial solution (Taxotere) [82]. CryoTEM studies indicated that the NCs were coated on the passage through the enterocytes by lipoproteins (LP), in a similar way to chylomicrons. These ‘coated’ NCs were then secreted basolaterally and are likely transported via the lymphatic system. Inhibition of lymphatic transport, using cycloheximide to block chylomicron synthesis, dramatically decreased the plasma levels thus supporting this proposed mechanism



9

Table 3 Whole body biodistribution of nanoparticles following oral administration. Material

Cargo

Main site of biodistribution

Detection method

Ref.

Chitosan and poly(r-glutamic acid)

Aspart-insulin

GI tract and urinary bladder

[53]

Chitosan and poly(r-glutamic acid)

Insulin

GI tract

Single-photon emission computed tomography fused computed tomography Single-photon emission computed tomography fused computed tomography Single-photon emission computed tomography fused computed tomography Single-photon emission computed tomography fused computed tomography Single-photon emission computed tomography fused computed tomography Single-photon emission computed tomography fused computed tomography Fluorescent confocal microscope

Chitosan and poly(r-glutamic acid) Chitosan and poly(r-glutamic acid) Chitosan and poly(r-glutamic acid) Chitosan and poly(r-glutamic acid) Chitosan and poly(r-glutamic acid)

Insulin Insulin Exendin-4 Insulin Insulin

GI tract GI tract GI tract and urinary bladder GI tract and urinary bladder Duodenum, jejunum and ileum

b

[49] b

[83] a

[55] b

[51] b

[54] b

[48] b

Chitosan and poly(r-glutamic acid) Chitosan

G-CSF BSA

GI tract, urinary bladder and bone marrow Liver and intestine

Single-photon emission computed tomography fused computed tomography Spectrofluorimetry

[84] b

[17] c

PLGA

Insulin

Kidney, spleen, pancreas and intestine

Insulin-specific ELISA kit

[59] b

PLGA PLGA

Docetaxel N/A

Intestine, spleen and mesenteric ymph node Kidney and liver

LC–MS/MS

[82] d

Fluorescent plate reader

[85] c

Chitosan-PLGA

Insulin

GI tract

Kodak® In Vivo Imager

[86] c

Liposomes

Ovalbumin

Gut mucosa and Peyer's patches

Automatic scintillation apparatus

[87] c

Porous silicon

N/A

Lower GI tract and urine

Gamma counter

[88] b

3-Aminopropyl functionalized magnesium phyllosilicate (aminoclay) ZM4 nanoparticles

N/A N/A

GI tract Abdominal lymph nodes and ileum

CCD camera with a special C-mount lens and a long wave emission filter Odyssey Infrared Imaging System

[89] c

[90] c

Poly(lactic acid)-b-PEG-antibody

N/A

Liver and spleen

Fluorescent confocal microscope

[38] c

Oleoyl-carboxymethyl-chitosan

N/A

Liver and kidney

Fluorescent plate reader

[91] e

Cyclodextrin–poly(anhydride) nanoparticles Poly-(1,4-phenyleneacetone dimethylene thioketal)

N/A siRNA

Intestine

Single-photon emission computed tomography fused computed tomography Fluorometer

Colon

[92] b

[93] d

Polyacrylic acid with spermine

FD4

Duodenum, jejunum, and ileum

Fluorescent confocal microscope

[94] b

Poly(lactic acid)-PEG

Tetanus toxoid

GI tract and lymphoid nodes

Packard Cobra II Auto-Gamma Counter

[95] b

a b c d e

Diabetic rats. Healthy rats. Healthy mice. Diseased mice. Carp.

of transport. Plasma levels of the drug following oral administration of the NCs displayed profiles consistent with controlled release from the formulation. In addition, the ‘coated’ NCs altered the biodistribution to the organs studied (Table 3). Therapeutic efficacy in a tumour model was also superior to that seen following IV administration and the authors suggest that this is due to accumulation of the LP ‘coated’ NCs in the lung tumours via the EPR effect. Recently, Reineke et al., following local administration or oral gavage to rats, employed a range of quantitative techniques including TEM, confocal microscopy and gel permeation chromatography to investigate the mechanisms of intestinal absorption, transit and biodistribution of non-biodegradable polystyrene microspheres (MSs) (500 nm–5 μm) [96]. The study indicates rapid intestinal uptake of the MSs within 5 min, with 30–45% of the dose absorbed 5 h post-administration. While most particles were detected in the liver, widespread distribution to all tissues sampled including the brain was found. Local administration to the jejunum versus the ileum altered the biodistribution pattern. The higher levels of MSs in the heart and spleen following administration to the ileum may be related to the higher proportion of GALT in

that area and the subsequent transport of the MSs in the lymphatic system. However, mechanistic studies at the cellular level employing a range of endocytosis inhibitors decreased uptake indicating that uptake was not solely associated with M cells but that non-phagocytic processes including endocytosis were also involved. In conclusion, these studies indicate the key role of the intestinal lymphatics in transporting NPs from the intestine to the systemic circulation. Although these studies have employed drug cargos that have poor oral bioavailability, but are not peptides/proteins, they do shed new insights on NP mediated lymphatic transport to overcome pharmacokinetic limitations. The targeting of peptide loaded NPs to the mesenteric lymph vessels would be advantageous for p/p prone to first pass metabolism and would also be attractive for peptides that treat diseases of the lymphatics. However, to date the extent of lymphatic transport of peptide loaded NP's has been shown to be relatively, low, and is likely to vary widely depending on the nature of the NP, the drug loading, and the hydrophobic versus hydrophilic properties of the p/p [79]. In addition, in the absence of direct cannulation and sampling from the mesenteric lymph vessels it is difficult to quantify the extent of NP uptake via


10


the lymphatics. While new approaches to study lymphatic transport, such as cryo-TEM and confocal microscopy, provide interesting insights and can provide useful mechanistic data, it should be borne in mind that these are indirect predictors of lymphatic transport and therefore the conclusions drawn must be interpreted with caution [77]. 5.2. Whole body distribution In some studies whole body distribution has been investigated, using a variety of detection techniques, following oral delivery of NP formulations [97]. Selected studies are summarised in Table 3. In most studies, the NPs were retained in the GIT with some distribution reported in the bladder, kidney, liver and lymph nodes. As discussed above, following oral administration of oil filled PLGA NCs containing docetaxel, biodistribution to the lymphatics but also to other major organs was reported as follows: intestine N spleen N mesenteric lymph nodes N lungs N liver N kidneys N heart N fat N brain [82]. Sharma et al. [59] also reported distribution of insulin to the spleen, kidneys and pancreas persisting for up to 72 h following oral administration in PLGA NPs. This distribution pattern was similar to that seen after SC administration, albeit at lower levels. In contrast, following IV administration insulin levels were only detected up to 24 h. A recent study examined the biodistribution and toxicity of oleoylcarboxymethyl-chitosan (OCMCS) in vivo following oral administration to carp [91]. The OCMCS was soluble at neutral pH and self-assembled to form NP with an average size of 215 nm. The particles were non-toxic (MTT) in vitro and displayed uptake into Caco-2 cells. In vivo, histological examination of a range of tissues following administration of the NPs to carp over 7 days indicated no toxicity. Tissue distribution and retention of particles at Days 1, 2 and 3 post-dosing was performed by tracking FITC labelled NPs. Particles were detected mainly in the liver (40%), kidneys (26%) and heart (12%) and detectable levels persisted for 3 days indicating the ability of the particles to escape the RES in spite of the relatively large size. These NPs may have potential for p/p oral delivery provided adequate drug loading and protection from enzymatic degradation was achieved. 6. Physiologically-based pharmacokinetic modelling (PBPK) as a predictive tool for oral administration of NPs To date, most of the advances in understanding of oral NP-mediated p/p delivery have been derived from varied studies, with differing methodologies, nanoparticle technology and p/p. As a result, making reliable predictions for newer peptide drugs, using these current isolated studies, is limited by both the lack of consistency between studies and gaps in knowledge that facilitate direct extrapolations between studies. This suggests a need for more reliable in silico predictive models that take into account the variety of parameters that can influence NP mediated oral p/p delivery. Whole-body PBPK modelling is a method to predict and describe drug disposition in a range of interconnecting compartments representing organs/tissues as well as in the interconnecting vasculature [98]. PBPK modelling integrates drug data (physicochemical data, membrane permeability, intrinsic clearance), systems data (physiological properties of population) and trial design data in a physiologically relevant structural model [99]. PBPK modelling has many advantages, including the facilitation of interspecies extrapolation to mechanistically translate animal data to human populations, predicting tissue as well as plasma concentrations including tissues that are inaccessible for sampling, extrapolation to diseased population from healthy volunteer data, providing insights into mechanisms underpinning disposition and identifying important covariates, facilitating selection of first-in-human dose and optimisation of clinical trial design [100–103]. While there are many advantages, PBPK modelling is also challenging as comprehensive physiological, biochemical and physicochemical data is required to build models and accurate interpretation of model outputs is essentially

dependent on accurate model inputs, which may not be readily available [100]. In addition, model predictions need to be validated against clinical data, which should preferably be prospectively gathered [100]. When attempting to model pharmacokinetics of oral p/p NP formulation, it is important to remember, as stated above, that the formulation may be absorbed across the GI membrane as intact peptide-loaded nanoparticle, alternatively the peptide may be absorbed alone following release from the NP in the gut lumen, or thirdly the peptide may be absorbed following fusion of the NP with the cells of the gut membrane [101]. While a number of studies have utilised PBPK models for predicting therapeutic p/p (drug load) or nanoparticles (drug carrier) pharmacokinetics following parenteral administration, few models have been developed to describe oral absorption of NP formulations. Thus, it is important to remember that modelling oral administration of p/p-loaded NPs requires a further level of complexity compared to the current models. There is a need to develop newer models that take account of the impact of NP on oral p/p PK, specifically the altered absorption, biodistribution and clearance, as discussed previously. PBPK modelling of small molecules is more advanced compared to simulation of therapeutic protein disposition [104]. In the case of small molecules modelling and validation of IV administration are encouraged prior to attempting simulation of oral/extravascular routes of drug administration [105]. This approach should also be applied to any investigations of therapeutic proteins in NP formulations. In terms of accurately modelling oral absorption of therapeutic proteins and/or nanoparticles, the information required would include: nanoformulation degradation in simulated gastric fluids from in vitro tests, the size and shape of the nanoformulation, endothelium pore sizes, the role of the lymphatic system, and membrane permeability including mechanisms of transport such as paracytosis, clathrin-mediated and calveolae-mediated endocytosis and micropinocytosis [101]. Other factors, including drug efflux transporters and gut wall metabolism may also need to be taken into account [106]. A ‘predict, learn and confirm’ paradigm would be essential to evaluate predictions of bioavailability by comparing simulations with preclinical in vivo/clinical data [106,107]. PBPK modelling may be useful to optimise the peptide formulation or to predict bioavailability of orally administered NPs containing therapeutic peptides [106]. While the literature shows that PBPK modelling of oral drug delivery, NPs and therapeutic proteins is possible [98], many of these models are relatively novel and a plethora of information remains to be elucidated before the models can be routinely applied. For example, PBPK models may need to be modified to account for lymphatic absorption and distribution, transportation mechanisms of NP movement from blood to tissue and within tissues, active targeting by including preferential transportation kinetics for the molecular target, removal of NPs by phagocytosis and metabolism of the parent NP compound as well as its metabolites [101,104,108]. It has been proposed that PBPK models of the NP and its drug load could be combined, with drug release kinetics from NPs forming the link between both NP and drug kinetics [104]. In addition, progress toward accurately predicting the pharmacokinetics of orally administered peptide-loaded NPs is currently limited by a series of unknowns, these poorly defined parameters include, but are not limited to: composition of luminal contents, GI transit and hydrodynamics, permeability, influx and efflux transport at the GI membrane, intestinal wall metabolism and many of the physiological processes governing both p/p and NP pharmacokinetics. A PBPK approach to oral delivery of nanoparticles loaded with p/p is described in Fig. 3. In summary, PBPK modelling of therapeutic proteins and NPs is relatively new and additional information is required to facilitate the prediction of pharmacokinetics from orally administered p/p-loaded NPs; this may be feasible given appropriate in vitro and in vivo preclinical models to elucidate the many unknown parameters [102].



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Fig. 3. A schematic depicting oral absorption of peptide-loaded nanoparticles within a simple physiologically-based pharmacokinetic model (PBPK). Peptide-loaded nanoparticles can exist in the gut lumen as peptide-loaded nanoparticles or the peptide can be released from the nanoparticle. Peptide-loaded nanoparticles can be absorbed across the gut wall in two ways; 1) as intact peptide-loaded nanoparticles or 2) nanoparticle components may fuse with enterocytes and release the peptide load into endocytotic vesicles which traverse the enterocyte. Peptide alone would be highly degraded within the gut lumen (kdeg). Peptide-loaded nanoparticles may be absorbed through enterocytes or M cells and are transported into either the systemic circulation or the mesenteric lymphatic system. The absorption rate constant, ka, for a given peptide-loaded nanoparticle is the sum of the absorption rate constants described by: transmucosal transport (kat) and M cells transport (kam) of peptide-loaded nanoparticles (PLN), nanoparticle-assisted peptide transport (NAP) and peptide alone (P). These absorption rate constants can be further broken down based on absorption into the portal vein (katp) or absorption into lymph (katl or kaml).

7. Regulatory considerations It is now well established among regulatory agencies that the use of nanotechnology to improve clinical efficacy of medicines adds an additional regulatory challenge in terms of demonstrating the quality, safety and efficacy of such nanomedicines. Anticipating the need to open discussion in an evolving scientific field, the European Medicines Agency published a Reflection paper in 2006 on ‘Nano-technology based medicinal products for human use’. In 2009, the Committee for Human Medicinal products (CHMP) established an ad hoc expert group on nano-medicines to provide specialist input on new scientific knowledge and contribute to new guidelines on nanomedicines. The FDA have adopted a similar approach with the establishment of a Nanotechnology Task Force and published regulatory guidelines relating nano-technology derived medicines. For example, in 2011, the FDA issued a draft guidance entitled ‘Considering whether an FDA regulated product involves the application of nanotechnology’ to outline the agency's current perspectives on nanotechnology and seek industry comment. The final approved guidance was published in 2014. Interestingly, the guidance outlines the two key questions which the FDA will ask when considering whether a medicine involves the use of nanotechnology, as follows: (1) Whether a material or end product is engineered to have at least one external dimension, or an internal or surface structure, in the nanoscale range (approximately 1 nm to 100 nm); (2) whether a material or end product is engineered to exhibit properties or phenomena, including physical or chemical properties or

biological effects, that are attributable to its dimension(s), even if these dimensions fall outside the nanoscale range, up to 1000 nm. A number of nanomedicines have been approved by various regulatory agencies, including Caelyx®, (Doxorubicin liposomes) and Abraxane® (paclitaxel albumin bound nanoparticles), demonstrating that favourable benefit/risk ratios can be successfully demonstrated for parenterally administered nanomedicines. While there are a number of nanotechnology derived oral medicines licensed for use, such as Rapamune ® (sirolimus nanocrystals) and Neoral (cyclosporine nanoemulsion), it should be noted that in these cases the nanotechnology employed is primarily focused on enhancing drug solubility within the GIT as opposed to a specific effect of the NPs on drug pharmacokinetics/biodistribution. Therefore, while there are various regulatory guidance and existing frameworks for approval of oral nanomedicines, scientific questions remain to be fully explored relating to the influence of NP characteristics (size, surface characteristics etc.) on the quality, safety and efficacy of the nanomedicines. In particular, the safety and toxicological assessment of the NPs remain central to evaluation of the benefit/risk analysis [109]. However, there is a lack of thorough systematic toxicity studies on NPs which are needed to reliably assess toxicity/efficacy and support the regulatory decisions regarding risks associated with NP. Araujo et al. in 2015 reviewed the safety and toxicity of NPs following oral administration. A range of materials including chitosan, PLGA, lipid-based systems, carbon, silica and gold were discussed [110]. The authors highlight that the potential for toxicity following oral administration will be influenced


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not only by the nature of the biomaterial used but also by features including size, charge and shape, all of which may influence the PK of the NPs. In the case of materials classified as absorption enhancers, including chitosan, which act mainly via opening of TJ, the long term consequences of this mechanism and the potential for reversibility would need to be investigated. Due to variations in organ distribution and surface effects, the toxicity of nanosystems may be very different to that of micro or macro systems manufactured from the same material thus indicating the need for more toxicity studies on materials when formulated as NPs, even those classified as biocompatible or biodegradable [109]. In addition, features such as shape and zeta potential for synergistic toxicities between a drug and the biomaterials have to date been poorly investigated. Finally, existing toxicological methods may need to be adapted, or new biorelevant tools employed, to provide a comprehensive understanding of the risk of toxicity of such NPs. Due to the potential for variation in the ADME profiles of nanomedicines and the complex nature of the formulations, often containing a variety of excipients, new regulatory frameworks are required to assess the quality, safety, and efficacy of these complex products. Many of the clinical evaluation studies to date have utilised ‘generally recognised as safe’ (GRAS) listed excipients with the hope of facilitating the licensing process of resulting products. However, in many cases these excipients have shown disappointing results and it may be timely to explore more novel and ‘bioactive’ excipients. Newer regulatory initiatives to include science- and risk-based regulatory frameworks, regulatory tools for facilitating early access to medicines [e.g. “Adaptive Pathways” (www.ema.europa.eu)], and the development of innovative technical tools (i.e. in vitro and in silico methods) will be needed to facilitate regulatory evaluation of such products [111,112]. In addition, the manufacturing process, and reproducibility in particular with respect to scale-up, has largely been ignored. It is essential to identify the critical quality attributes (CQAs) which dictate the performance of the nanomedicine and to design the process to ensure these parameters are consistently and reliably achieved. The benefits of establishing such a validated, robust process will be easier manufacturing and process control as well as production of optimised nanomedicines with maximum quality and therapeutic efficacy. 8. Conclusions and future perspectives A detailed understanding of the pharmacokinetics and pharmacodynamics of orally administered p/p is currently limited by a lack of studies that comprehensively assess PK/BD/PD in reliable in vivo models. Furthermore such analysis of p/p formulated as oral NPs is almost completely absent. The lack of comprehensive studies makes a critical analysis of the mechanisms of absorption difficult. For example, it is challenging to quantify whether intact NP uptake is a relevant mechanism as many studies suggesting this are performed using non-biodegradable versus degradable NPs. To help address these deficits validated analytical techniques which directly trace and quantify the disposition of the NPs are required. On the contrary, many of the current studies trace only the cargo and not the NP and this indirect method can often be misleading and fail to fully elucidate the influence of the NP formulation on PK/PD. In addition, a review of the literature shows that most studies are confined to a limited number of p/p, with most work focused on insulin, cyclosporin and calcitonin. Studies on a wider range of p/p would help expand the knowledge base and facilitate comparative studies on the influences of protein structure and stability. Also the range of excipients employed to date tends to be limited to those listed as GRAS and potential exists to improve the performance of oral NPs by development of novel more effective ‘bioactive’ excipients. In conclusion, it is widely accepted that the growth of biopharmaceutical products is likely to continue, currently 7 of the 10 top selling drugs are biologics (www.genengnews.com). New regulatory frameworks are required to keep pace with these developments. In the field of

biopharmaceuticals, nano-formulations undoubtedly have advantages as drug delivery systems and the application of such technologies to the oral route is exciting. However, in order to facilitate regulatory assessment and ensure timely access to market of these products for the patient, it will be necessary to show detailed mechanistic studies illustrating the influence of the NP on ADME profiles and the consequent effects on safety and efficacy. The need to illustrate a positive benefit to risk ratio for orally administered NPs will become even more critical as newer more ‘bioactive’ excipients are employed in these formulations. Acknowledgements The authors are part of the TRANS-INT European Consortium, which has received funding from the European Union's Seventh Framework Programme for research, technological development and demonstration under grant agreement No. 281035. References [1] J. Gordon Still, Development of oral insulin: progress and current status, Diabetes Metab. Res. Rev. 18 (Suppl. 1) (2002) S29–S37. [2] C. Lautenschlager, C. Schmidt, C.M. Lehr, D. Fischer, A. Stallmach, PEG-functionalized microparticles selectively target inflamed mucosa in inflammatory bowel disease, Eur. J. Pharm. Biopharm. 85 (2013) 578–586. [3] Q. Xu, L.M. Ensign, N.J. Boylan, A. Schon, X. Gong, J.C. Yang, N.W. Lamb, S. Cai, T. Yu, E. Freire, J. Hanes, Impact of surface polyethylene glycol (PEG) density on biodegradable nanoparticle transport in mucus ex vivo and distribution in vivo, ACS Nano 9 (2015) 9217–9227. [4] V. Bourganis, T. Karamanidou, E. Samaridou, K. Karidi, O. Kammona, C. Kiparissides, On the synthesis of mucus permeating nanocarriers, Eur. J. Pharm. Biopharm. 97, Part A (2015) 239–249. [5] X.Q, J.S. Suk, N. Kim, J. Hanes, L.M. Ensign, PEGylation as a strategy for improving nanoparticle-based drug and gene delivery, Adv. Drug Deliv. Rev. 99, Part A (2015) 28–51. [6] P. Jani, G.W. Halbert, J. Langridge, A.T. Florence, Nanoparticle uptake by the rat gastrointestinal mucosa: quantitation and particle size dependency, J. Pharm. Pharmacol. 42 (1990) 821–826. [7] C.M. O'Driscoll, Lipid-based formulations for intestinal lymphatic delivery, Eur. J. Pharm. Sci. 15 (2002) 405–415. [8] A.T. Florence, Nanoparticle uptake by the oral route: fulfilling its potential? Drug Discov. Today Technol. 2 (2005) 75–81. [9] A. des Rieux, V. Fievez, M. Garinot, Y.J. Schneider, V. Preat, Nanoparticles as potential oral delivery systems of proteins and vaccines: a mechanistic approach, J. Control. Release 116 (2006) 1–27. [10] A. des Rieux, E.G. Ragnarsson, E. Gullberg, V. Preat, Y.J. Schneider, P. Artursson, Transport of nanoparticles across an in vitro model of the human intestinal follicle associated epithelium, Eur. J. Pharm. Sci. 25 (2005) 455–465. [11] A. des Rieux, V. Fievez, I. Theate, J. Mast, V. Preat, Y.J. Schneider, An improved in vitro model of human intestinal follicle-associated epithelium to study nanoparticle transport by M cells, Eur. J. Pharm. Sci. 30 (2007) 380–391. [12] B. He, P. Lin, Z. Jia, W. Du, W. Qu, L. Yuan, W. Dai, H. Zhang, X. Wang, J. Wang, X. Zhang, Q. Zhang, The transport mechanisms of polymer nanoparticles in Caco-2 epithelial cells, Biomaterials 34 (2013) 6082–6098. [13] M.S. Cartiera, K.M. Johnson, V. Rajendran, M.J. Caplan, W.M. Saltzman, The uptake and intracellular fate of PLGA nanoparticles in epithelial cells, Biomaterials 30 (2009) 2790–2798. [14] M. Gaumet, R. Gurny, F. Delie, Localization and quantification of biodegradable particles in an intestinal cell model: the influence of particle size, Eur. J. Pharm. Sci. 36 (2009) 465–473. [15] M.S. Verma, S.Y. Liu, Y.Y. Chen, A. Meerasa, F.X. Gu, Size-tunable nanoparticles composed of dextran-b-poly(D,L-lactide) for drug delivery applications, Nano Res. 5 (2012) 49–61. [16] J.L. Madara, Regulation of the movement of solutes across tight junctions, Annu. Rev. Physiol. 60 (1998) 143–159. [17] C. He, L. Yin, C. Tang, C. Yin, Size-dependent absorption mechanism of polymeric nanoparticles for oral delivery of protein drugs, Biomaterials 33 (2012) 8569–8578. [18] J.F. Guo, L. Bourre, D.M. Soden, G.C. O'Sullivan, C. O'Driscoll, Can non-viral technologies knockdown the barriers to siRNA delivery and achieve the next generation of cancer therapeutics? Biotechnol. Adv. 29 (2011) 402–417. [19] J. Guo, J.C. Evans, C.M. O'Driscoll, Delivering RNAi therapeutics with non-viral technology: a promising strategy for prostate cancer? Trends Mol. Med. 19 (2013) 250–261. [20] M. Longmire, P.L. Choyke, H. Kobayashi, Clearance properties of nano-sized particles and molecules as imaging agents: considerations and caveats, Nanomedicine-Uk 3 (2008) 703–717. [21] V. Bourganis, T. Karamanidou, E. Samaridou, K. Karidi, O. Kammona, C. Kiparissides, On the synthesis of mucus permeating nanocarriers, Eur. J. Pharm. Biopharm. 97 (2015) 239–249. [22] J. Guo, K.A. Fisher, R. Darcy, J.F. Cryan, C. O'Driscoll, Therapeutic targeting in the silent era: advances in non-viral siRNA delivery, Mol. BioSyst. 6 (2010) 1143–1161.


B.T. Griffin et al. / Advanced Drug Delivery Reviews xxx (2016) xxx–xxx [23] C. Prego, D. Torres, E. Fernandez-Megia, R. Novoa-Carballal, E. Quinoa, M.J. Alonso, Chitosan-PEG nanocapsules as new carriers for oral peptide delivery — effect of chitosan pegylation degree, J. Control. Release 111 (2006) 299–308. [24] S. Jevsevar, M. Kunstelj, V.G. Porekar, PEGylation of therapeutic proteins, Biotechnol. J. 5 (2010) 113–128. [25] V.K. Pawar, J.G. Meher, Y. Singh, M. Chaurasia, B. Surendar Reddy, M.K. Chourasia, Targeting of gastrointestinal tract for amended delivery of protein/peptide therapeutics: strategies and industrial perspectives, J. Control. Release 196 (2014) 168–183. [26] F.M. Veronese, G. Pasut, PEGylation, successful approach to drug delivery, Drug Discov. Today 10 (2005) 1451–1458. [27] C. Prego, M. Garcia, D. Torres, M.J. Alonso, Transmucosal macromolecular drug delivery, J. Control. Release 101 (2005) 151–162. [28] C. Prego, M. Fabre, D. Torres, M.J. Alonso, Efficacy and mechanism of action of chitosan nanocapsules for oral peptide delivery, Pharm. Res. Dordr. 23 (2006) 549–556. [29] G. Ponchel, J. Irache, Specific and non-specific bioadhesive particulate systems for oral delivery to the gastrointestinal tract, Adv. Drug Deliv. Rev. 34 (1998) 191–219. [30] Y. Huang, W. Leobandung, A. Foss, N.A. Peppas, Molecular aspects of muco- and bioadhesion: tethered structures and site-specific surfaces, J. Control. Release 65 (2000) 63–71. [31] J. Reineke, D.Y. Cho, Y.L. Dingle, P. Cheifetz, B. Laulicht, D. Lavin, S. Furtado, E. Mathiowitz, Can bioadhesive nanoparticles allow for more effective particle uptake from the small intestine? J. Control. Release 170 (2013) 477–484. [32] J.F. Guo, W.P. Cheng, J.X. Gu, C.X. Ding, X.Z. Qu, Z.Z. Yang, C. O'Driscoll, Systemic delivery of therapeutic small interfering RNA using a pH-triggered amphiphilic poly-L-lysine nanocarrier to suppress prostate cancer growth in mice, Eur. J. Pharm. Sci. 45 (2012) 521–532. [33] L. Ye, R. Zeng, Y. Bai, D.C. Roopenian, X. Zhu, Efficient mucosal vaccination mediated by the neonatal Fc receptor, Nat. Biotechnol. 29 (2011) 158–163. [34] P. Zhang, Y. Xu, X. Zhu, Y. Huang, Goblet cell targeting nanoparticle containing drug-loaded micelle cores for oral delivery of insulin, Int. J. Pharm. 496 (2015) 993–1005. [35] Y. Jin, Y. Song, X. Zhu, D. Zhou, C. Chen, Z. Zhang, Y. Huang, Goblet cell-targeting nanoparticles for oral insulin delivery and the influence of mucus on insulin transport, Biomaterials 33 (2012) 1573–1582. [36] T. Fan, C. Chen, H. Guo, J. Xu, J. Zhang, X. Zhu, Y. Yang, Z. Zhou, L. Li, Y. Huang, Design and evaluation of solid lipid nanoparticles modified with peptide ligand for oral delivery of protein drugs, Eur. J. Pharm. Biopharm. 88 (2014) 518–528. [37] B. Malik, A.K. Goyal, F. Zakir, S.P. Vyas, Surface engineered nanoparticles for oral immunization, J. Biomed. Nanotechnol. 7 (2011) 132–134. [38] E.M. Pridgen, F. Alexis, T.T. Kuo, E. Levy-Nissenbaum, R. Karnik, R.S. Blumberg, R. Langer, O.C. Farokhzad, Transepithelial transport of Fc-targeted nanoparticles by the neonatal fc receptor for oral delivery, Sci. Transl. Med. 5 (2013), 213ra167. [39] K.B. Chalasani, G.J. Russell-Jones, S.K. Yandrapu, P.V. Diwan, S.K. Jain, A novel vitamin B12–nanosphere conjugate carrier system for peroral delivery of insulin, J. Control. Release 117 (2007) 421–429. [40] A. Makhlof, S. Fujimoto, Y. Tozuka, H. Takeuchi, In vitro and in vivo evaluation of WGA-carbopol modified liposomes as carriers for oral peptide delivery, Eur. J. Pharm. Biopharm. 77 (2011) 216–224. [41] N. Zhang, Q.N. Ping, G.H. Huang, W.F. Xu, Y.N. Cheng, X.Z. Han, Lectin-modified solid lipid nanoparticles as carriers for oral administration of insulin, Int. J. Pharm. 327 (2006) 153–159. [42] N. Zhang, Q.N. Ping, G.H. Huang, W.F. Xu, Investigation of lectin-modified insulin liposomes as carriers for oral administration, Int. J. Pharm. 294 (2005) 247–259. [43] N. Mishra, K. Khatri, M. Gupta, S.P. Vyas, Development and characterization of LTAappended chitosan nanoparticles for mucosal immunization against hepatitis B, Artif. Cells Nanomed. Biotechnol. 42 (2014) 245–255. [44] N. Mishra, S. Tiwari, B. Vaidya, G.P. Agrawal, S.P. Vyas, Lectin anchored PLGA nanoparticles for oral mucosal immunization against hepatitis B, J. Drug Target. 19 (2011) 67–78. [45] Y.M. Zhao, L.P. Gao, H.L. Zhang, J.X. Guo, P.P. Guo, Grape seed proanthocyanidin extract prevents DDP-induced testicular toxicity in rats, Food Funct. 5 (2014) 605–611. [46] S. Jain, V.V. Rathi, A.K. Jain, M. Das, C. Godugu, Folate-decorated PLGA nanoparticles as a rationally designed vehicle for the oral delivery of insulin, Nanomedicine UK 7 (2012) 1311–1337. [47] Y. Yun, Y.W. Cho, K. Park, Nanoparticles for oral delivery: targeted nanoparticles with peptidic ligands for oral protein delivery, Adv. Drug Deliv. Rev. 65 (2013) 822–832. [48] K. Sonaje, Y.H. Lin, J.H. Juang, S.P. Wey, C.T. Chen, H.W. Sung, In vivo evaluation of safety and efficacy of self-assembled nanoparticles for oral insulin delivery, Biomaterials 30 (2009) 2329–2339. [49] K. Sonaje, Y.J. Chen, H.L. Chen, S.P. Wey, J.H. Juang, H.N. Nguyen, C.W. Hsu, K.J. Lin, H.W. Sung, Enteric-coated capsules filled with freeze-dried chitosan/poly(gammaglutamic acid) nanoparticles for oral insulin delivery, Biomaterials 31 (2010) 3384–3394. [50] C.B. Woitiski, R.J. Neufeld, F. Veiga, R.A. Carvalho, I.V. Figueiredo, Pharmacological effect of orally delivered insulin facilitated by multilayered stable nanoparticles, Eur. J. Pharm. Sci. 41 (2010) 556–563. [51] H.N. Nguyen, S.P. Wey, J.H. Juang, K. Sonaje, Y.C. Ho, E.Y. Chuang, C.W. Hsu, T.C. Yen, K.J. Lin, H.W. Sung, The glucose-lowering potential of exendin-4 orally delivered via a pH-sensitive nanoparticle vehicle and effects on subsequent insulin secretion in vivo, Biomaterials 32 (2011) 2673–2682.

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[52] A. Lalatsa, V. Lee, J.P. Malkinson, M. Zloh, A.G. Schatzlein, I.F. Uchegbu, A prodrug nanoparticle approach for the oral delivery of a hydrophilic peptide, leucine(5)enkephalin, to the brain, Mol. Pharm. 9 (2012) 1665–1680. [53] K. Sonaje, K.J. Lin, S.P. Wey, C.K. Lin, T.H. Yeh, H.N. Nguyen, C.W. Hsu, T.C. Yen, J.H. Juang, H.W. Sung, Biodistribution, pharmacodynamics and pharmacokinetics of insulin analogues in a rat model: oral delivery using pH-responsive nanoparticles vs. subcutaneous injection, Biomaterials 31 (2010) 6849–6858. [54] F.Y. Su, K.J. Lin, K. Sonaje, S.P. Wey, T.C. Yen, Y.C. Ho, N. Panda, E.Y. Chuang, B. Maiti, H.W. Sung, Protease inhibition and absorption enhancement by functional nanoparticles for effective oral insulin delivery, Biomaterials 33 (2012) 2801–2811. [55] E.Y. Chuang, K.J. Lin, F.Y. Su, H.L. Chen, B. Maiti, Y.C. Ho, T.C. Yen, N. Panda, H.W. Sung, Calcium depletion-mediated protease inhibition and apical–junctionalcomplex disassembly via an EGTA-conjugated carrier for oral insulin delivery, J. Control. Release 169 (2013) 296–305. [56] X.Y. Li, S.Y. Guo, C.L. Zhu, Q.L. Zhu, Y. Gan, J. Rantanen, U.L. Rahbek, L. Hovgaard, M.S. Yang, Intestinal mucosa permeability following oral insulin delivery using core shell corona nanolipoparticles, Biomaterials 34 (2013) 9678–9687. [57] F. Cui, K. Shi, L. Zhang, A. Tao, Y. Kawashima, Biodegradable nanoparticles loaded with insulin-phospholipid complex for oral delivery: preparation, in vitro characterization and in vivo evaluation, J. Control. Release 114 (2006) 242–250. [58] F.D. Cui, A.J. Tao, D.M. Cun, L.Q. Zhang, K. Shi, Preparation of insulin loaded PLGAHp55 nanoparticles for oral delivery, J. Pharm. Sci. 96 (2007) 421–427. [59] G. Sharma, C.F. van der Walle, M.N. Ravi Kumar, Antacid co-encapsulated polyester nanoparticles for peroral delivery of insulin: development, pharmacokinetics, biodistribution and pharmacodynamics, Int. J. Pharm. 440 (2013) 99–110. [60] Z.M. Wu, L. Zhou, X.D. Guo, W. Jiang, L. Ling, Y. Qian, K.Q. Luo, L.J. Zhang, HP55coated capsule containing PLGA/RS nanoparticles for oral delivery of insulin, Int. J. Pharm. 425 (2012) 1–8. [61] K. Wang, J. Qi, T. Weng, Z. Tian, Y. Lu, K. Hu, Z. Yin, W. Wu, Enhancement of oral bioavailability of cyclosporine a: comparison of various nanoscale drug-delivery systems, Int. J. Nanomedicine 9 (2014) 4991–4999. [62] S. Onoue, H. Suzuki, Y. Kojo, S. Matsunaga, H. Sato, T. Mizumoto, K. Yuminoki, N. Hashimoto, S. Yamada, Self-micellizing solid dispersion of cyclosporine A with improved dissolution and oral bioavailability, Eur. J. Pharm. Sci. 62 (2014) 16–22. [63] J. Lai, Y. Lu, Z. Yin, F. Hu, W. Wu, Pharmacokinetics and enhanced oral bioavailability in beagle dogs of cyclosporine A encapsulated in glyceryl monooleate/poloxamer 407 cubic nanoparticles, Int. J. Nanomedicine 5 (2010) 13–23. [64] W. Qu, Y. Li, L. Hovgaard, S. Li, W. Dai, J. Wang, X. Zhang, Q. Zhang, A silicabased pH-sensitive nanomatrix system improves the oral absorption and efficacy of incretin hormone glucagon-like peptide-1, Int. J. Nanomedicine 7 (2012) 4983–4994. [65] W.B. Dai, Y.L. Guo, H. Zhang, X.Q. Wang, Q. Zhang, Sylysia 350/Eudragit S100 solid nanomatrix as a promising system for oral delivery of cyclosporine A, Int. J. Pharm. 478 (2015) 718–725. [66] Z.H. Zhang, Y.L. Zhang, J.P. Zhou, H.X. Lv, Solid lipid nanoparticles modified with stearic acid-octaarginine for oral administration of insulin, Int. J. Nanomedicine 7 (2012) 3333–3339. [67] S.V. Rao, K. Yajurvedi, J. Shao, Self-nanoemulsifying drug delivery system (SNEDDS) for oral delivery of protein drugs: III. In vivo oral absorption study, Int. J. Pharm. 362 (2008) 16–19. [68] F. Hintzen, G. Perera, S. Hauptstein, C. Muller, F. Laffleur, A. Bernkop-Schnurch, In vivo evaluation of an oral self-microemulsifying drug delivery system (SMEDDS) for leuprorelin, Int. J. Pharm. 472 (2014) 20–26. [69] A.K. Agrawal, H. Harde, K. Thanki, S. Jain, Improved stability and antidiabetic potential of insulin containing folic acid functionalized polymer stabilized multilayered liposomes following oral administration, Biomacromolecules 15 (2014) 350–360. [70] D. Chen, D. Xia, X. Li, Q. Zhu, H. Yu, C. Zhu, Y. Gan, Comparative study of Pluronic((R)) F127-modified liposomes and chitosan-modified liposomes for mucus penetration and oral absorption of cyclosporine A in rats, Int. J. Pharm. 449 (2013) 1–9. [71] C. Fu, T. Liu, L. Li, H. Liu, D. Chen, F. Tang, The absorption, distribution, excretion and toxicity of mesoporous silica nanoparticles in mice following different exposure routes, Biomaterials 34 (2013) 2565–2575. [72] T. Andreani, A.L. de Souza, C.P. Kiill, E.N. Lorenzon, J.F. Fangueiro, A.C. Calpena, M.V. Chaud, M.L. Garcia, M.P. Gremiao, A.M. Silva, E.B. Souto, Preparation and characterization of PEG-coated silica nanoparticles for oral insulin delivery, Int. J. Pharm. 473 (2014) 627–635. [73] M. Mahkam, F. Hosseinzadeh, M. Galehassadi, Preparation of ionic liquid functionalized silica nanoparticles for oral drug delivery, J. Biomater. Nanobiotechnol. 3 (2012) 391–395. [74] X.H. Zhao, C. Shan, Y.G. Zu, Y. Zhang, W.G. Wang, K.L. Wang, X.Y. Sui, R.Q. Li, Preparation, characterization, and evaluation in vivo of Ins-SiO2-HP55 (insulinloaded silica coating HP55) for oral delivery of insulin, Int. J. Pharm. 454 (2013) 278–284. [75] D.R. Bhumkar, H.M. Joshi, M. Sastry, V.B. Pokharkar, Chitosan reduced gold nanoparticles as novel carriers for transmucosal delivery of insulin, Pharm. Res. Dordr. 24 (2007) 1415–1426. [76] J.F. Guo, K. Rahme, K.A. Fitzgerald, J.D. Holmes, C.M. O'Driscoll, Biomimetic gold nanocomplexes for gene knockdown: will gold deliver dividends for small interfering RNA nanomedicines? Nano Res. 8 (2015) 3111–3140. [77] N.L. Trevaskis, L.M. Kaminskas, C.J. Porter, From sewer to saviour — targeting the lymphatic system to promote drug exposure and activity, Nat. Rev. Drug Discov. 14 (2015) 781–803. [78] C.M. O'Driscoll, Anatomy and Physiology of the Lymphatics, in: W.N. Charman, V.J. Stella (Eds.), Lymphatic Transport of Drugs, Taylor and Francis Group 1992, pp. 1–35.


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[79] B.T. Griffin, C.M. O'Driscoll, Opportunities and challenges for oral delivery of hydrophobic versus hydrophilic peptide and protein-like drugs using lipid-based technologies, Ther. Deliv. 2 (2011) 1633–1653. [80] H.K. Shete, N. Selkar, G.R. Vanage, V.B. Patravale, Tamoxifen nanostructured lipid carriers: enhanced in vivo antitumor efficacy with reduced adverse drug effects, Int. J. Pharm. 468 (2014) 1–14. [81] A. Estella-Hermoso de Mendoza, M.A. Campanero, H. Lana, J.A. Villa-Pulgarin, J. de la Iglesia-Vicente, F. Mollinedo, M.J. Blanco-Prieto, Complete inhibition of extranodal dissemination of lymphoma by edelfosine-loaded lipid nanoparticles, Nanomedicine (London) 7 (2012) 679–690. [82] S. Attili-Qadri, N. Karra, A. Nemirovski, O. Schwob, Y. Talmon, T. Nassar, S. Benita, Oral delivery system prolongs blood circulation of docetaxel nanocapsules via lymphatic absorption, Proc. Natl. Acad. Sci. U. S. A. 110 (2013) 17498–17503. [83] E.Y. Chuang, G.T. Nguyen, F.Y. Su, K.J. Lin, C.T. Chen, F.L. Mi, T.C. Yen, J.H. Juang, H.W. Sung, Combination therapy via oral co-administration of insulin- and exendin-4loaded nanoparticles to treat type 2 diabetic rats undergoing OGTT, Biomaterials 34 (2013) 7994–8001. [84] F.Y. Su, E.Y. Chuang, P.Y. Lin, Y.C. Chou, C.T. Chen, F.L. Mi, S.P. Wey, T.C. Yen, K.J. Lin, H.W. Sung, Treatment of chemotherapy-induced neutropenia in a rat model by using multiple daily doses of oral administration of G-CSF-containing nanoparticles, Biomaterials 35 (2014) 3641–3649. [85] B. Semete, L. Booysen, L. Kalombo, B. Ramalapa, R. Hayeshi, H.S. Swai, Effects of protein binding on the biodistribution of PEGylated PLGA nanoparticles post oral administration, Int. J. Pharm. 424 (2012) 115–120. [86] X. Zhang, M. Sun, A. Zheng, D. Cao, Y. Bi, J. Sun, Preparation and characterization of insulin-loaded bioadhesive PLGA nanoparticles for oral administration, Eur. J. Pharm. Sci. 45 (2012) 632–638. [87] A.C. Alves, C.M. Gontijo, M.C. Oliveira, S.O.F. Diniz, F.M. Oliveira, V.N. Cardoso, G.A. Ramaldes, Biodistribution of free 99mTc-ovalbumin and 99MTc-ovalbumin encapsulated in liposomes, Braz. Arch. Biol. Technol. 48 (2005) 235–241. [88] L.M. Bimbo, M. Sarparanta, H.A. Santos, A.J. Airaksinen, E. Makila, T. Laaksonen, L. Peltonen, V.P. Lehto, J. Hirvonen, J. Salonen, Biocompatibility of thermally hydrocarbonized porous silicon nanoparticles and their biodistribution in rats, ACS Nano 4 (2010) 3023–3032. [89] L. Yang, Y.C. Lee, M. Il Kim, H.G. Park, Y.S. Huh, Y.T. Shao, H.K. Han, Biodistribution and clearance of aminoclay nanoparticles: implication for in vivo applicability as a tailor-made drug delivery carrier, J. Mater. Chem. B 2 (2014) 7567–7574. [90] M.S. Falzarano, C. Passarelli, E. Bassi, M. Fabris, D. Perrone, P. Sabatelli, N.M. Maraldi, S. Dona, R. Selvatici, P. Bonaldo, K. Sparnacci, M. Laus, P. Braghetta, P. Rimessi, A. Ferlini, Biodistribution and molecular studies on orally administered nanoparticle–AON complexes encapsulated with alginate aiming at inducing dystrophin rescue in mdx mice, Biomed. Res. Int. (2013). [91] Y. Liu, M. Kong, C. Feng, K.K. Yang, Y. Li, J. Su, X.J. Cheng, H.J. Park, X.G. Chen, Biocompatibility, cellular uptake and biodistribution of the polymeric amphiphilic nanoparticles as oral drug carriers, Colloids Surf. B 103 (2013) 345–353. [92] P. Areses, M.T. Agueros, G. Quincoces, M. Collantes, J.A. Richter, L.M. Lopez-Sanchez, M. Sanchez-Martinez, J.M. Irache, I. Penuelas, Molecular imaging techniques to study the biodistribution of orally administered Tc-99m-labelled naive and ligand-tagged nanoparticles, Mol. Imaging Biol. 13 (2011) 1215–1223. [93] D.S. Wilson, G. Dalmasso, L.X. Wang, S.V. Sitaraman, D. Merlin, N. Murthy, Orally delivered thioketal nanoparticles loaded with TNF-alpha-siRNA target inflammation and inhibit gene expression in the intestines, Nat. Mater. 9 (2010) 923–928. [94] A. Makhlof, M. Werle, Y. Tozuka, H. Takeuchi, A mucoadhesive nanoparticulate system for the simultaneous delivery of macromolecules and permeation enhancers to the intestinal mucosa, J. Control. Release 149 (2011) 81–88.

[95] M. Tobio, A. Sanchez, A. Vila, I. Soriano, C. Evora, J.L. Vila-Jato, M.J. Alonso, The role of PEG on the stability in digestive fluids and in vivo fate of PEG–PLA nanoparticles following oral administration, Colloids Surf. B 18 (2000) 315–323. [96] J.J. Reineke, D.Y. Cho, Y.T. Dingle, A.P. Morello, J. Jacob, C.G. Thanos, E. Mathiowitz, Unique insights into the intestinal absorption, transit, and subsequent biodistribution of polymer-derived microspheres, Proc. Natl. Acad. Sci. U. S. A. 110 (2013) 13803–13808. [97] S.H. Bakhru, S. Furtado, A.P. Morello, E. Mathiowitz, Oral delivery of proteins by biodegradable nanoparticles, Adv. Drug Deliv. Rev. 65 (2013) 811–821. [98] P. Thygesen, P. Macheras, A. Van Peer, Physiologically-based PK/PD modelling of therapeutic macromolecules, Pharm. Res. 26 (2009) 2543–2550. [99] A. Rostami-Hodjegan, Physiologically based pharmacokinetics joined with in vitroin vivo extrapolation of ADME: a marriage under the arch of systems pharmacology, Clin. Pharmacol. Ther. 92 (2012) 50–61. [100] F. Khalil, S. Laer, Physiologically based pharmacokinetic modeling: methodology, applications, and limitations with a focus on its role in pediatric drug development, J. Biomed. Biotechnol. (2011). [101] D.M. Moss, M. Siccardi, Optimizing nanomedicine pharmacokinetics using physiologically based pharmacokinetics modelling, Brit. J. Pharmacol. 171 (2014) 3963–3979. [102] L. Diao, B. Meibohm, Tools for predicting the PK/PD of therapeutic proteins, Expert Opin. Drug Metab. Toxicol. 11 (2015) 1115–1125. [103] E. Offman, A.N. Edginton, A PBPK workflow for first-in-human dose selection of a subcutaneously administered pegylated peptide, J. Pharmacokinet. Pharmacodyn. 42 (2015) 135–150. [104] M. Li, K.T. Al-Jamal, K. Kostarelos, J. Reineke, Physiologically based pharmacokinetic modeling of nanoparticles, ACS Nano 4 (2010) 6303–6317. [105] F. Khalil, S. Laer, Physiologically based pharmacokinetic models in the prediction of oral drug exposure over the entire pediatric age range-sotalol as a model drug, AAPS J. 16 (2014) 226–239. [106] E.S. Kostewicz, L. Aarons, M. Bergstrand, M.B. Bolger, A. Galetin, O. Hatley, M. Jamei, R. Lloyd, X. Pepin, A. Rostami-Hodjegan, E. Sjogren, C. Tannergren, D.B. Turner, C. Wagner, W. Weitschies, J. Dressman, PBPK models for the prediction of in vivo performance of oral dosage forms, Eur. J. Pharm. Sci. 57 (2014) 300–321. [107] H. Jones, K. Rowland-Yeo, Basic concepts in physiologically based pharmacokinetic modeling in drug discovery and development, CPT Pharmacometrics Syst Pharmacol. 2 (2013), e63. [108] D. Li, C. Emond, G. Johanson, O. Jolliet, Using a PBPK model to study the influence of different characteristics of nanoparticles on their biodistribution, J. Phys. Conf. Ser. 429 (2013). [109] D.J. Crommelin, A.T. Florence, Towards more effective advanced drug delivery systems, Int. J. Pharm. 454 (2013) 496–511. [110] F. Araujo, N. Shrestha, P.L. Granja, J. Hirvonen, H.A. Santos, B. Sarmento, Safety and toxicity concerns of orally delivered nanoparticles as drug carriers, Expert Opin. Drug Metab. Toxicol. 11 (2015) 381–393. [111] H.G. Eichler, L.G. Baird, R. Barker, B. Bloechl-Daum, F. Borlum-Kristensen, J. Brown, R. Chua, S. Del Signore, U. Dugan, J. Ferguson, S. Garner, W. Goettsch, J. Haigh, P. Honig, A. Hoos, P. Huckle, T. Kondo, Y. Le Cam, H. Leufkens, R. Lim, C. Longson, M. Lumpkin, J. Maraganore, B. O'Rourke, K. Oye, E. Pezalla, F. Pignatti, J. Raine, G. Rasi, T. Salmonson, D. Samaha, S. Schneeweiss, P.D. Siviero, M. Skinner, J.R. Teagarden, T. Tominaga, M.R. Trusheim, S. Tunis, T.F. Unger, S. Vamvakas, G. Hirsch, From adaptive licensing to adaptive pathways: delivering a flexible life-span approach to bring new drugs to patients, Clin. Pharmacol. Ther. 97 (2015) 234–246. [112] F. Ehmann, K. Sakai-Kato, R. Duncan, D. Hernan Perez de la Ossa, R. Pita, J.M. Vidal, A. Kohli, L. Tothfalusi, A. Sanh, S. Tinton, J.L. Robert, B. Silva Lima, M.P. Amati, Nextgeneration nanomedicines and nanosimilars: EU regulators' initiatives relating to the development and evaluation of nanomedicines, Nanomedicine (London) 8 (2013) 849–856.
