Current Challenges in Non-Invasive Insulin Delivery Systems a Comparative Review

Available online at www.sciencedirect.com

ws 59 (2007) 1521–1546www.elsevier.com/locate/addr

Advanced Drug Delivery Revie

Current challenges in non-invasive insulin delivery systems:A comparative review

El-Sayed Khafagy, Mariko Morishita ⁎, Yoshinori Onuki, Kozo Takayama

Department of Pharmaceutics, Hoshi University, Ebara 2-4-41, Shinagawa, Tokyo 142-8501, Japan

Received 23 March 2007; accepted 16 August 2007Available online 22 August 2007

Abstract

The quest to eliminate the needle from insulin delivery and to replace it with non- or less-invasive alternative routes has driven rigorouspharmaceutical research to replace the injectable forms of insulin. Recently, various approaches have been studied involving many strategies usingvarious technologies that have shown success in delivering insulin, which are designed to overcome the inherent barriers for insulin uptake acrossthe gastrointestinal tract, mucosal membranes and skin. This review examines some of the many attempts made to develop alternative, moreconvenient routes for insulin delivery to avoid existing long-term dependence on multiple subcutaneous injections and to improve thepharmacodynamic properties of insulin. In addition, this article concentrates on the successes in this new millennium in developing potential non-invasive technologies and devices, and on major new milestones in modern insulin delivery for the effective treatment of diabetes.© 2007 Elsevier B.V. All rights reserved.

Keywords: Non-invasive delivery system; Modern insulin delivery; Administration routes; Marketed products; Formulation technologies; Future patents

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15222. Routes of insulin administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1522

2.1. Oral administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15222.1.1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15222.1.2. Absorption enhancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15232.1.3. Enzyme inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15242.1.4. Mucoadhesive polymeric systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15242.1.5. Particulate carrier delivery systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15252.1.6. Targeted delivery systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1526

2.2. Buccal administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15262.3. Nasal administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15282.4. Pulmonary administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1529

2.4.1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15292.4.2. Dry powder inhalation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15302.4.3. Absorption enhancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15302.4.4. Particulate carrier systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1531

2.5. Ocular administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15322.6. Rectal administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15332.7. Transdermal administration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1534

⁎ Corresponding author. Tel./fax: +81 3 5498 5783.E-mail address: [email protected] (M. Morishita).

0169-409X/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.addr.2007.08.019

http://dx.doi.org/10.1016/j.addr.2007.08.019

mailto:[email protected]

1522 E.-S. Khafagy et al. / Advanced Drug Delivery Reviews 59 (2007) 1521–1546

2.7.1. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15342.7.2. Absorption enhancers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15342.7.3. Transdermal drug delivery systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15342.7.4. Iontophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15352.7.5. Sonophoresis (ultrasound) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15362.7.6. Microneedles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1536

3. Recent marketed and developed formulations for non-invasive insulin delivery . . . . . . . . . . . . . . . . . . . . . . . . 15374. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1539References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1540

1. Introduction

Advances in biotechnology have meant that a wide variety ofpeptide drugs are now produced on a commercial scale. Withthe advent of biotechnology, particularly advances in recombi-nant protein technology, the therapeutic roles of peptides andproteins have received a great deal of attention [1–3]. Proteinand peptide drug delivery has been an area of intensive researchbecause of their efficacy in several disease conditions. However,despite rapid progress in the large-scale manufacture oftherapeutic proteins, the convenient and effective delivery ofthese drugs to the body remains a major challenge. Most ofthese therapeutic peptides are still administered by the parenteralroute because of their poor bioavailability when delivered viaother routes. Peptide drugs are usually indicated for chronicconditions, and the use of injections on a daily basis duringlong-term treatment has obvious drawbacks [4]. The past decadesaw increased interest in the formulation and delivery ofbiological drugs for a range of diseases. Unlike conventionaldrugs, the clinical development of these types of drugs has notbeen possible without some sort of sophisticated pharmaceuticaltechnology. Among the most important therapeutic proteins andpeptides being explored is insulin. Insulin was isolated frombovine pancreas in 1922 by Frederick Banting and Charles Best,who received the 1923 Nobel Prize for Medicine with JohnMcLeod. Conventional insulin treatment is basically a replace-ment therapy, in which exogenous insulin is administeredsubcutaneously to mimic, as closely as possible, the insulinsecretion of the healthy pancreas. The subcutaneous route hasbeen the mainstay of insulin delivery until now. Although theseparenteral routes are satisfactory in terms of efficacy in the greatmajority of cases, they can result in peripheral hyperinsulinemia,the stimulation of smooth muscle cell proliferation, and theincorporation of glucose into the lipid of arterial walls, and theymight therefore be the causative factor in diabetic micro- andmacroangiopathy [5]. Moreover, the burden of daily injections,physiological stress, pain, inconvenience, cost, risks, infection,inability to handle insulin, and the localized deposition ofinsulin, leading to local hypertrophy and fat deposition at theinjection sites remain problems [6]. The synthesis of insulin byrecombinant DNA technology represented an important scien-tific milestone and made large quantities of the protein availableat an affordable price, a factor that led insulin to become one ofthe most popular proteins to be studied for non-parenteraldelivery. Consequently, the results of research into several

aspects of the delivery of the insulin are available. In recentyears, there has been a great deal of interest in the exploitationof non-invasive routes for insulin delivery, and their develop-ment by the pharmaceutical industry, including oral, nasal,buccal, pulmonary, transdermal, rectal, and ocular drug deliverysystems [7–9]. The objective of this review is to provide anupdate on the most promising advances in non-invasivelydelivery systems for insulin that may overcome the barriers toits absorption.

2. Routes of insulin administration

2.1. Oral administration

2.1.1. BackgroundThe gastrointestinal tract (GIT) is the route of choice for the

administration of most drugs, regardless of their molecularstructure or weight. The manufacture of an oral dosage formdoes not have tomeet specialized regulatory requirements relatingto such issues as sterility, pyrogenicity, and particulate contam-ination. Insulin has an important place in drug therapies forinsulin-dependent diabetes mellitus (type I) and for many patientswith non-insulin-dependent diabetes mellitus (type II). However,it is still generally delivered via injections. It would be highlyadvantageous if insulin could be administered orally [10], becausethe oral delivery of insulin can mimic the physiological fate ofinsulin and may provide better glucose homeostasis. This wouldalso lessen the incidence of peripheral hyperinsulinemia, which isassociated with neuropathy, retinoendopathy, and so forth [11].Various challenges are usually evaluated by determining the fateof insulin in the GIT. The main challenges reported involveovercoming the enzymatic degradation of insulin and theinsufficient permeation of insulin through the GIT [12]. Successin the oral delivery of therapeutic insulin would improve thequality of life of many people who must routinely receiveinjections of this drug. In the last few decades, various attemptshave been made to overcome the limitations and drawbacks ofconventional oral insulin therapy. The successful oral delivery ofinsulin involves overcoming the barrier of enzymatic degradation,achieving epithelial permeability, and conserving the bioactivityof the drug during formulation processing. Pharmaceuticalstrategies have been proposed to maximize oral insulin bioavail-ability in insulin delivery systems, to overcome barriers, and todevelop safe and effective therapies. Table 1 presents a summaryof recent oral insulin delivery systems.

Table 1Oral insulin delivery systems

System Application Observation Refs.

Absorption enhancersBile salt/fatty acid mixed micellar system In vivo/rats Improved paracellular absorption [13]Insulin solution/N-lauryl-β-D-maltopyranoside In vitro/Ussing chamber Enhanced colon permeability [14]W/O/W emulsion/DHA or EPA In situ/rat intestine PAa was 43.2% [15]Enteric-coated capsule/Witepsol W35+NaSal In vivo/dogs PAa was 12.6% [16]Insulin solution/AP, SGC, SC or Na2EDTA In situ/rat intestine PAb was 0.1%–5.5% [17]

Enzyme inhibitorsDrug–carrier matrix/BBI and elastatinal In vitro/artificial intestinal fluid Significant reduction in enzymatic degradation [20]Insulin solution/AP, SGC, STI, CM or BAC In situ/rat intestine PAb was 0.1%–5.1% [21]Insulin solution/SGC, BTT, LPT, CTT or BAC In situ/rat intestine PAb was 0.1%–2.3% [22]Insulin solution/CkOVM or DkOVM In vitro/diffusion chamber Two fold increase in insulin stability and flux values [25]Insulin solution/hyaluronidase In situ/rat intestine Significant reduction in blood glucose levels [27]

Mucoadhesive polymeric systemsP(MAA-g-EG) hydrogel microparticles In vivo/rats PAa was 9.5% [47]Lectin-conjugated alginate microparticles In vivo/rats Hypoglycemic effect lasted for ∼8 h [48]Chitosan NPs In vivo/rats PAa was 14.9% [49]Chitosan–TBA–insulin tablets In vivo/rats PAa was 1.69% [61]

Particulate carrier delivery systemsLecithin-based microemulsion In vivo/rats 30% reduction in blood glucose levels [74]Double liposomes In vivo/rats PAb was 0.39%–5.5% [76]Fusogenic liposomes In situ/rat intestine PAa was 10.1%–15.7% [78]Eudragit S100 microspheres In vivo/rabbits 24% reduction in blood glucose levels [82]Insulin–phospholipid complex NPs In vivo/rats PAa was 7.7% [84]

Targeted delivery systemsColon-targeted delivery system (CODES™) In vivo/dogs BA was 0.5% [90]Colon-targeted delivery system (Azopolymer-coated pellets) In vivo/rats PAb was 0.89%–3.38% [91]Insulin–transferrin conjugate In vivo/rats 70% reduction in blood glucose levels [96]

Abbreviations: DHA, docosahexaenoic acid; EPA, eicosapentaenoic acid; PA, pharmacological availability; NaSal, sodium salicylate; RH, relative hypoglycemia;AP, aprotinin; SGC, sodium glycocholate; SC, sodium caprate; Na2EDTA, ethylenediaminetetraacetic acid disodium salt; BBI, Bowman–Birk inhibitor;STI, soybean trypsin inhibitor; CM, camostat mesilate; BAC, bacitracin; BTT, bestatin; LPT, leupeptin; CTT, cystatin; CkOVM, chicken ovomucoid;DkOVM, duck ovomucoid; P(MAA-g-EG), poly(methacrylic acid-g-ethylene glycol); NPs, nanoparticles; chitosan–TBA–insulin, chitosan–4-thiobutylamidineinsulin conjugate; BA, absolute bioavailability. PA of insulin systems was determined based on the extent of the hypoglycemic response relative to that achievedwith subcutaneousa or intravenousb insulin injection.

1523E.-S. Khafagy et al. / Advanced Drug Delivery Reviews 59 (2007) 1521–1546

2.1.2. Absorption enhancersAbsorption enhancers improve the absorption of drugs by

increasing their paracellular and transcellular transport. Theyinvolve several different mechanisms of action, including changesin membrane fluidity, decrease in mucus viscosity, the leakage ofproteins through membranes, and the opening of tight junctions[4]. Common examples of non-specific permeation enhancers arebile salts, fatty acids, surfactants, salicylates, chelators, and zonulaoccludens toxin.

Bile salts in mixed micellar systems increase the permeationof insulin by accessing a paracellular pathway [13]. A study ofN-lauryl-β-D-maltopyranoside also suggested that this enhancermay open the tight junctions of the epithelium, thereby increasingthe permeation of insulin via a paracellular pathway [14]. Inanother interesting study,water-in-oil-in-watermultiple emulsionsincorporating 2% docosahexaenoic acid or eicosapentaenoic acidhad dose-related pharmacological effects on insulin and maypotentially become the formulations for the enteral delivery ofinsulin [15]. Another report demonstrated the hypoglycemiceffects of enteric-coated capsules containing insulin formulated inWitepsol W35 with sodium salicylate, which significantly

decreased plasma glucose levels and increased hypoglycemiarelative to the effects of a subcutaneous injection of regular solubleinsulin [16].Morishita et al. evaluated the administration of insulinsolution to the various colonic and rectal loops of fasted ratsin situ, with or without sodium caprate, Na2EDTA, or sodiumglycocholate as an absorption enhancer, and with or withoutprotease inhibitors such as aprotinin [17]. Their results suggestedthat absorption enhancers increase insulin efficacy more effec-tively in the colon than in the small intestine. An innovativestrategy involving themodulation of tight junctions to improve thetransport of paracellular drugs and proteins normally not absorbedthrough the intestine is a very attractive solution [18]. In diabeticrats, the bioavailability of oral insulin coadministered with zonulaoccludens toxin was sufficient to lower serum glucose concentra-tions to levels similar to those achieved after the parenteralinjection of insulin [19].

However, the use of absorption enhancers is limited bythe fact that once cell membranes are permeabilized or tightjunctions opened, transport is enhanced not only for peptide andprotein drugs but also for undesirable molecules present in theGIT [9].


2.1.3. Enzyme inhibitorsRecent studies have evaluated the use of enzyme inhibitors to

slow the rate of insulin degradation. The coadministration ofenzyme inhibitors provides a viable means to circumvent theenzymatic barrier to the delivery of peptide and protein drugs.Insulin is strongly degraded by trypsin, α-chymotrypsin, andelastase, and to a lesser extent, by brush-border membrane-boundenzymes [20].

In an interesting study, Yamamoto et al. evaluated the effects offive different enzyme inhibitors—sodium glycocholate, camostatmesilate, bacitracin, soybean trypsin inhibitor, and aprotinin—onthe intestinal metabolism of insulin in rats [21]. Among theseenzyme inhibitors, sodium glycocholate, camostat mesilate, andbacitracin were effective in improving the physiological avail-ability of insulin in the large intestine. However, none of theseenzyme inhibitors was effective in the small intestine, possiblybecause of the numerous enzymes secreted there. Liu et al. alsoevaluated the potential utility of various enzyme inhibitors inimproving the intestinal absorption of insulin and investigatedtheir efficacy in different intestinal regions [22].

Recently, chicken and duck ovomucoids have been identifiedas a new class of enzyme inhibitors [23,24]. Ovomucoids derivedfrom the egg whites of avian species were tested for their efficacyin preventing insulin degradation in the presence of trypsin andα-chymotrypsin. A formulation containing insulin and chickenovomucoid (CkOVM) or duck ovomucoid (DkOVM) wasevaluated for its dissolution stability in the presence of trypsinandα-chymotrypsin [25]. The results suggested that the inhibitoryeffect of DkOVM was due to the inactivation of the enzymeα-chymotrypsin, which is mainly responsible for the breakdownof insulin.

Polymer–inhibitor conjugates have been shown to offerin vitro protection against trypsin, α-chymotrypsin, and elastase[20]. A drug–carrier matrix has been developed that protectsembedded insulin from in vitro degradation by the luminallysecreted serine proteases trypsin (EC 3.4.21.4), chymotrypsin(EC 3.4.21.1), and elastase (EC 3.4.21.36). Increasing amountsof the Bowman–Birk inhibitor (BBI) and elastatinal werecovalently bound to the mucoadhesive polymer sodiumcarboxymethylcellulose (Na-CMC). All polymer–BBI conju-gates showed strong inhibitory activity toward trypsin andchymotrypsin, whereas this was markedly lower towardelastase. The polymer–elastatinal conjugates displayed higherinhibitory activity toward elastase. Even after 4 h of incubation,33.6%±3.2% of the therapeutic agent remained stable againstenzymatic attack. These conjugates, when combined withpolycarbophil–cysteine, induced a 20%–40% reduction inbasal glucose levels for more than 80 h [26].

Recent evidence has elegantly demonstrated that the mucusand glycocalyx layers, extracellular domains directly attachedto the intestinal epithelium, function significantly as absorptionand/or proteolytic impedimental compartments for insulin[27,28].

However, the use of enzyme inhibitors may also affect theabsorption of other peptides or proteins that would normally bedegraded. A major drawback of these inhibitors is their hightoxicity, especially during chronic drug therapy. Furthermore, the

non-site-specific intestinal application of such compounds maychange the metabolic pattern in the GIT, because of the reduceddigestion of food proteins.

2.1.4. Mucoadhesive polymeric systemsThe term ‘mucoadhesion’ refers to the adhesion between

polymeric carriers and the mucosa and is exhibited by certainpolymers, which become adhesive upon hydration [29,30]. Thus,the goals of mucoadhesive drug delivery systems are to extend theresidence time at the site of drug absorption, to intensify contactwith the mucus to increase the drug concentration gradient, toensure immediate absorption without dilution or degradation inthe luminal fluid, and to localize the drug delivery system to acertain site [31,32]. Delivery systems containing mucoadhesivepolymers provide intimate contact with the mucosa, therebyreducing drug degradation between the delivery system and theabsorbing membrane. They are controlled release systems thatprovide the simultaneous release of both drug and inhibitor, andallow the immobilization of enzyme inhibitors in the deliverysystems [33]. Novel polymers have shown excellent inhibitoryactivity against proteolytic enzymes and reasonable mucoadhe-sivity, and might therefore be a useful tool in overcoming theenzymatic barrier to oral peptide therapeutics. The binding ofhydrophilic polymers, such as polyacrylates, cellulose derivatives,and chitosan derivatives, to biological surfaces is based onhydrogen bonding and ionic interactions. In the last few years, alarge number of mucoadhesive systems have been developed,including superporous hydrogel-composite-based systems [34],lipid-based nanocarriers [35], thiolated polymers [36], andchitosan-based carriers [37,38]. Some mucoadhesive polymershave also been shown to act as absorption enhancers or inhibitorsof proteolytic enzymes [39].

Several research groups have attempted to integrate pHsensitivity into mucoadhesive polymeric carriers [40–42]. Theaim of these is to protect the sensitive drug from proteolyticdegradation in the stomach and the upper part of the smallintestine. The feasibility of systemic insulin delivery by an oralroute using graft copolymer networks of poly(methacrylic acid-g-ethylene glycol) [P(MAA-g-EG)] has been studied, andMorishitaet al. found the polymer system to be a very promising candidatefor oral drug delivery [43,44]. The smaller-sized microparticularinsulin-loaded polymer showed a rapid burst-type insulin releaseand higher insulin absorption compared with that achieved withlarger microparticles, resulting in a greater hypoglycemic effectwithout detectable mucosal damage [44]. The combined effects ofthese approaches have been demonstrated in the development ofmultifunctional P(MAA-g-EG) hydrogels, a smart polymer thathas sharp pH-dependent release, Ca2+-deprivation ability, andmucoadhesive characteristics [45,46]. This system shows veryhigh (∼10%) pharmacological availability of insulin after oraladministration [47].

In another reported study, lectin-conjugated alginate micro-particles enhanced the intestinal absorption of insulin to facilitate adrop in glucose levels in the blood [48]. In a similar study,mucoadhesive polysaccharide (chitosan) nanoparticles seemed toenhance the intestinal absorption of many peptides and proteins[49].


In recent years, thiolated polymers, designated ‘thiomers’,which are mucoadhesive-based polymers with thiol-bearing sidechains, have appeared as a promising alternative in the arena ofnon-invasive peptide delivery [50]. So far, the cationic thiomerschitosan–cysteine [51], chitosan–thiobutylamidine [52,53], andchitosan–thioglycolic acid [54,55], and the anionic thiomers poly(acrylic acid)–cysteine [56], poly(acrylic acid)–cysteamine [57],carboxymethylcellulose–cysteine [58], and alginate–cysteine[59] have been generated. Because inter- and intramoleculardisulfide bonds form within the thiomer itself, dosage forms suchas tablets and microparticles display strong cohesive properties,resulting in higher stability, prolonged disintegration times, and amore controlled release of the embedded peptide drug [60]. Afterthe oral administration of insulin tablets based on thiolatedpolymer to non-diabetic conscious rats, their blood glucose levelsdecreased significantly for 24 h relative to the decrease achievedwith subcutaneous injection [61].

As described above, numerous mucoadhesive polymericdelivery systems have been proposed. These delivery systemsare thought to be effective in enhancing the intestinal absorption ofbiological molecules vulnerable to proteolytic enzymes [62,63].However, their toxicity over a long period is still unclear, andhuman data are required to regulate their potential use for clinicalapplications.

2.1.5. Particulate carrier delivery systemsMost oral delivery strategies for insulin based on particulate

carriers have been developed to circumvent the barriers to oralpeptide delivery. They efficiently protect protein and peptidedrugs against enzymatic degradation in the harsh environment ofthe GIT, provide high transfer of drugs across the epithelialmucosa, control the release rate, and target drug delivery tospecific intestinal sites [64]. Pathogens and microparticles smallerthan 10 μm enter the gut-associated lymphoid tissues (GALT),which include Peyer's patches, the appendix, and small solitarylymphoid nodules. Peyer's patches are follicles of lymphoid tissuecoveredwith a specialized epithelium containingMcells [65]. Thepotential modes of entry of submicron particles from the intestineinclude via M cells and enterocytes, and by paracellular routes.Histological evaluation of tissue sections has demonstrated that100 nm particles diffuse throughout the submucosal layers,whereas larger particles (10 μm) are predominantly localized inthe epithelial lining of the tissue. Similar particle size-dependentuptake was also confirmed in an experiment with Caco-2 cells[66]. Lysosomal degradation is normally associated with theendocytotic uptake ofmicroparticles, but because this can interferewith the antigen-sampling role of the M cells, Peyer's patches aredeficient in lysosomes. These favorable characteristics of theGALT have stimulated research into targeting Peyer's patches forpeptide and protein delivery [67]. Colloidal carrier systems thathave already been studied to improve peptide delivery includemicroemulsions [68], liposomes [69], polymeric nano- andmicro-particles [70], and polymeric micelles [71].

A novel oral dosage formulation of dry insulin emulsionresponded to changes in the external environment, simulatinggastrointestinal conditions, suggesting that this new enteric-coateddry emulsion formulation is potentially applicable to the oral

delivery of peptide and protein drugs [72]. In another related study,a new solid-in-oil-in-water emulsion for the oral administration ofinsulin has been developed using surfactant-coated insulin [73]. Amicroemulsion of recombinant human (rh)-insulin has alsodemonstrated an improved efficacy of orally administered insulin[74,75].

Liposomes are also a potential alternative carrier for the oraldelivery of proteins. In one particular study, double liposomescontaining insulin were examined in combination with aprotinin[76]. In a similar approach, Zelihagül et al. investigated thepenetration properties of various liposome formulations contain-ing insulin through a Caco-2 cell monolayer. They found that theoral administration of insulin- and sodium-taurocholate-incorpo-rated liposomes significantly decreased blood glucose levels.Furthermore, a high in vitro/in vivo correlation was observedusing the Caco-2 cell monolayer model [77]. In one specific study,fusogenic liposomes were shown to be unique delivery vehiclescapable of introducing their contents directly into the cytoplasmwith the aid of the envelope glycoproteins of Sendai virus [78].The results indicated that fusogenic liposomes are useful carrierswith which to improve the absorption of insulin via the intestinaltract.

Recently, a layer-by-layer self-assembly technique has beenapplied to chitosan and sodium alginate microencapsulation [79].Alginate–chitosan microcapsules provide a simple method forcontrolling the loading and release of protein molecules withinthese polysaccharide microcapsules. Nanocapsules based on poly(ethyl 2-cyanoacrylate) containing insulin to form biocompatiblemicroemulsions represent a convenientmethod for the entrapmentof bioactive peptides [80]. Research in this area has also shed newlight on the potential use of chitosan microspheres in orallyadministered and other mucosally administered protein andpeptide drugs, because they show excellent mucoadhesive andpermeation-enhancing effects across biological surfaces [81].Control of the size and size distribution of chitosan microspheresis necessary to improve their reproducibility, bioavailability, andrepeatable release behavior.

Polyacrylic acid, a pH-dependent material, has revived somehope of achieving an oral insulin formulation. Eudragit S100microspheres have the potential to act as an oral carrier for peptidedrugs like insulin [82]. In a similar approach, novel pH-sensitivepolymethacrylic acid–chitosan–polyethylene glycol nanoparti-cles were prepared under mild aqueous conditions by polyelec-trolyte complexation [83]. A preliminary investigation indicatedthat these particles are a good candidate for oral peptide delivery.In this context, biodegradable nanoparticles loaded with aninsulin–phospholipid complex were prepared by a novel reversemicelle–solvent evaporation method, in which soybean phospha-tidylcholinewas used to improve the liposolubility of insulin, withbiodegradable polymers used as the carrier material to controldrug release [84]. Serum insulin levels following the intragastricadministration of insulin–soybean phosphatidylcholine (Ins–SPC) nanoparticles are illustrated in Fig. 1.

However, difficulties have been encountered that must beovercome to achieve success in carrier delivery systems: the lowincorporation efficiency of hydrophilic drugs; the precise controlof drug release; the avoidance of particle aggregation; and the


possible accumulation of non-degradable particles in tissues. Evenwith degradable particles, the use of unreasonably high quantitiesof carrier can lead to harmful carrier toxicity [85].

2.1.6. Targeted delivery systemsThe desire to deliver protein and peptide biopharmaceuticals

conveniently and effectively has led to the intense investigation oftargeted delivery systems. Despite various challenges, progresstoward the convenient non-invasive delivery of proteins andpeptides has been made through specific routes of administration.The delivery of proteins and peptides to specific sites of action hasbeen used to lower the total dose delivered and to concentrate thetherapeutic dose at specific sites of pharmacological action [86].Absorption is not uniform throughout the GIT, and site-specificabsorption occurs because of differences in the composition andthickness of the mucus layer, pH, surface area, and enzymeactivity [87]. Drug delivery to the colon, for instance, has severalattractive features, including a prolonged residence time, reducedenzymatic activity, increased tissue responsiveness to absorptionenhancers, and natural absorptive characteristics [88]. Oral admin-istration offers a potential portal to the superficial layers of the GIT(local delivery) and to the blood and lymphatic systems (systemicdelivery). However, the harsh hydrolytic environment of the GITand the epithelial barriers to absorption pose major challenges tothe success of this mode of drug delivery for peptide and proteindrugs.

Insulin administration in a colon-targeted delivery system hasbeen developed extensively over the past few years [89]. Thecolon-targeted delivery of insulin with sodium glycocholate wasmore effective in increasing hypoglycemic effects after oraladministration. The combination of sodium glycocholate and poly(ethylene oxide) tended to prolong the absorption of insulin afteroral administration using the colon-targeted delivery system [90].Tozaki et al. also reported that novel azopolymer-coated pelletsmay be useful carriers for the colon-targeted delivery of peptides,including insulin and (Asu1,7)eel–calcitonin [91].

Fig. 1. Plasma glucose levels after oral administration of Ins–SPC nanoparticlesto diabetic rats: NPs 20 IU/kg (■), Ins solution control 20 IU/kg (▲), Ins solution1 IU/kg s.c. (●). The serum insulin levels after oral administration of Ins–SPCnanoparticles in diabetic rats: NPs 20 IU/kg (□), Ins solution control 20 IU/kg(△), Ins solution 1 IU/kg s.c. (○). Data represent means±SD (n=6) [84].

The release of a peptide in a specific region of the GIT, whereuptake into the lymph system is maximized or where enzymeactivity is low, has been used to increase the absorption of drugsafter oral administration [92]. Surface charge and particle size arethe main factors that control the uptake of particulates by Peyer'spatches [93]. Proteins such as lectins and transferrin have alsobeen suggested as transport carriers in the gastrointestinalabsorption of polypeptides. The covalent attachment of tomatolectin molecules to polystyrene particles significantly enhancedtheir uptake by Peyer's patches and normal intestinal tissues [94].The total percentage of the administered dose taken up through thelymphoid tissue was statistically much greater than that absorbedthrough non-lymphoid tissue. It was estimated that 60% of theuptake in the small intestine occurred through the Peyer's patches,even though the patches comprise a small percentage of the totalsurface area of the small intestinal tissue. A significant amount oftotal uptake was also shown to occur in the large intestine, par-ticularly in the lymphoid sections of this tissue. These results wereconfirmed by fluorescence microscopy. Joseph et al. used poly(lactide-co-glycolide) (PLGA) microspheres for the delivery oforal peptides in diabetic mice [95]. The microsphere formulationlowered the glycemic response to oral glucose challenge in themice. Themicrospheres used in this studywere∼1 μm in size andwere supposedly absorbed through the Peyer's patches. Thetransferrin-receptor-mediated transcytosis of an insulin–transfer-ring (In–Tf) conjugate has been demonstrated in Caco-2 cellmonolayers [96]. The results indicated that transepithelialtransport by Tf-receptor-mediated transcytosis is a feasibleapproach to the development of an oral delivery system forinsulin, as well as other peptide drugs. Tyrphostin-8, as anenhancer of Tf-receptor-mediate transcytosis, enhanced hypogly-cemic effects of In-Tf conjugate in diabetic rats, especially at 7 hafter oral administration [97].

Although there have been promising results with agents thatincrease targeted delivery or specific-receptor-mediated transcy-tosis, insufficient quantities of drug-loaded particles wereabsorbed through the intestinal epithelium. Whether the physico-chemical properties of polymers or ligand conjugates favor thenon-specific uptake by enterocytes or M cells remains controver-sial. Furthermore, toxicity problemsmight arise as the result of thecontinued absorption of particles by M cells into Peyer's patches,which could induce an immune response.

2.2. Buccal administration

In the various transmucosal routes of insulin delivery, manystrategies have been used by scientists, and they are compiled inTable 2.

The buccal mucosa has excellent accessibility, an expanse ofsmooth muscle, and a relatively immobile mucosa, and is hencesuitable for the administration of retentive dosage forms. Directaccess to the systemic circulation through the internal jugular veinallows drugs to bypass the hepatic first-pass metabolism, leadingto high bioavailability. Other advantages include low enzymaticactivity, suitability for drug excipients that mildly and reversiblydamage or irritate the mucosa, painless administration, easy drugwithdrawal, facility to include a permeation enhancer/enzyme

Table 2Transmucosal routes of insulin delivery systems

Administrationroute

System Model Results Refs.

BuccalInsulin solution/lysalbinic acid Hamster cheek pouch model Significantly increased insulin permeability [101]Deformable lipid vesicles Rabbits BRa was 19.78% [103]

NasalNasal powder formulation Rabbits BA was 11.1%–22.4% [113]Insulin solution/SDC+CDs Rats 48%–72% reduction in blood glucose levels [114]Insulin solution/Alkylglucosides Rats Significantly enhanced insulin absorption [116]Insulin–chitosan powder Sheep BRa was 17% [126]Chitosan–TBA–insulin microparticles Rats BA was 7.24% [128]AGMS Rats PAb was 8.6% [130]

PulmonaryInsulin microcrystals/Zn2+ Rats 17% reduction in blood glucose levels [137]Insulin/DPPC physical mixture Rats Significant reduction in blood glucose levels [144]Insulin/TDM or DMβCD Rats BRa was 0.19%–0.84% [146]Insulin liposomes Mice Significant reduction in blood glucose levels [147]Insulin–CAP–PEG particles Rats BRa was increased 1.8-fold [148]Insulin PLGA nanospheres Guinea pigs Long-lasting hypoglycemic response [152]Insulin–polybutylcyanoacrylate NPs Rats BRa was 57.2% [159]

OcularInsulin solution/SGC, SDC, STC or POELE Rabbits BAa was 3.6%–8.2% [174]Insulin solution/Alkylglucosides Rats Significantly stimulated systemic insulin

absorption[176]

Insulin solution/sucrose cocoate Rats BA was 5.2% [178]Gelfoam® ocular device Rabbits 60% reduction in blood glucose levels over 8 h [184]

RectalInsulin suppositories/Witepsol W35, SDC, SC, STDCor STC

Dogs PAa was 50% [187]

Insulin suppositories/WitepsolW35, NaSal and POELE Dogs PAa was 49%–55% [188]Insulin glycerol–gelatin suppositories/snail mucin Rats 44% reduction in blood glucose levels within 2 h [190]

Abbreviations: BR, relative bioavailability; BA, absolute bioavailability; SDC, sodium deoxycholate; CDs, cyclodextrins; chitosan–TBA–insulin, chitosan–4-thiobutylamidine insulin conjugate; AGMS, aminated gelatin microspheres; PA, pharmacological availability; DPPC, 1,2-dipalmitoylphosphatidylcholine; TDM,tetradecyl-β-maltoside; DMβCD, dimethyl-β-cyclodextrin; insulin–CAP–PEG, insulin–calcium phosphate polyethylene glycol; PLGA, poly(lactide-co-glycolide); NPs,nanoparticles; SGC, sodium glycocholate; STC, sodium taurocholate; POELE, polyoxyethylene-9-lauryl ether; SC, sodium cholate; STDC, sodium taurodeoxycholate;NaSal, sodium salicylate. PA and BR of insulin systems were determined based on the extent of the hypoglycemic response relative to that achieved with subcutaneousa orintravenousb insulin injection.


inhibitor or pH modifier in the formulation, and versatility in thedesign of multidirectional or unidirectional release systems forlocal or systemic action. The mucosa lining the oral cavityrepresents a potentially important topical route for the delivery ofproteins and therapeutic peptides [98]. It has been shown that thebuccal administration of proteins, such as insulin, interferons, andinterleukins, has some advantages and reduces many related sideeffects. For example, the buccal route provides a constant,predictable drug concentration to the blood. Veuillez et al. haveshown that peptide transport across the buccal mucosa occurs viapassive diffusion and is often accompanied by varying degrees ofmetabolism [99].

Various approaches have been taken to improve the buccalabsorption of peptides, including the use of absorption enhancersto increase membrane permeability and/or the addition of enzymeinhibitors to increase drug stability. From this point of view, therole of absorption enhancers in the buccal transport of proteins iscrucial. Many substances can function as absorption enhancers,

the most popular being detergents such as bile acid salts, sodiumlauryl sulfate, etc. However, many absorption enhancers havesome side effects, often causing irritation of the buccal mucosa.An additional problem is the taste of buccal compositions. Themost efficient absorption enhancers, bile acids salts, have a strongbitter taste, so the regular use of compounds containing bile acidsis hardly acceptable for long-term administration.

Pluronic F-127 (PF-127) gel containing insulin and unsaturatedfatty acids, such as oleic acid (18:1), eicosapentaenoic acid (20:5),or docosahexaenoic acid (22:6), showed a continuous hypogly-cemic effect following its buccal administration in normal rats[100]. PF-127 gels containing oleic acid showed the highestpharmacological availability (15.9%±7.9%). Comparative anal-yses indicated that 20% PF-127 gels containing unsaturated fattyacids are potential formulations for the buccal delivery of insulin.In particular, a good candidate for an effective absorption enhancerseems to be lysalbinic acid [101]. Lysalbinic acid, a product of thealkaline hydrolysis of egg albumin and a mild detergent, meets


those requirements. The experimental data suggest that, using thismethod, it is possible to determine quickly and qualitatively thecomposition of the formulation to be used for the buccal deliveryof large medicinal molecules like α-interferon and insulin. Thus,lysalbinic acid has been shown to increase significantly theparacellular permeability of the hamster oral mucosa to peptidecompounds of low to medium molecular weights. The molecularmechanism by which lysalbinic acid increases transmucosaltransport is not yet completely clear. However, it may be similar tothat of other detergent enhancers (sodium dodecyl sulfate, bilesalts, etc.), based on intercellular lipid solubilization. Histologicalinvestigation of the rat mucosa has shown that lysalbinic acid hasno irritating or sensibilizing effects during buccal use [102]. Inrecent years, a novel type of highly deformable lipid vesicle(transferosome), composed of soybean phosphotidylcholine,cholesterol, and sodium deoxycholate, has been developed toenhance insulin bioavailability [103]. Compared with the sub-cutaneous administration of insulin solution, the relative pharma-cological bioavailability and the relative bioavailability in theexperimental group treated with insulin-deformable vesicles were15.59% and 19.78%, respectively, which were higher than thoseof rabbits treated with conventional insulin vesicles (Pb0.05),blank deformable vesicles, or insulin mixture (Pb0.05). Deform-able vesicles may be a better carrier than conventional vesicles forbuccal insulin delivery.

It is anticipated that the effects of salivary scavenging andaccidental swallowing of the delivery system, in addition to thebarrier properties of the buccal mucosa, will remain majorlimitations in the development of buccal drug delivery systems.Therefore, various buccal mucoadhesive delivery systems havebeen used inmany different dosage forms, in an attempt to achievethe delivery of drugs through the buccal mucosa and to overcomethe side effects of absorption enhancers. By creating an adhesiveinteraction between the delivery system and the oral mucosa, theresidence time of the drug at the absorption site is extended, whichallows therapeutic drug levels to be maintained for the desiredlength of time [104]. Nagai was among the first to pioneer themucoadhesive drug delivery system in the early 1980s by admin-istering insulin across the buccal mucosa in beagle dogs [105].Pelleted mucoadhesive polymeric nanoparticles for the buccaldelivery of insulin were an attempt to develop an alternativebuccal delivery system for insulin [106]. A significant hypogly-cemic response was observed after 7 h, without any detectablefluctuation in the blood glucose profile or risk of hypoglycemia. Inanother related work, insulin was formulated into mucoadhesivebuccal tablets using Carbopol 934, hydroxypropyl cellulose, orhydroxypropylmethyl cellulose and different absorption promo-ters [107].

Furthermore, Insulin Buccal Spray (IBS), a formulation withsoybean lecithin and propanediol combined as an absorptionenhancer, improved the hypoglycemic effect of insulin in diabeticrabbits and rats [108]. The hypoglycemic effect lasted over 5 h and4 h in diabetic rabbits and rats, respectively, and blood glucoselevels decreased significantly in both species compared with thoseof the control groups. Pharmacodynamic and pharmacokineticresults showed that IBS is a promising buccal delivery system forclinical trials and future clinical applications.

In the many buccal delivery systems for numerous peptide/protein drugs, the exciting challenge remains to increase thebioavailability of the therapeutic drugs across the oral buccalmucosa. So far, efficacy studies have only been presented asabstracts, and safety reports of buccal insulin are scare. Themajority of the above mentioned clinical studies did not assess theside effects of treatments.

2.3. Nasal administration

Nasal insulin delivery has been widely investigated as analternative to subcutaneous injection for the treatment of diabetesand is considered to be a promising technique for the followingreasons: the nose has a large surface area available for drugabsorption because the epithelial surface is covered with numer-ous microvilli; the subepithelial layer is highly vascularized, andthe venous blood from the nose passes directly into the systemiccirculation, thereby avoiding the loss of drug by first-passmetabolism in the liver; it allows lower doses, more rapid attain-ment of therapeutic blood levels, quicker onset of pharmacolog-ical activity, fewer side effects, high total blood flow per cm3, anda porous endothelial basement membrane; it is easily accessible;and the drug is delivered directly to the brain along the olfactorynerves [109,110].

The pharmacokinetic profile of intranasal insulin is similar tothat achieved with intravenous injection and, in contrast tosubcutaneous insulin delivery, bears a close resemblance to the‘pulsatile’ pattern of endogenous insulin secretion during mealtimes [111]. To date, attempts to implement this approach haveindicated that intranasal insulin therapy has considerable potentialfor the control of postprandial hyperglycemia, especially in thetreatment of patients with insulin-dependent diabetes mellitus[112]. Despite the potential of the nasal route, a number of factorslimit the intranasal absorption of drugs, especially peptide andprotein drugs. Mucociliary clearance, enzymatic activity, and theepithelium combined with the mucus layer constitute barriers tothe nasal absorption of high-molecular-weight and hydrophilicpeptides. Therefore, the use of absorption enhancers and pro-teolytic enzyme inhibitors, and the design of suitable dosageformulations, such as mucoadhesive and dry powder deliverysystems, have been investigated to enhance the nasal bioavail-ability of these drugs [113].

The effects of sodium deoxycholate (SDC) in combinationwith cyclodextrins (CD) as enhancers of the nasal absorption ofinsulin have been determined by measuring blood glucose levels[114]. Combining SDC with beta-CD lowered the serious nasalciliotoxicity of SDC and had a marked absorption-promotingeffect, which was due not to the low concentration of SDC but tothe inhibition of leucine aminopeptidase activity. The effects of asoybean-derived sterol mixture and of a steryl glucoside mixtureas enhancers of the nasal absorption of insulin in rabbits have beeninvestigated [115]. A series of new glycosides with extended alkylside chains (C13–16) linked to maltose or sucrose were synthesizedand used effectively to enhance nasal insulin absorption inanesthetized rats [116]. Cross comparisons of alkylmaltoses andalkanoylsucroses showed that the alkyl chain length had a greatereffect than the glycoside moiety in determining the potency of


potential insulin absorption-enhancing agents. When tetradecyl-maltoside was applied to the nasal mucosa 15 min before insulinwas applied, enhanced insulin absorption was observed. Anotherstudy reported that lipid-emulsion-based formulations weredevised to enhance insulin absorption through the nasal cavity,although at lower insulin doses, but showed no statistically sig-nificant enhancement [117].

Unfortunately, many absorption enhancers cause significantdamage to the nasal mucosa or other side effects when used at veryeffective concentrations, particularly with long-term exposure.Most of the traditional absorption enhancers, such as surfactantsand bile salts, have limited clinical use because of the irreversibledamage to the nasal mucosa that accompanies their absorption-enhancing effects [118–120].

In another approach, the mucociliary clearance rate can bereduced with the use of mucoadhesive systems. These drugdelivery systems can control the rate of drug clearance from thenasal cavity and protect the drug from enzymatic degradation bynasal secretions. Increasing the residence time of the drugformulation in the nasal cavity, and hence prolonging the periodof contact with the nasal mucosa, may improve drug absorption[121,122]. Approaches that increase the residence times of drugformulations in the nasal cavity usually involve the use of micro-spheres, liposomes, or gels that have mucoadhesive properties.These nasal drug delivery systems absorb water, swell, and form agel-like layer in contact with the nasal mucosa, which is clearedslowly from the nasal cavity. Therefore, absorption occurs rapidly,often with high bioavailability. Mucoadhesive polymers exert adirect effect on the mucosa by absorbing water from the mucusand swelling. The epithelial cells are dehydrated, causing the tightjunctions to separate, increasing the absorption of drugs that aretransported via the paracellular pathway [123]. The physicochem-ical properties of the drugs are also important factors affectingnasal drug absorption. A number of lipophilic drugs are com-pletely or almost completely absorbed from the nasal mucosa. Themechanisms and effectiveness of these drug delivery systems aredescribed to guide the development of specific and effectivetherapies for the future development of preparations of peptidesand other drugs that would otherwise be administered parenterally.

The insulin gel formulation produced a significant hypogly-cemic response in rabbits, and insulin bioavailability from thenasal gel formulationwas 20.6%of that achievedwith intravenousinjection. These results suggest that the carbopol nasal gel can beconsidered as a preferred platform in macromolecular nasaladministration [124].

Recently, Varshosaz et al. studied the development of achitosan bioadhesive gel for the nasal delivery of insulin [125]. A2%medium-molecular-weight chitosan gel with EDTA caused anincrease in insulin absorption and a reduction in glucose levels ofas much as 46% relative to those achieved via the intravenousroute. Therefore, insulin–chitosan nanoparticles are widelyaccepted as a nasal drug delivery system [126]. Other resultsshowed that 400 mg of chitosan and 70 mg of ascorbyl palmitateused as a cross-linker in insulin–chitosan microspheres, caused a67% reduction in blood glucose comparedwith that achievedwithdelivery via the intravenous route, and the absolute bioavaliabilityof insulin was 44% [127]. Furthermore, chitosan–4-thiobutyla-

midine (chitosan–TBA) microparticles showed the controlledrelease of insulin over 6 h, with an absolute bioavailability of7.24%±0.76% in conscious rats [128]. These data indicated thatthe potential of chitosan–TBA microparticles in nasal insulinadministration is substantially higher than that of unmodifiedchitosan [129].

Aminated gelatin microspheres (AGMS) were recentlyinvestigated as a nasal drug delivery system for peptide drugs[130]. AGMS significantly increased the nasal absorption ofinsulin in rats when administered as a dry powder formulation, butno significant hypoglycemic effect was observed when given insuspension. One of the proposed mechanisms for the increasedinsulin absorption involved the hydrogel nature of the micro-spheres, which can absorb water from the nasal mucosa, thusresulting in the temporary dehydration of the epithelial membraneand the opening its tight junctions. The electrostatic interactionsbetween model drugs and the microspheres were also consideredto be major factors controlling release behavior. Therefore, thepositive charge on the AGMS also evidently contributes to theirabsorption-enhancing effect. Therefore, AGMS might be a newcandidate carrier for the nasal delivery of peptide drugs.

Manypreclinical and clinical studies of the intranasal delivery ofproteins, peptides, and DNA have been completed, and theyindicate that efficacious delivery can be achieved systematically.Despite the promising results, the development of nasal insulinbiotherapeutics is beset with problems that require an integratedand rational approach. To date, relatively limited clinical ex-perience with intranasal insulin indicates a need for high andrepeated doses to achieve glycemic control. The occurrence ofnasal irritation in up to 25%of patients and the potential for damageto the nasal mucosa and nasal-ciliary function are causes forconcern, especially when viewed in the context of the requirementfor long-term exposure. Another problem of the nasal mode ofinsulin application is the considerable intra- and interindividualvariability in bioavailability. The amount of insulin that can beapplied at one time is also limited, because high-frequency nasalinsulin applications reduce the bioavailability of the applied insulin.

2.4. Pulmonary administration

2.4.1. BackgroundThe lungs offer a large surface area for drug absorption, of

approximately 140 m2. The alveolar epithelium is very thin(approximately 0.1–0.5 mm thick), thereby permitting rapid drugabsorption. The alveoli can be effectively targeted for drugabsorption by delivering the drug as an aerosol, with a massmedian aerodynamic diameter of less than 5 μm. Furthermore, thefirst-pass metabolism of the GIT is avoided. Although metabolicenzymes are found in the lungs, the metabolic activities andpathways may differ from those observed in the GIT, and thismakes the pulmonary administration of many peptides andproteins very promising [131].

Inhaled insulin is a novel approach to delivering insulin non-invasively and is emerging as a viable alternative to injectableinsulin. Intensive insulin therapy has been shown to have sig-nificant benefits in patients with type 1 or type 2 diabetes [132].The reality of pulmonary insulin becoming a viable alternative to


injections is, in large measure, due to the inherent anatomicaladvantages that make it an ideal route for the administrationof insulin. The availability of inhaled insulin will open newpossibilities in treating patients with diabetes. Patients who arereluctant to accept insulin injections can now be offered a realalternative. This should facilitate the earlier initiation of insulintherapy where appropriate and the intensification of existingregimens. Inhaled insulinwill provide an alternative even for thosepatients who may not be resistant to the idea of injections. Thepulmonary route of administration was targeted to increase thecompliance and life quality of patients with diabetes, who mayotherwise have to endure multiple daily subcutaneous injectionsof insulin. Increased compliance is important in decreasing thesevere and debilitating long-term effects of poorly controlleddiabetes [133].

As a consequence, the permeability of insulin as a modelpeptidewas examined to characterize the tracheal epithelial barrierin in vitro experiments using excised rabbit tracheae [134]. Insulinshowed slight degradation during the 150min duration of trachealpermeation, and the apparent permeability coefficient for insulinwas 7×10−9 cm/s. The tracheal permeability to insulin wassignificantly increased by 10 mM glycocholate, 1 mM bestatin(aminopeptidase B and leucine aminopeptidase inhibitor), and10,000 kIU/mL aprotinin (inhibitor of trypsin and chymotrypsin).The peptidase activity of the rabbit tracheal epithelium was asfollows: di-peptidyl-aminopeptidase IVN leucine aminopeptida-seNcathepsin BN trypsin. These activities were significantlylower than those of jejunal mucosal tissues. These results suggestthat the tracheal absorption of peptide drugs through therespiratory tract may contribute to the systemic delivery of thesedrugs following pulmonary administration by intratracheal insuf-flation or instillation.

2.4.2. Dry powder inhalationInsulin was used as a model protein to demonstrate the

feasibility of using protein crystals for the pulmonary delivery of asustained-release protein drug formulation [135]. The hypogly-cemic effects of the microcrystal suspension were prolonged over7 h. These results could be attributed to the sustained release ofinsulin from the microcrystals, which were deposited widelythroughout the entire lung. Insulin dry powder, made of insulinand other appropriate materials, was also insufflated into rat lungsfrom an incision in the throat [136]. The area above the curve(AAC) for insulin (5 U/kg) administered by pulmonary deliverywas very close to that of insulin given by subcutaneous admin-istration at the same dose. Thus, the pulmonary delivery of insulinacts effectively and rapidly. The feasibility of insulinmicrocrystalsas a long-acting formulation for pulmonary delivery wasexamined [137]. In an in vivo experiment with rats, zinc enhancedthe hypoglycemic effects of insulin microcrystals, with minimumreductions in blood glucose of 17%.

To investigate the enhancement effect of lanthanide ions (Ln3+)on the absorption of larger molecules from the pulmonarypathway, insulin (mol. wt.=5730) was chosen as the modelpeptide [138]. Lanthanum is an inhibitor of calcium flux andinhibits the insulin secretion induced by glucose and acetylcholineto basal levels but does not alter the stimulatory effects of insulin

[139]. The temporal changes in the gadolinium (Gd3+) contentof serum were also investigated because Gd3+ prevents thelipopolysaccharide-induced decrease in the expression of hepaticinsulin-like growth factor-I (IGF) and IGF-binding protein-3[140]. The effect of Ln3+ in promoting the bioavailability ofinsulin is closely related to its species, concentration, and order ofdelivery. The anionic form of gadolinium seems to be moreeffective than its cationic form. The coadministration ofGd3+ withinsulin was most effective in increasing insulin absorption in thelung.

Hyaluronic acid (HA) and recombinant human insulinwere co-spray-dried to form a dry powder suitable for inhalation [141].Several properties of HA support its unique utility and consequentselection for the pulmonary route of insulin administration. Somestudies have investigated the inhibition by HA of phagocytosisby macrophages [142]. This phenomenon, combined with themucoadhesive activity of HA, may prolong the time that HA-based microparticles remain close to the main absorption site inthe deep lung [143]. Systemic insulin levels and the correspondingglucose levels were monitored following the administration ofthesemicroparticles to the lungs ofmale beagle dogs. The additionof Zn2+ or hydroxypropyl cellulose improved the mean residencetime, AUC/dose, and Tmax, compared with those of pure spray-dried insulin.

2.4.3. Absorption enhancersSeveral approaches have been used to increase the bioavail-

ability of inhaled insulin, including the coadministration ofabsorption enhancers or delivery agents and the encapsulation ofinsulin in proprietary particles. Mitra et al. investigated theenhancement of insulin absorption in the presence of phospho-lipids and lung lavage fluid in vivo and in vitro [144]. A sig-nificantly greater reduction in blood glucose was observed with aphysical mixture of 1,2-dipalmitoyl phosphatidylcholine–insulincompared with that observed with liposomes, suggesting apossible effect of the physical state of the phospholipid chain oninsulin absorption in vivo. In another study, several dry powderformulations of insulin were prepared using a spray-drying tech-nique to examine the effects of additives on insulin absorption[145]. The absolute bioavailability of insulin solution and drypowder containing bacitracin or Span 85 was almost 100% and20% of that of the insulin solution, respectively. Citric acid wasmore effective in increasing the hypoglycemic effect of the drypowder than that of the solution. When dry insulin powdercontaining 0.036 mg/dose of citric acid was administered, lactatedehydrogenase activity, a sensitive indicator of acute toxicity inlung cells, in the bronchoalveolar lavage was as low as thatobserved after saline administration. Thus, citric acid appears to bea safe and potent absorption enhancer for insulin in a dry powderform.

Cyclodextrin (CD) derivatives, such as tetradecyl-β-maltoside(TDM) and dimethyl-β-cyclodextrin (DMβCD), enhance thepulmonary absorption of insulin and have a reversible action onthe respiratory epithelium through complexation [146]. CDsenhance the transmucosal absorption of insulin, by forming aninclusion complex with insulin or by direct action on the mem-brane. When insulin formulated with increasing concentrations


(0.06%–0.25%) of TDM or DMβCD was administered toanesthetized rats, there was a concentration-dependent decreasein plasma glucose and an increase in plasma insulin concentra-tions. The relative bioavailability of insulin formulations contain-ing TDM was higher (0.34%–0.84%) than that of formulationscontainingDMβCD (0.19%–0.48%). This study also implied thatTDM enhances absorption, perhaps by solubilizing membranecomponents or loosening cell–cell tight junctions.

2.4.4. Particulate carrier systemsThe pulmonary delivery of peptides and proteins is compli-

cated by the complexity of the anatomical structure of the humanrespiratory system and the effects on the disposition of the drugcaused by the respiration process. A novel nebulizer-compatibleliposomal carrier for the aerosol pulmonary delivery of insulinwasdeveloped and characterized [147]. Experimental results showedthat insulin could be efficiently encapsulated in liposomes using amethod involving preformed vesicles and detergent dialysis. Theoptimal encapsulation efficiency was achieved when 40% ethanolwas used. The particle size of the liposomal aerosols expressedfrom an ultrasonic nebulizer approximated 1 μm. Animal studiesshowed that plasma glucose levels were effectively reduced whenliposomal insulin was delivered by the inhalation route usingaerosolized insulin-encapsulated liposomes, as shown in Fig. 2.By including a fluorescent probe (phosphatidylethanolamine–rhodamine) into the liposome, researchers found that the lipo-somal carriers were effectively and homogeneously distributed inthe lung aveoli. Liposome-mediated pulmonary drug deliverypromotes an increase in the drug retention time in the lungs, andmore importantly, a reduction in extrapulmonary side effects,which invariably results in enhanced therapeutic efficacy.

The influence of calcium phosphate (CAP) and polyethyleneglycol (PEG) particles on the systemic delivery of insulin admin-istered by the pulmonary route appears to be a crucial factor [148].Insulin–CAP–PEG particles in suspension (1.2 U/kg, 110–140 μL) were administered to the lungs of fasted rats byintratracheal instillation (INCAPEG) or spray instillation (SIN-CAPEG). Pharmacokinetic and pharmacodynamic analysesshowed that the presence of CAP–PEG particles positivelyinfluenced the disposition of the insulin administered to the lungsof rats.

Fig. 2. Result of blood glucose level reduction in diabetic mice (n=9) [147].

Poly(lactide-co-glycolide) (PLGA) and polylactide particleshave already been used for drug delivery to the lungs. In cases oflung infections, such as tuberculosis, particles with a meandiameter of 1–3 μm have been produced to target the residentalveolar macrophages [149,150]. In contrast, large porousparticles of PLGA have been shown to escape macrophageuptake because of their large size, and they therefore permit theefficient delivery of inhaled insulin into the systemic circulationover long periods [151].

To produce insulin-loaded particles, PLGA nanospheres with amean diameter of 400 nm were prepared by a modified emulsionsolvent diffusion method in water [152]. After the administrationof 3.9 IU/kg of insulinwith the PLGAnanospheres, blood glucoselevels were reduced significantly and hypoglycemia was pro-longed for over 48 h, compared with the effects of a nebulizedaqueous solution of insulin used as a reference (6 h). This resultcould be attributed to the sustained release of insulin from nano-spheres deposited widely throughout the whole lung. Conversely,the use of CD in PLGAmicroparticles for the controlled release ofproteins has primarily been considered as a way to stabilize theencapsulated macromolecule, improving its therapeutic efficacy[153–155], and in some cases, to modulate the release featuresof the particles [156,157]. For this purpose, hydroxypropyl-β-cyclodextrin was used to produce the large porous particles ofPLGA intended for the pulmonary delivery of insulin [158]. Thesystem developed appears to have great potential for the combineddelivery of the protein and an adsorption promoter to therespiratory tract.

In another related investigation, insulin-loaded polybutylcya-noacrylate nanoparticles were prepared by the emulsion polymer-ization of various doses of insulin-loaded nanoparticles and givenintratracheally to normal rats [159]. A significant decrease inglucose levels of from 5 IU/kg to 20 IU/kg was achieved in eachdose group. The relative pharmacological bioavailability ofinsulin-loaded nanoparticles given by pulmonary administrationwas 57.2% greater than that achieved with the same formulationadministered subcutaneously.

Spray-drying is a valuable technique for producing drypowders suited to the pulmonary delivery of drugs. Chitosan–tripolyphosphate nanoparticles promote peptide absorption acrossmucosal surfaces [160]. Themicroencapsulation of protein-loadedchitosan nanoparticles using typical aerosol excipients, such asmannitol and lactose, produces microspheres as carriers ofprotein-loaded nanoparticles to the lung. Protein-loaded nanopar-ticles can also be successfully incorporated into microspheres toreach the deep lung.After contact with their aqueous environment,they are expected to release the nanoparticles and thus the thera-peutic macromolecules.

The great challenge for researchers remains the full optimiza-tion of the delivery system, which is the culmination of all thoseparticle properties required for therapeutic applications: goodencapsulation efficiency, the prevention of protein degradation,and the predictable release of the drug. This breakthrough in theavailability of inhaled insulin is certain to have exceptions. It mustbe recognized that inhaled insulin may not be ideally suited to allpatients. Enthusiasm for novelty must not override clinical pri-orities, and a number of issues remain to be resolved. Patients


receiving inhaled insulin had more episodes of hypoglycemia andgained more weight than did patients treated with oral agents[161,162].Mild tomoderate coughwas also reported in up to 25%of patients receiving inhaled insulin [163,164]. Uncontrollablefactors also affect pulmonary absorption, and smokers need lower[165] and asthmatics higher doses [166]. The pulmonary insulindose required for a similar glycemic effect is approximately 20times that required for a subcutaneous injection [167], and insulin-directed antibodies are an issue [168]. However, as a substitute forshort-acting insulin, inhaled insulin appears to be safe, efficient,and satisfactory for clinical use and acceptable to patients at thisearly stage in its development [169].

2.5. Ocular administration

Numerous research groups have reported early exploratorywork on systemic drug absorption via the ocular route. Thisefficient systemic absorption can be utilized as a non-invasivemeans of delivering drugs systemically. It also offers the advan-tages that it is much easier to administer than is an injection; therate of systemic absorption through the ocular route is as fast asvia an injection; eye tissues are much less sensitive to thedevelopment of immunological reactions than are other tissues;it bypasses first-pass gastrointestinal and liver effects, which areresponsible for the low oral bioavailability of peptides and otherdrugs; and no tolerance and ocular side effects have beendetected after long-term (3 months) daily administration ofinsulin eyedrops [170]. The eye presents unique opportunitiesand challenges when it comes to the delivery of pharmaceuticals,and it is very accessible to the application of topical medications.The potential route for insulin delivery to the anterior segment ofthe eye has been the conjunctival sac [171]. More recentinvestigations have shown that the conjunctival route of entryplays an important role in the penetration of drugs into theanterior segment. Furthermore, topically applied drugs havebeen shown to have access to the sclera from the conjunctiva.Therefore, it is conceivable that such drugs could find their wayto the posterior segment. It has been shown that even a high-molecular-weight peptide like insulin can accumulate in theretina and optic nerve after topical application, supporting thecontention that topically applied drugs can both reach theposterior segment and be therapeutic. Finally, topically appliedinsulin also accumulates in both the contralateral eye and thecentral nervous system [172].

After the pioneering work of Christie and Hanzal (1931),numerous investigations of the systemic delivery of insulin via theocular route were undertaken. Bartlett et al. investigated thefeasibility of using insulin eyedrops in humans by studying thelocal toxicity and efficacy of insulin administered without sur-factant to the eyes of healthy volunteers [173]. The results of thisstudy suggest that single-dose insulin at concentrations of up to100 U/mL, formulated in saline, has no detectable clinical toxicityon the anterior structures of the human eye. Not surprisingly, thistherapy was abandoned in humans because of its low bioavail-ability. Since then, the development of ophthalmic drug deliverysystems has always been hindered by local irritation, rapid loss indrainage, blinking, and tearing.

The results of previous studies suggest that the use ofabsorption enhancers should be introduced. A wide variety ofabsorption enhancers have been evaluated in the delivery ofinsulin via the ocular route. Yamamoto et al. determined theextent, pathways, and effects of absorption enhancers on thesystemic absorption of insulin after the instillation of a topicalsolution to albino rabbit eyes [174]. The absorption enhancersused were polyoxyethylene-9-lauryl ether (POELE), sodiumglycocholate, sodium taurocholate, and sodium deoxycholate,all at a concentration of 1%. The nasal mucosa contributed aboutfour times more than the conjunctival mucosa to the systemicabsorption of ocularly applied insulin. However, the conjunctivalmucosa was more discriminating in its sensitivity to the nature ofthe bile salts used than was the nasal mucosa. Collectively, thesefindings indicate that it is feasible to achieve hypoglycemia withocularly administered insulin. Consequently, eyedrops of 0.25%insulin plus 0.5% POELE or polyoxyethylene-20-stearyl ether(Brij-78) were instilled into rabbit eyes twice a day for 3 months[175]. The efficacy of the insulin in lowering the blood glucoseconcentration and the uptake of insulin into the systemic circu-lation remained the same throughout the experimental period. Noallergic responses or local side effects were detected, indicatingthat both insulin and the absorption enhancers (POELE and Brij-78) are safe for instillation into the eyes over long periods. A seriesof alkylglycosides with various alkyl chain lengths and carbo-hydrate moieties were tested for their ability to enhance thesystemic absorption of insulin after topical ocular delivery inanesthetized rats [176]. All the reagents were effective only whenused at concentrations above their critical micelle concentrations,and the most hydrophobic alkylglycoside reagents were the mostefficacious in promoting systemic insulin absorption. Regularporcine insulin was administered as eyedrops, either alone or incombination with several different absorption enhancers, to eighthealthy euglycemic dogs [177]. No ocular symptoms occurredwith the administration of insulin alone or together with 0.5%solutions of Brij-78, fusidic acid, POELE, dodecylmaltoside, ortetradecylmaltoside. This study demonstrated that short-actinginsulin is systemically absorbed in dogs via the ocular route whenapplied with certain emulsants. Sucrose cocoate (SL-40) is anemulsifier used in emollients and skin-moisturizing cosmeticformulations that contains a mixture of sucrose esters of coconutfatty acids in an aqueous ethanol solution. Sucrose cocoate wasexamined to determine its potential usefulness and enhancingeffects in nasal and ocular drug delivery [178]. When insulin wasadministered ocularly in the presence of 0.5% sucrose cocoate,significant increases in plasma insulin levels and a decrease inblood glucose levels were observed. To prolong the retention timeof the formulation in the precorneal area, a positively chargedinsulin-containing liposome was prepared [179,180]. Thisformulation reduced the blood glucose concentrations of rabbitsto 65%–70% of the initial levels for up to 5 h.

Commercially available Gelfoam®, an absorbable gelatinsponge, is used in the fabrication of an ocular insert in the formof a matrix system. Both in vitro flow-through and in vivomethods of device removal were examined to determine thedissolution rate of insulin from a Gelfoam®-based eye device[181]. Two eyedrop formulations and 13 eye device formulations

Fig. 3. Effect of sodium taurodeoxycholate (NaTDC; 100 mg) or sodiumtaurocholate (NaTC; 100 mg) alone or in combination with sodium cholate(NaC; 50 mg) on the mean plasma glucose levels (% of initial values) inhyperglycemic beagle dogs after rectal administration of Witepsol W35suppositories containing human insulin (5 U/kg) compared with the effects ofsubcutaneously injected insulin (Ins s.c., 4 U/kg) [188].


were evaluated [182]. The in vivo results for devices containing0.5 or 1.0 mg of insulin with 20 μg of Brij-78 showed substantialimprovement in insulin activity and a significantly prolongedsystemic delivery of insulin within the desired therapeutic levels,with no risk of hypoglycemia. The prolonged activity of theinsulin was due to its gradual release from the device, whichslowed tear production. Furthermore, the mean blood glucoseconcentration returned to a nearly normal level within 60 min ofthe removal of the device. Although it is known that enhancers canpromote the systemic absorption of insulin via the ocular route,little is known about the long-term toxicity of these enhancers. Forthis practical reason, sodium insulin and zinc insulin Gelfoam®ocular devices have been developed as insulin carriers for thesystemic delivery of insulin, and they do not contain any surfactantor absorption enhancer [183,184]. Lee and Yalkowsky investi-gated the role of acid in the enhancer-free absorption of insulinfrom Gelfoam® ocular devices [185]. A Gelfoam® device con-taining 0.2 mg of insulin is sufficient to control blood glucoselevels in a uniform manner (60% of the initial concentration) forover 8 h. These results suggest that a change in the Gelfoam®when treated with acid is responsible for the efficient systemicabsorption of insulin from these enhancer-free devices.

Whereas all ocular preparations must be compatible with theiris, corneal, and conjunctival tissues, these factors are especiallyimportant for ocular devices. Because the device must remain inone place for several hours, irritation of the local tissue can bemore problematic with an eye device than with an eyedrop, whichis eliminated within seconds. A device must also be comfortableand designed so that it does not fall out during sleep. Mostimportantly, the device must release the drug in a constant andreproduciblemanner. Ultimately, all the preclinical studies suggestthe feasibility of delivering insulin systemically via the ocularroute. No toxic effects were observed in several preliminaryhuman studies. However, the application of this approach stillrequires further investigation to be clinically useful.

2.6. Rectal administration

During the past few years, considerable interest has arisen inthe rectal route for insulin administration. This route is regarded asa more physiological route for the application of insulin. Rectalinsulin delivery offers several advantages over some of the otherenteral routes. First, the rectal route is independent of intestinalmotility, gastric-emptying time, and diet. It is most likely that thepresence of degrading enzymes in the gut wall decreases from theproximal end to the distal end of the small intestine and rectum.The most important advantage suggested for the rectal adminis-tration of insulin is the possibility of avoiding, to some extent, thehepatic first-pass metabolism.

Hosny [186] found that insulin suppositories containing 50 Uof insulin incorporated with 50 mg of deoxycholic acid, sodiumtaurocholate, or both, placed in the rectum of alloxan-inducedhyperglycemic rabbits, caused a large decrease in plasma glucoseconcentrations, and the relative hypoglycemia was calculated tobe 38.0%, 34.9%, and 44.4%, respectively, compared with thatobserved for insulin (40 U) injected subcutaneously. The mostpronounced effect was observed with the addition of polycarbo-

phil to a suppository formulation containing a combination ofdeoxycholic acid and sodium taurocholate, which produced 56%relative hypoglycemia compared with that achieved with a sub-cutaneous injection. These suppository formulations are verypromising alternatives to current insulin injections, because theyare roughly half as efficacious as subcutaneous injections. Insulinsuppositories were formulated usingWitepsolW35 as the base, toinvestigate the effects of various bile salts/acids on the plasmaglucose concentrations of diabetic beagle dogs [187]. A relativehypoglycemia of about 50% was achieved using insulin sup-positories containing Witepsol W35 as the base and sodiumdeoxycholate (100 mg) plus sodium cholate (50 mg), sodiumtaurodeoxycholate (100 mg), or sodium taurocholate (100 mg) asenhancers of the rectal absorption of insulin, as shown in Fig. 3. Adesirable hypoglycemia, expressed as Cmax, and/or AUC, can beachieved by adjusting the insulin dose in the formulation accord-ing to the degree of initial hyperglycemia. Investigation of theeffects of insulin suppositories on the plasma glucose concentra-tions of diabetic beagle dogs showed that a relative hypoglycemiceffect of about 50%–55% can be achieved using insulin sup-positories containing Witepsol W35 as the base, insulin (5 U/kg),and sodium salicylate (50mg) or POELE (1%) as rectal absorptionenhancers [188].

Studies have recently shown that the formation of an adhesiveinteraction between the delivery system and the rectal mucosa canbe harnessed as an absorption modifier because it increases thecontact time of the coadministered drug and possibly acts as asustained-release polymer. A thermoreversible liquid insulinsuppository, which undergoes a phase transition to a bioadhesivegel at body temperature, enhances the bioavailability of insulin[189]. The thermoreversible liquid insulin suppository (containing100 IU/g insulin, 15% poloxamer P407, 20% poloxamer P188,0.2% polycarbophil, and 10% sodium salicylate) could potentiallybe developed as a more convenient, safe, and effective rectaldelivery system for insulin. Adikwu [190] evaluated snail mucinmotifs as rectal absorption enhancers for insulin. Themucins were


extracted from the giant African snail Archachatina marginata bydifferential precipitation with acetone. Mucin administeredexogenously formed disulphide linkages between its cysteinedomain and those of endogenous mucin, increasing the gel net-work and hence the viscosity of the mucus, probably conferringsustained- or prolonged-release properties to the dosage form. Abatch with 7% mucin reduced the basal glucose level to 44%within 2 h of administration of the glycero-gelatin loaded mucinsuppository.

The efficacy and bioavailability of rectally delivered peptideremained low compared with those of peptide delivered by intra-venous or subcutaneous injection. These delivery systems ap-peared to significantly increase the uptake of high-molecular-weight polar drugs, such as insulin, before surfactant and otherabsorption enhancers were introduced. This is a concern in long-term therapy, because of the absorption of unwanted toxic mole-cules present in the GIT and disturbance to the permeability ofthe mucosal membrane. Patient compliance and the pain thataccompanies rectal administration are both challenges for itswidespread use as a therapeutic dosage form. Its irreproduciblebioavailability and the special storage conditions required are alsogreat obstacles to the rectal delivery of insulin.

2.7. Transdermal administration

2.7.1. BackgroundBecause the skin is the largest organ of the human body,

transdermal drug delivery is an appealing alternative to sub-cutaneous delivery. It offers good patient compliance and thepossibility of controlled release over time, while avoiding possibledrug degradation resulting fromGITor first-pass liver effects. Theskin also provides a painless interface for systemic drug delivery[191,192]. Despite these advantages, the human skin is anextremely effective barrier that protects against and is imperme-able to foreign molecules, especially large hydrophilic molecules.The low permeability of the skin is caused mainly by the stratumcorneum, the skin's outermost layer [193]. The development of asophisticated new approach to overcome the skin permeabilityobstacle has challenged the pharmaceutical community to developnew delivery methods. Attempts to overcome this skin barrier toallow the transfer of large drugs have included techniques that

Table 3Transdermal formulation technologies of insulin delivery systems

Formulation/technology Application Outcome

Synthetic peptides STZ-induced diabetic rats SustaineFlexible lecithin vesicles Mice More thaNanoinsulin Mice SignificaIontophoresis STZ-induced diabetic rats SignificaIontophoresis/penetration enhancer Rabbits SignificaIontophoresis/LA, OA, LOA or LLA Excised rat skin SignificaIontophoresis/Poloxamer P407 gel STZ-induced diabetic rats 36%–40Sonophoresis Hairless rats SignificaSonophoresis Rabbits SignificaMicroneedles STZ-induced diabetic hairless rats 0.5–7.4

Abbreviations: STZ, streptozotocin; LA, lauric acid; OA, oleic acid; LOA, linoleic

weaken the barrier with skin absorption enhancers [194],iontophoresis [195], ultrasound [196], or microneedles [197].These are reviewed in Table 3.

2.7.2. Absorption enhancersIn the last 50 years, a large number of chemical absorption

enhancers (CAEs) belonging to several different classes, such assurfactants, fatty acids, fatty esters, and azone-like compounds,have allowed limited skin absorption enhancement [198]. CAEshave two major limitations: skin irritation and an inability todeliver large molecules. CAEs are generally unable to delivertherapeutic levels of relatively large drugs (molecular mass,≥4500 Da) through intact skin to the systemic circulation [199].Recent progress in high-throughput screening to identify enhancercombinations [200] and in the application of CAE designprinciples [201] may help to overcome these two limitations,but the transdermal delivery of large hydrophilic proteins remainsa formidable task.

The short synthetic cyclic peptide, TD-1, identified by in vivophage display, facilitates efficient transdermal protein drugdelivery through intact skin [202]. The coadministration of thepeptide and insulin to the abdominal skin of diabetic rats resultedin elevated systemic levels of insulin and suppressed serumglucose levels for at least 11 h. The transdermal-enhancing activityof the peptide was sequence specific and dose dependent, did notinvolve direct interaction with insulin, and allowed the penetrationof insulin into hair follicles beyond a depth of 600 μm. Time-lapsestudies have suggested that the peptide creates a transient openingin the skin barrier that allows macromolecular drugs to reach thesystemic circulation.

2.7.3. Transdermal drug delivery systemsTransdermal drug delivery systems can be generally divided

into physical, biochemical, and chemical methods. Furtherstrategies have been investigated for the carriage of insulinusing the enhancing effects of flexible lecithin vesicles containinginsulin, designed for the transdermal delivery of hydrophilicproteins [203]. The entrapment efficiencies of conventional andflexible vesicles were 35% and 81%, respectively. When flexiblevesicles were non-occlusively applied to the abdominal skin ofmice at a dose of 0.90 IU/cm2, an in vivo hypoglycemic study

Refs.

d suppression of blood glucose levels for 11 h [202]n 50% reduction in blood glucose levels in 18 h [203]nt reduction in blood glucose levels [204]nt reduction in blood glucose levels [211]nt reduction in stratum corneum barrier [215]nt insulin flux enhancement [217]% reduction in blood glucose levels [219]nt reduction in blood glucose levels for 60 min [252]nt hypoglycemic response [253]ng/mL plasma insulin levels and 80% reduction in blood glucose levels [267]

acid; LLA, linolenic acid.


showed a percentage decrease in blood glucose of 21.42%±10.19% at 1 h, which reached 61.48%±8.97% at 5 h and was stillgreater than 50% at 18 h.

An advanced study evaluated the pharmacokinetic andpharmacodynamic effects of transdermally delivered insulinusing novel CaCO3 nanoparticles in normal mice and those withdiabetes [204]. CaCO3 nanoparticles encapsulating insulin(nanoinsulin) were transdermally applied to the back skin ofnormal ddYmice and to dB/dB and kkAymice with diabetes afterfasting for 1 h. Maximum serum insulin was 67.1±25.9 μIU/mLat 4 h in ddY mice administered 200 μg of transdermal nano-insulin, whereas it was 462±20.9 μIU/mL 20 min after thesubcutaneous injection of 3 μg of monomer insulin. Transdermalnanoinsulin reduced glucose levels in a dose-dependent manner.In ddY mice, insulin bioavailability was 0.9% with transdermalnanoinsulin for 6 h, based on serum insulin levels, and 2.0%based on pharmacodynamic blood glucose-lowering effects. ThisCaCO3 nanoparticle system successfully delivered insulin trans-dermally, as demonstrated by significant sustained decreases inblood glucose in both normal and diabetic mice. These resultssupport the feasibility of developing transdermal nanoinsulin forhuman applications.

2.7.4. IontophoresisMore recently, one of themost advanced technologies that have

been developed in the 20th century to overcome low skinpermeability to insulin is iontophoresis [205]. Iontophoresis is atechnique used to enhance the transdermal delivery of compoundsthrough the skin by the application of a small electric current.Using the processes of electromigration and electro-osmosis,iontophoresis increases the permeation of charged and neutralcompounds. It offers the option of a programmed drug deliverytechnique that physically facilitates the transport of permeatesacross the skin [206]. Transdermal iontophoresis is one suchtechnique that shows promise and is believed to be a future optionin the controlled and enhanced delivery of peptides and proteins[207]. It allows non-invasive, continuous, pulsatile delivery, aswell as preprogrammed complex dosing regimens. Combinationsof iontophoresis with absorption enhancers, electroporation, andsonophoresis have been tested to increase transdermal drugpermeation and decrease possible side effects further. At present,research is focused on resolving skin toxicity issues and otherproblems, to make the technology a commercial reality [208].

Mao et al. [209] reported that in the transdermal delivery ofinsulin by pulse-current iontophoresis, the pulse permeation ratecorrelates positively with the reservoir insulin concentration andwith skin penetration by insulin. In another iontophoretic deliverystudy, Zakzewski et al. [210] reported experimental evidenceindicating a substantial increase in the penetration by insulin withthe same-day application of a depilatory lotion, in conjunctionwith iontophoretic enhancement. Kanikkannan et al. [211] studiedthe delivery of bovine insulin to streptozotocin-induced diabeticrats by iontophoresis. The iontophoresis of a monomeric humaninsulin analogue (B9 Asp, B27 Glu) through intact skin produceda significant decrease in plasma glucose levels in the diabetic rats.Insulin was used as the model for large peptides, to understandbetter the effect of peptide concentration, NaCl concentration, and

buffer type and concentration on the transport efficiency ofiontophoresis [212]. Overall, the findings revealed that thetransport efficiency of large peptides like insulin might beimproved by the optimization of the competing ions in solution.Pillai et al. [213] investigated the role of electronic parameters inthe iontophoretic transport of large peptides using insulin as themodel peptide. Ex vivo permeation experiments were conductedusing excised rat skin and the influence of varying currentstrengths, durations, on/off ratios, and switching on iontophoreticinsulin permeation were studied. Insulin permeation was found toincrease as a function of current strength and the duration ofcurrent application, and switching iontophoresis reduced the pHshift, thereby improving the electrochemical stability of insulin atpH 3.6 and pH 7.4.

Significant permeation of large peptides like insulin has beenachieved using combination strategies involving absorptionenhancers and transdermal iontophoresis [214]. Recently, Choiet al. demonstrated the effectiveness and mechanism of a trans-dermal drug delivery system using iontophoresis plus an absorp-tion enhancer [215]. A combination of absorption enhancerpretreatment and iontophoresis delivered drugs more effectivelythan iontophoresis alone. The proposed theory is that iontopho-retic drug delivery may be easiest through the dilated intercellularspaces of the stratum corneum, which have lowered electricalimpedance following absorption enhancer pretreatment. Theeffects of pretreatment with commonly used vehicles, such asethanol, propylene glycol, water, and their binary combinations,dimethyl acetamide, 10% dimethyl acetamide in water, ethylacetate, and isopropyl myristate on insulin iontophoresis havebeen investigated. Another study compared the effects of aterpene–ethanol combination with ethanol and neat terpene ontransdermal iontophoretic permeation by insulin [216]. Terpene–ethanol synergistically enhanced insulin permeation whencombined with iontophoresis and was influenced by the typeand concentration of terpene. The combination of fatty acids withiontophoresis was predicted to result in greater enhancement thanthat achieved with either of them alone [217]. The saturated fattyacid lauric acid (LA), and the cis-unsaturated fatty acids oleic acid(OA), linoleic acid (LOA), and linolenic acid (LLA), were studiedin combination with iontophoresis using excised rat skin.Unsaturated fatty acids showed greater enhancement than LA,and the flux enhancement of unsaturated fatty acids was increasedwith the number of double bonds in the following (decreasing)order: LOANOANLLA. Conversely, in the presence of ionto-phoresis, LA–ethanol showed the greatest enhancement. Trans-dermal transport data for insulin under anodal iontophoresis(electro-osmosis) following electroporation in the presence of 1,2dimyristoylphophatidylserine (DMPS) have been reported [218].When electro-osmosis was applied across the epidermis followingelectroporation with DMPS, the enhancement of insulin transportwas ∼18-fold higher than that achieved with electroporationalone. It was suggested that DMPS increased the lifetime of theelectropores in the epidermis, resulting in the enhanced transportof permeates.

Gels are considered the most suitable delivery vehicle foriontophoresis, because they can easily be amalgamated with theiontophoretic delivery system and match the contours of the skin


[219]. The poloxamer P407 gel formulation of insulin wasevaluated by ex vivo and in vivo skin permeation studies in rats,with absorption enhancer and/or iontophoresis. Iontophoresiseither alone or in combination with linoleic acid produced areduction in blood glucose levels of 36%–40%.

The major challenges in this area are the development ofportable, cost-effective devices and suitable semi-solid formula-tions that are compatible with both the device and the skin,because the combination of absorption enhancers and iontopho-resis causes greater skin irritation than is caused when either isused alone.

2.7.5. Sonophoresis (ultrasound)For the past two decades, sonophoresis (ultrasound) has been

used to enhance the delivery and activity of drugs [220]. Ultra-sound has an ever-increasing role in the delivery of therapeuticagents, including genetic material, proteins, and chemotherapeuticagents. There is a tremendous corpus of literature on the useof ultrasound to enhance the permeability of the skin fortransdermal drug delivery [221–228]. Therapeutic levels ofultrasound (1–3 MHz, 1–3 W/cm2) have been used for years todrive small hydrophobic molecules, like steroids, into or throughthe skin [229–232]. Absorption enhancers have sometimes beenused to increase skin permeability further [233–245]. However,no significant transport of protein was achieved until 10 years ago,when Mitrogotri et al. showed that low-frequency ultrasound wasmuch more effective than higher frequencies, and providedevidence of the mechanism involved [233–245]. Skin permeabil-ity increased with decreasing frequency, and with increasing timeof exposure and intensity (beyond a certain threshold), thusidentifying collapse cavitation as a causative mechanism [236–238,241,242,246]. The current theory is that cavitation eventsopen reversible channels in the lipid layers of the stratum corneumand provide less tortuous paths of transport for proteins such asinsulin [221,236–238,241]. Electron microscopy of skin exposedto low-frequency ultrasound revealed the removal of surface cellsand the formation of pores and pockets large enough (∼20 μm) toaccommodate the transport of proteins and other large molecules[247–250]. Tezel and Mitrogotri have formulated a model ofshock-wave andmicroject cavitation events and their impact uponskin permeability [251]. Although their model can be fitted to theirdata, there aremany assumptions and parameters in themodel, andmore direct evidence is needed to identify conclusively themechanisms of ultrasound-enhanced transdermal protein delivery.

Boucaud et al. investigated the dependence of ultrasound-induced transdermal delivery of insulin on ultrasound parameters[252]. Insulin was delivered in vivo to hairless rats using 20 kHzultrasound applied over a range of ultrasound intensities, appli-cation times, and pulse lengths. The most significant effect inlowering glycemia was achieved with the application of less than15 min ultrasound and was similar to that achieved with asubcutaneous injection of 0.5U of insulin. Pretreatment of hairlessrat skin with ultrasound followed by the application of insulinresulted in no significant modification in blood glucose levels,indicating that the transdermal transport of insulin occurredmainly during sonication. Therefore, sonophoresis may poten-tially be used for the non-invasive and painless delivery of insulin

in the treatment of insulin-dependent diabetes. A recent studydemonstrated the ultrasonic transdermal delivery of insulin in vivoin rabbits with a novel, low-profile two-by-two ultrasound arraybased on the cymbal transducer [253]. Glucose levels decreasedby 132.6±35.7 mg/dL from the initial baseline for 60 min. Evenafter the array and insulin reservoir were removed, the bloodglucose levels of the ultrasound–insulin group continued todecrease by 208.1±29 mg/dL from the initial baseline.

Although the future of ultrasound-enhanced transdermalprotein delivery is brimming with potential, it has not yet ap-peared in the clinic. Because large pores and channels are openedthrough natural skin barriers, many hormones and proteins arecandidates for transdermal delivery [236,237,254–259]. Howev-er, the effect of ultrasound on protein conformation and/or activityneeds to be addressed in more detail.

2.7.6. MicroneedlesA novel approach that increases transdermal transport involves

the use ofmicroneedles that pierce the skin and createmicrometer-scale openings. Although still extremely small on a clinical level,channels of micrometer dimensions are much larger than macro-molecules and therefore should dramatically increase skinpermeability to large drugmolecules.Microneedles ofmicrometerdimensions can create transport pathways large enough for smalldrugs, macromolecules, nanoparticles, and fluid flow, but smallenough to avoid pain and facilitate highly localized and evenintracellular targeting. The microelectronic revolution has pro-vided tools for highly precise, reproducible, and scalable methodsto fabricate structures of micrometer dimensions. This lithogra-phy-based approach can produce large arrays of microneedlesthat can be inserted into cells, skin, or other tissues. Arrays ofmicrometer-scale needles could be used to deliver drugs, proteins,and particles across skin in a minimally invasive manner. Theincreased importance of macromolecular therapeutics, combinedwith the newly acquired power of microfabrication, has recentlyprompted interest in fabricating [260–263] and testing [264,265]microneedles for drug delivery.

Practical microfabrication techniques have been developed toyield microneedle arrays of silicon, metal, and biodegradablepolymers of micrometer dimensions in various geometries [266].Microneedle arrays having solid or hollow bores with tapered orbeveled tips may provide a minimally invasivemethod to increaseskin permeability for diffusion-based transport of large moleculessuch as proteins. Hollow microneedles have permitted the flow ofmicroliter quantities into the skin in vivo, including the micro-injection of insulin to reduce blood glucose levels in diabetic rats.These results suggest that microneedles are a useful approach totransdermal drug delivery. Building on microneedle transdermalstudies, Martanto et al. [267] designed and fabricated arrays ofsolid microneedles to be inserted into the skin of diabetic hairlessrats for the transdermal delivery of insulin to lower blood glucoselevels. Fig. 4 shows that the microneedles increased the perme-ability of the skin to insulin, which rapidly and steadily reducedblood glucose levels to an extent similar to that achieved with0.05–0.5 U of insulin injected subcutaneously. Plasma insulinconcentrations were measured directly as 0.5–7.4 ng/mL. Solidmetal microneedles are capable of increasing transdermal insulin

Fig. 4. Changes in blood glucose levels in diabetic hairless rats after insulindelivery using microneedles (▲), subcutaneous hypodermic injection of 0.05 U(◊), 0.5 U (□), or 1.5 U (○) of insulin, or passive delivery across untreated skin(×). Microneedles were inserted into the skin for 10 min and then removed.Insulin solution was applied to the skin immediately after microneedle insertionand left on the skin for 4 h (as shown by arrow). Data are expressed as meanvalues (n≥3) with average standard deviations associated with each data pointof 14% [267].


delivery and lowering blood glucose levels by as much as 80% indiabetic hairless rats in vivo. Hypodermic needles have providedthe gold standard for drugdelivery for over a century, but advancesin biotechnology make their limitations increasingly apparent.

It seems that a transdermal delivery system for insulin is anattractive option. Although progress in the transdermal delivery ofinsulin and other peptides seems encouraging, it is still doubtfulwhether this delivery route provides a general approach to the non-invasive delivery of insulin. Because of the poor permeationcapacity and large molecular size of insulin, the poor bioavail-ability of insulin during transdermal delivery compared with thatachieved with subcutaneous injection, and the high susceptibilityof the skin to adverse immunological reactions, more studies,including clinical studies, are required to assess the potentialimpact of these factors on novel transdermal insulin deliverytechniques.

3. Recent marketed and developed formulations fornon-invasive insulin delivery

Significant progress has been reported in the recent past in thedelivery of insulin using buccal and pulmonary routes, as has beenachieved for many other hormonal drugs, such as calcitonin andvasopressin, which are available in intranasal sprays. The field ofinsulin delivery took a tremendous step forward with the approvalof Exubera® from Pfizer and Nektar Therapeutics. Insulin haslong been a target of drug delivery companies because of thenumber of patients using it and the difficulties and issuessurrounding the reliance on injections. Pharmaceutical companiesare confident that ease of use and the elimination of the needle willbe enough to counter the added costs of alternative insulin deliverysystems, and Exubera® is expected to reinforce this notion. Theapproval of this drug has legitimized alternative forms of drugdelivery, providing patients with increased hope of eliminating or

reducing their dependence on injections and instilling confidencein numerous drug delivery companies that alternative forms ofinsulin delivery systems can win the approval of federalauthorities. Marketed developed insulin formulations and tech-nologies for non-invasive routes are summarized in Table 4.

Researchers working in academic institutions and drugdelivery companies have begun collaborative work on severalnovel delivery technologies to introduce a viable and feasible oralinsulin dosage form that can potentially replace painful insulintherapy. The overwhelming attractiveness of oral administration isprompting numerous companies to develop technologies to over-come the challenges of oral peptide delivery. As a result, there is ahigh degree of innovation and competition, withmultiple productsalready in clinical trials.

One of the more unusual alternatives is from EmisphereTechnology, Inc. Emisphere Technology has very recently madepublic that it is undertaking clinical phase II trials in type 2 diabeticpatients in order to file an investigational new drug (IND)application with the U.S. Food and Drug Administration (FDA).This will be the first attempt by Emisphere Technology to test theEligen™ oral drug delivery strategy that delivers insulin withthe Emisphere® delivery agent or carrier in a capsule. Theirproprietary Eligen™ technology platform is based upon the use ofsynthetic non-acylated amino acids as carriers, which allow thepeptide to enter the bloodstream through the body's naturalpassive transcellular transport processes in the GIT. Non-acylatedamino acids, when used as carriers, do not alter the conformationof the delivered drug but do alter the physical properties at thepoint of transport, while not affecting the efficacy of the drug[268,269].

The interim results of a U.S. phase II trial of AI-401, an oraltolerance product of AutoImmune, Inc. developed for thetreatment of diabetes, were presented at a poster session at theAmericanDiabetesAssociationmeeting inChicago, in June 1998.The trial evaluated the feasibility of oral insulin therapy, delayingβ-cell destruction in the pancreas, and thus preserving endoge-nous insulin secretion in newly diagnosed type 1 diabetic patients.This study is one of four trials undertaken using AI-401, an oralformulation of recombinant human insulin. The Eli Lilly Com-pany is AutoImmune's worldwide partner in autoimmune (type 1)diabetes [272]. The final data from the oral insulin arm of theNIH-sponsored Diabetes Prevention Trial-Type 1 (DPT-1) showed astatistically significant benefit for patients with type 1 diabetes[273].

The Generex Biotechnology Corporation, a small Canadiandrug delivery firm, recently stole the march on the pharmaceuticalgiants by launching the oral insulin, Oral-lyn™, in the form of anoral spray for the treatment of patients with type 1 and type 2diabetes, in Ecuador last December [274,275]. Generex hasdeveloped a proprietary platform technology for the delivery ofdrugs into the human body through the oral cavity (with no depositin the lungs). The company's proprietary liquid formulationsallow drugs typically administered by injection to be absorbed intothe body through the lining of the inner mouth using thecompany's proprietary RapidMist™ device. The process involvesthe creation of a stable mixed micellar solution containing thepharmaceutical agent, absorption enhancers, and other excipients.

http://www.emisphere.com

http://www.autoimmuneinc.com

http://www.generex.com

Table 4Recent marketed and developed formulations for non-invasive insulin delivery

Route of administration Trade name/insulin product Company/Developer & Researcher Technology Status Refs.

OralEligen™ Emisphere Technology, Inc. Oral capsule using non-acylated

amino acids as carriersPhase II [268,269]

Macrulin™ Cortecs International/Provalis PLC Macrosol™ W/O microemulsiontechnology

Phase II [270,271]

AI-401 AutoImmune, Inc./Eli Lilly Oral formulation of recombinanthuman insulin

Phase II [272,273]

BuccalOral-lyn™ Generex Biotechnology Corp. Mixed micellar solution Market

available[274,275]

NasalNasulin™ Bentley Pharmaceutical, Inc. New physiological absorption

enhancerPhase II [276]

QDose™ Vectura, Ltd./MicroDose Technologies Inc. Dry powder Phase I [277]ChiSys™ West Pharmaceutical Services Chitosan-based system Phase I [278]

PulmonaryExubera® Nectar Therapeutics, Inc./Pfizer/

Sanofi–Aventis SAHuman insulin (rDNA origin)inhalation powder

Marketavailable

[279–286]

AERx®iDMS Aradigm Corp./Novo Nordisk A/S Liquid aerosol Phase III [287–297]HIIP® Alkermes, Inc./Eli Lilly AIR® technology Phase III [298–300]Technosphere® PDC/MannKind Corp. Encapsulated microsphere Phase III [301]Aerodose® AeroGen, Inc./Nektar Therapeutics, Inc. Liquid aerosol Phase IIa [302,303]Spiros® Dura Pharmaceuticals, Inc./Elan Corp. Dry powder Phase II [304]Insulin inhaler Kos Pharmaceuticals, Inc./Abbott

LaboratoriesCrystalline insulin Phase IIa [305]

MicroDose DPI® MicroDose Technologies, Inc./Novartis Dry powder Phase I [306]ProMaxx® Epic Therapeutics, Inc./

Baxter Healthcare Corp.Insulin microsphere Phase I [307]

TransdermalPassPort™ Altea Therapeutics Microporation technique Phase I [308]Transfersulin® IDEA AG Transferosome Phase I [309–311]U-Strip™ Encapsulation System, Inc. Iontophoresis/sonophoresis Phase II [312]Macroflux® Alza Corp. Passive transdermal/

electrotransport systemsMarketavailable

[313]


When the solution is introduced to the buccal mucosa, it results inthe increased bioavailability of the active drug and more rapidonset of its action. RapidMist™ is a small, lightweight, hand-held,easy-to-use aerosol applicator containing an oral formulation,which is aerosolized with a pharmaceutical-grade chemical pro-pellant. To promote Oral-lyn™ in USA and Europe, Generexis currently commencing clinical trials involving thousands ofpatients around the world.

West Pharmaceutical Services' ChiSys™ formulation tech-nology, a chitosan-based nasal delivery system, enhances formu-lations for nasal insulin delivery and acts as a bioadhesive to helpdrugs adhere to the nasal mucosa. Phase I trials for nasal insulinwere completed in 2001 [278].

Entering the era of systemic pulmonary delivery, the healthcarecommunity is awaiting a revolution in many insulin deliverytechnologies. Several pharmaceutical companies are developingpulmonary insulin delivery systems. These products fall into twomain groups: solution and drug powder formulations, which aredelivered through different patented inhaler systems.

The approval of Exubera®, a human insulin (rDNA origin)inhalation powder, by the U.S. FDA has opened up a whole worldof possibilities for the deep lung delivery of drugs [279,280]. Animportant milestone in the treatment of systemic diseases withpulmonary delivery, it is set to revolutionize the drug deliverymarket. Exubera® is the first new inhaled form of insulin and thefirst insulin option in the European Union and USA in more than80 years that does not need to be administered by injection. Indeveloping Exubera®, Pfizer and Sanofi–Aventis have collabo-rated with Nektar Therapeutics (formerly Inhale Therapeutics), acompany that specializes in finding delivery solutions for oral,injectable, and pulmonary drug administration, to create an inhalerthat will support the manufacture (including powder processing)of Exubera® insulin and of Inhance™ (Exubera® pulmonarydelivery inhaler). Inhance™ weighs about 115 g and, whenclosed, is about the size of an eyeglass case. Inhance™ produces acloud of insulin powder in a clear chamber visible to the patient.This insulin powder is designed to pass rapidly into thebloodstream to regulate the body's blood sugar levels [281].

http://sec.edgar

http://pfizer.com

http://exubra.com

http://www.emisphere.com

http://www.autoimmuneinc.com

http://www.generex.com

http://www.bentleypharm.com

http://www.vectura.com

http://sec.edgar

http://pfizer.com

http://aradigm.com

http://alkermes.com

http://www.mannkindcorp.com

http://www.aerogen.com

http://www.prnewswire.com

http://www.abbott.com

http://www.microdose-tech.com

http://www.baxterbiopharmasolutions.com

http://www.alteatherapeutics.com

http://www.openpr.com

http://www.encsys.com

http://www.alza.com


Exubera®, a rapid-acting insulin in powder form that is inhaledbefore each meal, has been studied extensively in patients withtype 1 and type 2 diabetes mellitus [282–285]. The powderpreparation is packed into blisters of different dosages (1 mg and3mg blisters). Inhance™ generates a pulse of compressed air, thusdeagglomerating the dry powdered insulin into a cloud anddelivering it from the blister into a reservoir, from which it isinhaled. The standing cloud of aerosol eliminates the high-velocity impact of the insulin on the throat and upper airway. Thebioavailability of this product is approximately 10%, comparedwith that of regular human insulin administered by subcutaneousinjection [286].

Aradigm Corporation reports that Novo Nordisk A/S ispursing additional clinical phase III trials of AERx® insulinDiabetes Management System (AERx® iDMS), which delivers aliquid form of human insulin [287]. The AERx Essence®microprocessor-controlled inhaler offers a high level ofperformance (efficiency and reproducibility) in a smaller andless expensive package. In AERx Strip™ technology, the single-use nozzle contained in each disposable AERx Strip™ makesdosage adjustment possible to the nearest unit, as is possiblewith insulin injections. The system has been studied in patientswith type 1 and type 2 diabetes mellitus, and the bioavailabilityof insulin following the use of AERx® iDMS was 13%–17%[288–297].

Alkermes, Inc. and its partner Eli Lilly are also utilizingproprietary technology to create large porous particles of lowmassthat consist of a biodegradable polymer of a phospholipid matrixthat contains fast-acting human insulin. Future developmentsmight involve the production of a sustained-release formulation[298,299]. Their partnership is built upon the AIR® (AdvancedInhalation Research) technology platform that creates small(5 μm) particles to be delivered through a small, easy-to-useinhaler as capsules containing human inhalable insulin powder(HIIP®) as a dry powder in two dose strengths, 0.9 mg or 2.6 mg,equivalent to 2 or 6 units of subcutaneous insulin, respectively[300]. This platform has produced inhalable insulin that isundergoing phase III testing as a substitute for meal-time insulininjections in patients with type 1 or type 2 diabetes.

MannKind Corporation announced the results of its phase IIIclinical study of inhaled insulin in patients with type 2 diabetesin September 2006 at the 42nd Annual Meeting of the EuropeanAssociation for the Study of Diabetes in Copenhagen [301].This study was designed to evaluate whether the MannKindTechnosphere® Insulin System shows safety and efficacy similarto those observed in treatments with insulin aspart (NovoLog®),an injected rapid-acting insulin analogue. Technosphere®insulin, which was developed by the Pharmaceutical DiscoveryCorporation (PDC), is an ordered lattice array of sphericalmicroparticles at low pH, with 18% insulin trapped as a drycrystalline powder, which uses a diketopiperazine derivative thatdissolves at the neutral pH of the alveolar surface, rapidlyreleasing the insulin. The bioavailability of Technosphere®insulin is 15%–26%. The formulation is designed for use withthe MedTone™ Dry Powder Inhaler, a capsule-based high-impedance inhaler that uses a passive powder deagglomerationmechanism.

AeroGen, Inc. (acquired by Nektar Therapeutics in August2005) developed the pocket-sized Aerodose® insulin inhaler,designed to deliver a proprietary liquid insulin formulation. It isbreath activated and incorporates a titratable cartridge that allowspatients to adjust their dose based on their insulin requirements[302]. The development of this product was placed on hold inearly 2003, after the completion of phase IIa trials. Nektar Thera-peutics is now continuing to evaluate partnering opportunities,which may permit further development and commercialization ofthis product. The product has been studied in a small group ofpatients with type 2 diabetes mellitus [303]. Clinical studiesshowed that the relative bioavailability of inhaled Aerodose®insulin was approximately 21%, yet its development has beenhalted.

Altea Therapeutics has announced that it has commencedphase I clinical trials in USA of PassPort™, the company's dailyinsulin skin patch designed to provide continuous basal levels ofinsulin for patients with diabetes [308].

4. Conclusion

There has been a long history of research directed toward thedevelopment of novel routes of insulin delivery since recombi-nant DNA technology made insulin available at a reasonablecost in an injectable dosage form. Needle phobia and stress haveencouraged scientists to investigate and exploit all promisingroutes of insulin delivery, ranging from oral to rectal, with awide variety of devices and delivery systems. Many approacheshave been used to study various strategies to overcome theinherent barriers to insulin uptake across the GIT and bytransmucosal and transdermal routes. Each of the various routesof insulin administration has its own set of favorable andunfavorable properties. Most of the approaches described aboverepresent long-term possibilities for insulin delivery, butdifficulties in achieving adequate blood insulin concentrationsare yet to be overcome. Over the last several decades, theseformidable tasks have focused on oral insulin delivery. Our finalachievement will be a clinically therapeutic bioavailable oralinsulin that bypasses the obstacles of the GIT and overcomes thechallenges inherent in the physicochemical properties of theinsulin molecule. In recent years, the development of innovativeoral insulin delivery carriers that improve oral insulin absorptionhas thrown some promising light on the new horizon of oralinsulin therapy. Although extensive human clinical studiesare still required, especially of long-term clinical applications,researchers in academic institutions and several drug deliverypharmaceutical companies are actively involved in the develop-ment of an oral insulin delivery system.

Significant progress has been reported in recent years in thedelivery of insulin using pulmonary and buccal routes, which haveled to market approval for Exubera® and Oral-lyn™, suggestingthat this breakthrough in oral insulin delivery marketing will soonbecome a larger reality. Some of the delivery systems discussed inthis review are likely to reach patients in the next few years. Thenew millennium promises a revolutionary change in the deliveryof insulin for billions of sufferers who are currently reliant onsubcutaneous administration.

http://aradigm.com

http://alkermes.com

http://www.mannkindcorp.com

http://www.aerogen.com

http://www.alteatherapeutics.com


References

[1] S. Frokjaer, D.D. Otez, Protein drug stability: a formulation challenge, Nat.Rev., Drug Discov. 4 (2005) 298–306.

[2] V.P. Torchillin, A.N. Lukyanov, Peptide and protein drug delivery to andinto tumors: challenge and solution, Drug Discov. Today 8 (2003)259–266.

[3] R.B. Shah, F. Ahsan, M.A. Khan, Oral delivery of proteins: progress andprognostication, Crit. Rev. Ther. Drug Carr. Syst. 19 (2005) 135–169.

[4] R.I. Mahato, A.S. Narang, L. Thoma, D.D. Miller, Emerging trends in oraldelivery of peptide and proteins, Crit. Rev. Ther. Drug Carr. Syst. 20 (2003)153–214.

[5] G. Gwinup, A.N. Elias, N.D. Vaziri, A case for oral insulin therapy inthe prevention of diabetic micro- and macroangiopathy, Int. J. Artif. Organs13 (1990) 393–395.

[6] F.P. Kennedy, Recent developments in insulin delivery techniques: currentstatus and future potential, Drugs 42 (1991) 213–227.

[7] D.R. Owens, New horizons-alternative routes for insulin therapy, Nat. Rev.,Drug Discov. 1 (2002) 529–540.

[8] W.T. Cefalu, Concept, strategies, and feasibility of non-invensive insulindelivery, Diabetes Care 27 (2004) 239–246.

[9] M. Gordberg, I. Gomez-Orellana, Challenge for the oral delivery ofmacromolecules, Nat. Rev., Drug Discov. 2 (2003) 289–295.

[10] N.M.K. Ghilzai, New development in insulin delivery, Drug Dev. Ind.Pharm. 29 (2003) 253–265.

[11] V. Agarwal, M.A. Khan, Current status of the oral delivery of insulin,Pharm. Technol. 10 (2001) 76–90.

[12] J.H. Hamman, G.M. Enslin, A.F. Kotze, Oral delivery of peptide drugs,BioDrugs 19 (2005) 165–177.

[13] E.M. Lane, C.M. O'driscoll, O.I. Corrigan, Quantitative estimation of theeffects of bile salts surfactant systems on insulin stability and permeabilityin the rat intestine using a mass balance model, J. Pharm. Pharmacol.57 (2005) 169–175.

[14] T. Uchiyama, T. Sugiyama, Y.S. Quan, A. Kotani, N. Okada, T. Fujita, S.Muranishi, A. Yamamoto, Enhanced permeability of insulin across the ratintestinal membrane by various absorption enhancers: their intestinalmucosal toxicity and absorption enhancing mechanism of n-lauryl-b-D-maltopyranoside, J. Pharm. Pharmacol. 51 (1999) 1241–1250.

[15] M. Morishita, M. Kajita, A. Suzuki, K. Takayama, Y. Chiba, S. Tokiwa, T.Nagai, The dose-related hypoglycemic effects of insulin emulsionsincorporating highly purified EPA and DHA, Int. J. Pharm. 201 (2000)175–185.

[16] E.A. Hosny, H.I. Al-Shora, M.M.A. Elmazar, Oral delivery of insulin fromenteric-coated capsules containing sodium salicylate: effect on relativehypoglycemia of diabetic beagle dogs, Int. J. Pharm. 237 (2002) 71–76.

[17] M. Morishita, I. Morishita, K. Takayama, Y. Machida, T. Nagai, Site-dependent effect of aprotinin, sodium caprate, Na2EDTA and sodiumglycocholate on intestinal absorption of insulin, Biol. Pharm. Bull.16 (1993) 68–72.

[18] N.N. Salama, N.D. Eddington, A. Fasano, Tight junction modulation andits relationship to drug delivery, Adv. Drug Deliv. Rev. 58 (2006) 15–28.

[19] A. Fasano, S. Uzzau, Modulation of intestinal tight junctions by zonulaoccludens toxin permits enteral administration of insulin and othermacromolecules in an animal model, J. Clin. Invest. 99 (1997) 1158–1164.

[20] M.K. Marschütz, A. Bernkop-Schnürch, Oral peptide drug delivery:polymer–inhibitor conjugates protecting insulin from enzymatic degrada-tion in vitro, Biomaterials 21 (2000) 1499–1507.

[21] A. Yamamoto, T. Taniguchi, K. Rikyuu, T. Tsuji, T. Fujita, M. Murakami,S. Muranishi, Effect of various protease inhibitors on the intestinalabsorption and degradation of insulin in rats, Pharm. Res. 11 (1994)1496–1500.

[22] H. Liu, R. Tang, W.S. Pan, Y. Zhang, H. Liu, Potential utility of variousprotease inhibitors for improving the intestinal absorption of insulin in rats,J. Pharm. Pharmacol. 55 (2003) 1523–1529.

[23] V. Agarwal, I.K. Reddy, M.A. Khan, Oral delivery of proteins: effect ofchicken and duck ovomucoid on the stability of insulin in the presence ofα-chymotrypsin and trypsin, Pharm. Pharmacol. Commun. 6 (2000)223–227.

[24] V. Agarwal, I.K. Reddy, M.A. Khan, Polmethylacrylate microcapsules ofinsulin for oral delivery: preparation and in vitro dissolution stability in thepresence of enzyme inhibitors, Int. J. Pharm. 225 (2001) 31–39.

[25] V. Agarwal, S. Nazzal, I.K. Reddy, M.A. Khan, Transport studies of insulinacross rat jejunum in the presence of chicken and duck ovomucoid,J. Pharm. Pharmacol. 53 (2001) 1131–1138.

[26] M.K. Marschütz, P. Caliceti, A. Bernkop-Schnürch, Design and in vivoevaluation of an oral delivery system for insulin, Pharm. Res. 17 (2000)1468–1474.

[27] M. Morishita, Y. Aoki, M. Sakagami, T. Nagai, K. Takayama, In situ ilealabsorption of insulin in rats: effect of hyaluronidase pretreatmentdiminishing the mucous/glycocalyx layers, Pharm. Res. 21 (2004)309–316.

[28] Y. Aoki, M. Morishita, K. Asai, B. Akikusa, S. Hosoda, K. Takayama,Region-dependent role of the mucous/glycocalyx layers in insulinpermeation across rat small intestine membrane, Pharm. Res. 22 (2005)1854–1862.

[29] H. Takeuchi, H. Yamamoto, Y. Kawashima, Mucoadhesive nanoparticulatesystems for peptide drug delivery, Adv. Drug Deliv. Rev. 47 (2001) 39–54.

[30] K.P.R. Chowdary, Y.S. Rao, Mucoadhesive microspheres for controlleddrug delivery, Biol. Pharm. Bull. 27 (2004) 1717–1724.

[31] R. Hejazi, M. Amiji, Chitosan-based gastrointestinal deliver system,J. Control. Release 89 (2003) 151–165.

[32] N.A. Peppas, Devices based on intelligent biopolymers for oral proteindelivery, Int. J. Pharm. 227 (2004) 11–17.

[33] A. Bernkop-Schnürch, A. Scerbe-Saiko, Synthesis and in vitro evaluationof chitosan–EDTA–protease–inhibitor conjugates whichmight be useful inoral delivery of peptides and proteins, Pharm. Res. 15 (1998) 263–269.

[34] F.A. Dorkoosh, M.P.M. Stokkel, D. Blok, G. Borchard, M. Rafiee-Tehrani,J.C. Verhoef, H.E. Junginger, Feasibility study on the retention ofsuperporous hydrogel composite polymer in the intestinal tract of manusing scintigraphy, J. Control. Release 99 (2004) 199–206.

[35] A. Lamprecht, J.L. Saumet, J. Roux, J.P. Benoit, Lipid nanocarriers as drugdelivery system for ibuprofen in pain treatment, Int. J. Pharm. 278 (2004)407–414.

[36] A. Bernkop-Schnürch, C.E. Kast, M.F. Richter, Improvement in themucoadhesive properties of alginate by the covalent attachment of cysteine,J. Control. Release 71 (2001) 277–285.

[37] H. Takeuchi, H. Yamamoto, T. Niwa, T. Hino, Y. Kawashima, Enteralabsorption of insulin in rats frommucoadhesive chitosan-coated liposomes,Pharm. Res. 13 (1996) 896–901.

[38] A. Bernkop-Schnürch, M.E. Krajicek, Mucoadhesive polymers as plat-forms for peroral peptide delivery and absorption: synthesis and evaluationof different chitosan–EDTA conjugates, J. Control. Release 50 (2001)215–223.

[39] C.M. Lehr, Lectin-mediated drug delivery: the second generation ofbioadhesive, J. Control. Release 65 (2000) 19–29.

[40] A.M. Lowman, M. Morishita, M. Kajita, T. Nagai, N.A. Peppas, Oraldelivery of insulin using pH-responsive complexation gels, J. Pharm. Sci.88 (1999) 933–937.

[41] K. Nakamura, R.J. Murray, J.I. Joseph, N.A. Peppas, M. Morishita, A.M.Lowman, Oral insulin delivery using P(MAA-g-EG) hydrogels: effects ofnetwork morphology on insulin delivery characteristics, J. Control. Release95 (2003) 589–599.

[42] A.C. Foss, T. Goto, M. Morishita, N.A. Peppas, Development of acrylic-based copolymers for oral insulin delivery, Eur. J. Pharm. Biopharm.57 (2003) 163–169.

[43] J.E. López, N.A. Peppas, Effect of poly (ethylene glycol) molecular weightand microparticle size on oral insulin delivery from P(MAA-g-EG)microparticles, Drug Dev. Ind. Pharm. 30 (2004) 497–504.

[44] M. Morishita, T. Goto, N.A. Peppas, J.I. Joseph, M.C. Torjman, C.Munsick, K. Nakamura, T. Yamagata, K. Takayama, A.M. Lowman,Mucosal insulin delivery systems based on complexation polymerhydrogels: effect of particle size on insulin enteral absorption, J. Control.Release 97 (2004) 115–124.

[45] M. Morishita, T. Goto, K. Takayama, N.A. Peppas, Oral insulin deliverysystems based on complexation polymer hydrogels, J. Drug Deliv. Sci.Tech. 16 (2006) 19–24.


[46] T. Yamagata, M. Morishita, N.J. Kavimandan, K. Nakamura, Y. Fukuoka,K. Takayama, N.A. Peppas, Characterization of insulin protectionproperties of complexation hydrogels in gastric and intestinal enzymefluids, J. Control. Release 112 (2006) 343–349.

[47] M. Morishita, T. Goto, K. Nakamura, A.M. Lowman, K. Takayama, N.A.Peppas, Novel oral insulin delivery systems based on complexationpolymer hydrogels: single and multiple administration studies in type 1 and2 diabetic rats, J. Control. Release 110 (2006) 587–594.

[48] B.Y. Kim, J.H. Jeong, K. Park, J.D. Kim, Bioadhesive interaction andhypoglycemic effect of insulin-loaded lectin–microparticle conjugates inoral insulin delivery system, J. Control. Release 102 (2005) 525–538.

[49] Y. Pan, Y. Li, H. Zhao, J. Zheng, H. Xu, G.Wei, J. Hao, F. Cui, Bioadhesivepolysaccharide in protein delivery system: chitosan nanoparticles improvethe intestinal absorption of insulin in vivo, Int. J. Pharm. 249 (2002)139–147.

[50] A. Bernkop-Schnürch, Thiomers: a new generation of mucoadhesivepolymers, Adv. Drug Deliv. Rev. 57 (2005) 1569–1582.

[51] A. Bernkop-Schnürch, U.M. Brandt, A.E. Clausen, Synthesis and in vitroevaluation of chitosan–cysteine conjugates, Sci. Pharm. 67 (1999)196–208.

[52] A. Bernkop-Schnürch, M. Hornof, T. Zoidl, Thiolated polymers–thiomers:synthesis and in vitro evaluation of chitosan–2-iminothiolane conjugates,Int. J. Pharm. 260 (2003) 229–237.

[53] M. Roldo, M. Hornof, P. Caliceti, A. Bernkop-Schnürch, Mucoadhesivethiolated chitosans as platforms for oral controlled drug delivery: synthesisand in vitro evaluation, Eur. J. Pharm. Biopharm. 57 (2004) 115–121.

[54] A. Bernkop-Schnürch, T.E. Hopf, Synthesis and in vitro evaluation ofchitosan–thioglycolic acid conjugates, Sci. Pharm. 69 (2001) 109–118.

[55] C.E. Kast, A. Bernkop-Schnürch, Thiolated polymers–thiomers: develop-ment and in vitro evaluation of chitosan–thioglycolic acid conjugates,Biomaterials 22 (2001) 2345–2352.

[56] A. Bernkop-Schnürch, S. Steininger, Synthesis and characterization ofmucoadhesive thiolated, Int. J. Pharm. 194 (2000) 239–247.

[57] A. Bernkop-Schnürch, A.E. Clausen, M. Hnatyszyn, Thiolated polymers:synthesis and in vitro evaluation of polymer–cysteamine conjugates, Int. J.Pharm. 226 (2001) 185–194.

[58] A. Bernkop-Schnürch, S. Scholler, R.G. Biebel, Development of controlleddrug release systems based on polymer–cysteine conjugates, J. Control.Release 66 (2000) 39–48.

[59] A. Bernkop-Schnürch, C.E. Kast, M.F. Richter, Improvement in themucoadhesive properties of alginate by covalent attachment of cysteine,J. Control. Release 71 (2001) 277–285.

[60] A. Bernkop-Schnürch, A.H. Krauland, V.M. Leitner, T. Palmberger,Thiomers: potential excipients for non-invasive peptide delivery systems,Eur. J. Pharm. Biopharm. 58 (2004) 253–263.

[61] A.H. Krauland, D. Guggi, A. Bernkop-Schnürch, Oral insulin delivery: thepotential of thiolated chitosan-insulin tablets on non-diabetic rats,J. Control. Release 95 (2004) 547–555.

[62] H.E. Junginger, Bioadhesive polymer systems for peptide delivery, ActaPharm. Technol. 36 (1990) 110–126.

[63] A. Bernkop-Schnürch, A.E. Clausen, Biomembrane permeability ofpeptides: strategies to improve their mucosal uptake, Mini Rev. Med.Chem. 2 (2002) 295–305.

[64] G. Ponchel, M.J. Montisci, A. Dembri, C. Durrer, D. Duchêne,Mucoadhesion of colloidal particulate systems in the gastro-intestinaltract, Eur. J. Pharm. Biopharm. 44 (1997) 25–31.

[65] D.J. Brayden, M.A. Jepson, A.W. Baird, Keynote review: intestinal Peyer'spatch M cells and oral vaccine targeting, Drug Discov. Today 10 (2005)1145–1157.

[66] M.P. Desai, V. Labhasetwar, E. Walter, R.J. Levy, G.L. Amidon, Themechanism of uptake of biodegradable microparticles in Caco-2 cells is sizedependent, Pharm. Res. 14 (1997) 1568–1573.

[67] I.M. van der Lubben, J.C. Verhoef, G. Borchard, H.E. Junginger, Chitosanand its derivatives in mucosal drug and vaccine delivery, Eur. J. Pharm. Sci.14 (2001) 201–207.

[68] S. Watnasirichaikull, T. Rades, I.G. Tucker, N.M. Davies, In vitro releaseand oral bioactivity of insulin in diabetic rats using nanocapsules dispersedin biocompatible microemulsion, J. Pharm. Pharmacol. 54 (2002) 473–480.

[69] M.A. Kisel, L.N. Kulik, I.S. Tsybovsky, A.P. Vlasov, M.S. Vorob'yov, E.A.Kholodova, Z.V. Zabarovskaya, Liposomes with phosphatidylethanolas a carrier for oral delivery of insulin: studies in the rat, Int. J. Pharm.216 (2001) 105–114.

[70] T. Jung, W. Kamm, A. Breitenbach, E. Kaiserling, J.X. Xiao, T. Kissel,Biodegradable nanoparticles for oral delivery of peptides: is there a role forpolymers to affect mucosal uptake, Eur. J. Pharm. Biopharm. 50 (2000)147–160.

[71] M.C. Jones, J.C. Leroux, Polymeric micelles: a new generation of colloidaldrug carrier, Eur. J. Pharm. Biopharm. 48 (1999) 101–111.

[72] E. Toorisaka, M. Hashida, N. Kamiya, H. Ono, Y. Kokazu, M. Goto, Anenteric-coated dry emulsion formulation for oral insulin delivery, J. Control.Release 107 (2005) 91–96.

[73] E. Toorisaka, H. Ono, K. Arimori, N. Kamiya, M. Goto, Hypoglycemiceffect of surfactant-coated insulin solubilized in a novel solid-in-oil-in-water (S/O/W) emulsion, Int. J. Pharm. 252 (2003) 271–274.

[74] A. Çilek, N. Çelebi, F. Tırnaksız, A. Tay, A lecithin-based microemulsionof rh-insulin with aprotinin for oral administration: investigation ofhypoglycemic effects in non-diabetic and STZ-induced diabetic rats,Int. J. Pharm. 298 (2005) 176–185.

[75] A. Čilek, N. Čelebi, F. Tirnaksiz, Lecithin-based microemulsion of apeptide for oral administration: preparation, characterization, and physicalstability of the formulation, Drug Deliv. 13 (2006) 19–24.

[76] K. Katayama, Y. Kato, H. Onishi, T. Nagai, Y. Machida, Double liposomes:hypoglycemic effects of liposomal insulin on normal rats, Drug Dev. Ind.Pharm. 29 (2003) 725–731.

[77] Z. Degim, N. Ünal, D. Eşsiz, U. Abbasoglu, The effect of various liposomeformulations on insulin penetration across Caco-2 cell monolayer, Life Sci.75 (2004) 2819–2827.

[78] T. Goto, M. Morishita, K. Nishimura, M. Nakanishi, A. Kato, J. Ehara, K.Takayama, Novel mucosal insulin delivery systems based on fusogenicliposomes, Pharm. Res. 23 (2006) 384–391.

[79] S. Ye, C. Wang, X. Liu, Z. Tong, B. Ren, F. Zeng, New loading processand release properties of insulin from polysaccharide microcapsulesfabricated through layer-by-layer assembly, J. Control. Release 112 (2006)79–87.

[80] S. Watnasirichaikul, N.M. Davies, T. Rades, I.G. Tucker, Preparation ofbiodegradable insulin nanocapsules from biocompatible microemulsions,Pharm. Res. 17 (2000) 684–689.

[81] L.Y. Wang, Y.H. Gu, Z.G. Su, G.H. Ma, Preparation and improvement ofrelease behavior of chitosan microspheres containing insulin, Int. J. Pharm.311 (2006) 187–195.

[82] D. Jain, A.K. Panda, D.K. Majumdar, Eudragit S100 entrapped insulinmicrospheres for oral delivery, AAPS PharmSciTech 6 (2005) 100–107.

[83] S. Sajeesh, C.P. Sharma, Novel pH responsive polymethacrylic acid–chitosan–polyethylene glycol nanoparticles for oral peptide delivery,J. Biomed. Mater. Res., B Appl. Biomater. 76 (2005) 298–305.

[84] F. Cui, K. Shi, L. Zhang, A. Tao, Y. Kawashima, Biodegradablenanoparticles loaded with insulin–phospholipid complex for oral delivery:preparation, in vitro characterization and in vivo evaluation, J. Control.Release 114 (2006) 242–250.

[85] M.Morishita, N.A. Peppas, Is the oral route possible for peptide and proteindrug delivery? Drug Discov. Today 11 (2006) 905–910.

[86] D.K. Pettit, W.R. Gombotz, The development of site-specific drug-deliverysystems for protein and peptide biopharmaceuticals, Trends Biotechnol.16 (1998) 343–349.

[87] A.L. Daugherty, R. Mrsny, Transcellular uptake mechanisms of intestinalepithelial barrier: part one, Pharm. Sci. Technol. Today 4 (1999) 144–151.

[88] R.J. Mrsny, The colon as a site for drug delivery, J. Control. Release22 (1992) 15–34.

[89] S. Haupt, A. Rubinstein, The colon as a possible target for orallyadministered peptide and protein drugs, Crit. Rev. Ther. Drug Carr. Syst.19 (2002) 499–551.

[90] M. Katsuma, S. Watanabe, H. Kawai, S. Takemura, K. Sako, Effect ofabsorption promoters on insulin absorption through colon-targeted delivery,Int. J. Pharm. 307 (2005) 156–162.

[91] H. Tozaki, J. Nishioka, J. Komoike, N. Okada, T. Fujita, S. Muranishi, S.I.Kim, H. Terashima, A. Yamamoto, Enhanced absorption of insulin and


(Asu1,7)eel-calcitonin using novel azopolymer-coated pellets for colon-specific drug delivery, J. Pharm. Sci. 90 (2001) 89–97.

[92] J.M. Sarciaux, L. Acar, P.A. Sado, Using microemulsion formulation fororal drug delivery of therapeutic peptides, Int. J. Pharm. 120 (1995)127–136.

[93] M. Shakweh, M. Besnard, V. Nicolas, E. Fattal, Poly (lactide-co-glycolide)particles of different physicochemical properties and their uptake byPeyer's patches in mice, Eur. J. Pharm. Biopharm. 61 (2005) 1–13.

[94] A.M. Hillery, P.U. Jani, A.T. Florence, Comparative, quantitative studyof lymphoid and non-lymphoid uptake of 60 nm particles, J. Drug Target.2 (1994) 151–156.

[95] J.W. Joseph, J. Kalitsky, S. St-Pierre, P.L. Brubaker, Oral delivery ofglucagon–like peptide-1 in a modified polymer preparation normalizesbasal glycaemia in diabetic db/db mice, Diabetologia 43 (2000) 1319–1328.

[96] C.Q. Xia, J. Wang, W.C. Shen, Hypoglycemic effect of insulin–transferrinconjugate in streptozotocin-induced diabetic rats, J. Pharmacol. Exp. Ther.295 (2000) 594–600.

C.Q. Xia, W.C. Shen, Tyrphostin-8 enhances transferring receptor-mediatetranscytosis in Caco-2 cells and increase hypoglycemic effect of orallyadministered insulin–transferring conjugate in diabetic rats, Pharm. Res. 18(2001) 191–195.[98] Y. Sudhakar, K. Kuotsu, A.K. Bandyopadhyay, Buccal bioadhesive drug

delivery ― a promising option for orally less efficient drugs, J. Control.Release 114 (2006) 15–40.

[99] F. Veuillez, Y.N. Kalia, Y. Jacques, J. Deshusses, P. Buri, Factors andstrategies for improving buccal absorption of peptides, Eur. J. Pharm.Biopharm. 51 (2001) 93–109.

[100] M. Morishita, J.M. Barichello, K. Takayama, Y. Chiba, S. Tokiwa, T.Nagai, Pluronic® F-127 gels incorporating highly purified unsaturated fattyacids for buccal delivery of insulin, Int. J. Pharm. 212 (2001) 289–293.

[101] P.L. Starokadomskyy, I.Ya. Dubey, New absorption promoter for the buccaldelivery: preparation and characterization of lysalbinic acid, Int. J. Pharm.308 (2006) 149–154.

[102] P.L. Starokadomskyy, New possibilities of chewing gum in local therapy,Proceedings of the Conference for Young Scientists, PhD Students andStudents. Institute of Molecular Biology and Genetics, Kyiv, Ukraine,September 25–27, 2003, p. 263.

[103] T.Z. Yang, X.T. Wang, X.Y. Yan, Q. Zhang, Phospholipid deformablevesicles for buccal delivery of insulin, Chem. Pharm. Bull. 50 (2002)749–753.

[104] S. Senel, M. Kremer, K. Nagy, C. Squier, Delivery of bioactive peptidesand proteins across oral (buccal) mucosa, Curr. Pharm. Biotechnol.2 (2001) 175–186.

[105] M. Ishida, Y. Machida, N. Nambu, T. Nagai, New mucosal dosage form ofinsulin, Chem. Pharm. Bull. 29 (1981) 810–816.

[106] P. Venugopalan, A. Sapre, N. Venkatesan, S.P. Vyas, Pelleted bioadhesivepolymeric nanoparticles for buccal delivery of insulin: preparation andcharacterization, Pharmazie 56 (2001) 217–219.

[107] E.A. Hosny, S.A. Elkheshen, S.I. Saleh, Buccoadhesive tablets for insulindelivery: in vitro and in vivo studies, Boll. Chim. Farm. 141 (2002)210–217.

[108] H.B. Xu, K.X. Huang, Y.S. Zhu, Q.H. Gao, Q.Z. Wu, W.Q. Tian, X.Q.Sheng, Z.X. Chen, Z.H. Gao, Hypoglycaemic effect of a novel insulinbuccal formulation on rabbits, Pharmacol. Res. 46 (2002) 459–467.

[109] T. Kissel, U. Werner, Nasal delivery of peptides: an in vitro cell culturemodel for the investigation of transport and metabolism in human nasalepithelium, J. Control. Release 53 (1998) 195–203.

[110] D. Ridley, A.C. Perkins, N. Washington, C.G. Wilson, M.L. Wastie, P.Oflynn, A. Blattman, G. Ponchel, D. Duchene, The effect of posture onnasal clearance of bioadhesive starch microspheres, S.T.P. Pharma. Sci. 5(1995) 442–446.

[111] M. Hinchcliffe, L. Illum, Intranasal insulin delivery and therapy, Adv. DrugDeliv. Rev. 35 (1999) 199–234.

[112] S. Türker, E. Onur, Y. Özer, Nasal route and drug delivery systems, Pharm.World Sci. 26 (2004) 137–142.

[113] E. Pringels, C. Callens, C. Vervaet, F. Dumont, G. Slegers, P. Foreman, J.P.Remon, Influence of deposition and spray pattern of nasal powders oninsulin bioavailability, Int. J. Pharm. 310 (2006) 1–7.

[114] Y. Zhang, X.G. Jiang, J. Yao, Nasal absorption enhancement of insulin bysodium deoxycholate in combination with cyclodextrins, Acta Pharmacol.Sin. 22 (2001) 1051–1056.

[115] T. Ando, Y. Maitani, T. Yamamoto, K. Takayama, T. Nagai, Nasal insulindelivery in rabbits using soybean-derived sterylglucoside and sterolmixtures as novel enhancers in suspension dosage forms, Biol. Pharm.Bull. 21 (1998) 862–865.

[116] D.J. Pillion, F. Ahsan, J.J. Arnold, B.M. Balusubramanian, O. Piraner, E.Meezan, Synthetic long-chain alkyl maltosides and alkyl sucrose esters asenhancers of nasal insulin absorption, J. Pharm. Sci. 91 (2002) 1456–1462.

[117] R. Mitra, I. Pezron, W.A. Chu, A.K. Mitra, Lipid emulsions as vehicles forenhanced nasal delivery of insulin, Int. J. Pharm. 205 (2000) 127–134.

[118] M.A. Wheatley, J. Dent, E.B. Wheeldon, P.L. Smith, Nasal drug delivery:an in vitro characterization of transepithelial electrical properties and fluxesin the presence or absence of enhancers, J. Control Release 8 (1988)167–177.

[119] Z. Shao, A.K. Mitra, Nasal membrane and intracellular protein release bybile salts and bile salt–fatty acid mixed micelles: correlation with facilitatednasal drug transport, Pharm. Res. 9 (1992) 1184–1189.

[120] N.G.M. Schipper, J. Verhoef, S.G. Romeijn, F.W.H.M.Merkus, Absorptionenhancers in nasal insulin delivery and their influence on nasal cilliaryfunctioning, J. Control. Release 21 (1992) 173–186.

[121] R.J. Soane, M. Frier, A.C. Perkins, N.S. Jones, S.S. Davis, L. Illum,Evaluation of the clearance characteristics of bioadhesive systems inhumans, Int. J. Pharm. 178 (1999) 55–65.

[122] R.J. Soane, M. Hinchcliffe, S.S. Davis, L. Illum, Clearence characteristicsof chitosan based formulations in the sheep nasal cavity, Int. J. Pharm.217 (2001) 183–191.

[123] L. Illum, Chitosan and its use as a pharmaceutical excipient, Pharm. Res.15 (1998) 1326–1331.

[124] A.R. Najafabadi, P. Moslemi, H. Tajerzadeh, Intranasal bioavailability ofinsulin from carbopol-based gel spray in rabbits, Drug Deliv. 11 (2004)295–300.

[125] J. Varshosaz, H. Sadrai, A. Heidari, Nasal delivery of insulin usingbioadhesive chitosan gels, Drug Deliv. 13 (2006) 31–38.

[126] A.M. Dyer, M. Hinchcliffe, P. Watts, J. Castile, I. Jabbal-Gill, R. Nankervis,A. Smith, L. Illum, Nasal delivery of insulin using novel chitosan basedformulations: a comparative study in two animal models between simplechitosan formulations and chitosan nanoparticles, Pharm. Res. 19 (2002)998–1008.

[127] J. Varshosaz, H. Sadrai, R. Alinagari, Nasal delivery of insulin usingchitosan microspheres, J. Microencapsul. 21 (2004) 761–774.

[128] A.H. Krauland, D. Guggi, A. Bernkop-Schnürch, Thiolated chitosanmicroparticles: a vehicle for nasal peptide drug delivery, Int. J. Pharm.307 (2006) 270–277.

[129] A.H. Krauland, V.M. Leitner, V. Grabovac, A. Bernkop-Schnürch, In vivoevaluation of a nasal insulin delivery system based on thiolated chitosan,J. Pharm. Sci. 95 (2006) 1–10.

[130] J. Wang, Y. Tabata, K. Morimoto, Aminated gelatin microspheres as a nasaldelivery system for peptide drugs: evaluation of in vitro release and in vivoinsulin absorption in rats, J. Control. Release 113 (2006) 31–37.

[131] R.U. Agu, M.I. Ugwoke, M. Armand, R. Kinget, N. Verbeke, The lung as aroute for systemic delivery of therapeutic proteins and peptides, Respir.Res. 2 (2001) 198–209.

[132] S. Bellary, A.H. Barnett, Inhaled insulin: new technology, new possibilities,Int. J. Clin. Pract. 60 (2006) 728–734.

[133] W.T. Cefalu, Rationale for and strategies to achieve glycemic control, in:J.L. Leahy, W.T. Cefalu (Eds.), Insulin Therapy, vol. 1, Marcel DekkerInc., New York, 2002, pp. 1–11.

[134] K. Morimoto, Y. Uehara, K. Iwanaga, M. Kakemi, Effects of sodiumglycocholate and protease inhibitors on permeability of TRH and insulinacross rabbit trachea, Pharm. Acta Helv. 74 (2000) 411–415.

[135] J.H. Kwon, B.H. Lee, J.J. Lee, C.W. Kim, Insulin microcrystal suspensionas a long-acting formulation for pulmonary delivery, Eur. J. Pharm. Sci.22 (2004) 107–116.

[136] X.J. Chen, J.B. Zhu, G.J. Wang, M.X. Zhou, F.M. Xin, N. Zhang, C.X.Wang, Y.N. Xu, Hypoglycemic efficacy of pulmonary delivered insulin drypowder aerosol in rats, Acta Pharmacol. Sin. 23 (2002) 467–470.


[137] H.K. Lee, J.H. Kwon, S.H. Park, C.W. Kim, Enhanced hypoglycemicactivity following intratracheal administration of insulin microcrystalsuspension with injection adjuvant, Biosci. Biotechnol. Biochem.70 (2006) 1003–1005.

[138] Z.C. Shen, Y. Cheng, Q. Zhang, S.L. Wei, R.C. Li, K. Wang, Lanthanidesenhance pulmonary absorption of insulin, Biol. Trace Elem. Res. 75 (2000)215–225.

[139] M. Korc, Effect of lanthanum on pancreatic protein synthesis instreptozotocin-diabetic rats, Am. J. Physiol.: Gastrointest. Liver Physiol.244 (1983) G321–G326.

[140] M. Granado, A.I. Martín, T. Priego, M.A. Villanúa, A. López-Calderón,Inactivation of Kupffer cells by gadolinium administration preventslipopolysaccharide-induced decrease in liver insulin-like growth factor-Iand IGF-binding protein-3 gene expression, J. Endocrinol. 188 (2006)503–511.

[141] K. Surendrakumar, G.P. Martyn, E.C.M. Hodgers, M. Jansen, J.A. Blair,Sustained release of insulin from sodium hyaluronate based dry powderformulations after pulmonary delivery to beagle dogs, J. Control. Release91 (2003) 385–394.

[142] Y. Suzuki, T. Yamaguchi, Effects of hyaluronic acid on macrophagephagocytosis and active oxygen release, Agents Actions 38 (1993) 32–37.

[143] F. Ahsan, I.P. Rivas, M.A. Khan, A.I.T. Suárez, Targeting to macrophages:role of physicochemical properties of particulate carriers–liposomes andmicrospheres–on the phagocytosis by macrophages, J. Control. Release79 (2002) 29–40.

[144] R. Mitra, I. Pezron, Y. Li, A.K. Mitra, Enhanced pulmonary delivery ofinsulin by lung lavage fluid and phospholipids, Int. J. Pharm. 217 (2001)25–31.

[145] H. Todo, H. Okamoto, K. Iida, K. Danjo, Effect of additives on insulinabsorption from intratracheally administered dry powders in rats, Int. J.Pharm. 220 (2001) 101–110.

[146] A. Hussain, T. Yang, A.A. Zaghloul, F. Ahsan, Pulmonary absorption ofinsulin mediated by tetradecyl-beta-maltoside and dimethyl-beta-cyclodex-trin, Pharm. Res. 20 (2003) 1551–1557.

[147] Y.Y. Huang, C.H. Wang, Pulmonary delivery of insulin by liposomalcarriers, J. Control. Release 113 (2006) 9–14.

[148] L. Garcia-Contreras, T. Morçöl, S.J.D. Bell, A.J. Hickey, Evaluation ofnovel particles as pulmonary delivery systems for insulin in rats, AAPSPharmSci 5 (2003) (Article 9).

[149] P. O'Hara, A.J. Hickey, Respirable PLGA microspheres containingrifampicin for the treatment of tuberculosis: manufacture and characteriza-tion, Pharm. Res. 17 (2000) 955–961.

[150] S. Suarez, P. O'Hara, M. Kazantseva, C.E. Newcomer, R. Hopfer, D.N.McMurray, A.J. Hickey, Respirable PLGA microspheres containingrifampicin for the treatment of tuberculosis: screening in an infectiousdisease model, Pharm. Res. 18 (2001) 1315–1319.

[151] D.A. Edwards, J. Hanes, G. Caponetti, J. Hrkach, A. Ben-Jebria, M.L.Eskew, J. Mintzes, D. Deaver, N. Lotan, R. Langer, Large porous particlesfor pulmonary drug delivery, Science 276 (1997) 1868–1871.

[152] Y. Kawashima, H. Yamamoto, H. Takeuchi, S. Fujioka, T. Hino, Pulmonarydelivery of insulin with nebulized DL-lactide/glycolide copolymer (PLGA)nanospheres to prolong hypoglycemic effect, J. Control. Release 62 (1999)279–287.

[153] M. Morlock, H. Knoll, G. Winter, T. Kissel, Microencapsulation of rh-erythropoietin using biodegradable poly(D,L-lactide-co-glycolide): proteinstability and the effects of stabilizing excipients, Eur. J. Pharm. Biopharm.43 (1997) 29–36.

[154] L. Meinel, O.E. Illi, J. Zapf, M. Malfanti, H.P. Merkle, B. Gander,Stabilizing insulin-like growth factor-I in poly(D,L-lactide-co-glycolide)microspheres, J. Control. Release 70 (2001) 193–202.

[155] M. Murillo, M.M. Goñi, J.M. Irache, M.A. Arangoa, J.M. Blasco, C.Gamazo, Modulation of the cellular immune response after oral orsubcutaneous immunization with microparticles containing Brucella ovisantigens, J. Control. Release 85 (2002) 237–246.

[156] F. Quaglia, G. De Rosa, E. Granata, F. Ungaro, E. Fattal, M.I. La Rotonda,Feeding liquid, non-ionic surfactant and cyclodextrin affect the propertiesof insulin-loaded poly(lactide-co-glycolide) microspheres prepared byspray-drying, J. Control. Release 86 (2003) 267–278.

[157] G. De Rosa, D. Larobina, M.I. La Rotonda, P. Musto, F. Quaglia, F.Ungaro, How cyclodextrin incorporation affects the properties of protein-loaded PLGA-based microspheres: the case of insulin/hydroxypropyl-beta-cyclodextrin system, J. Control. Release 102 (2005) 71–83.

[158] F. Ungaro, G. De Rosa, A. Miro, F. Quaglia, M.I. La Rotonda,Cyclodextrins in the production of large porous particles: development ofdry powders for the sustained release of insulin to the lungs, Eur. J. Pharm.Sci. 28 (2006) 423–432.

[159] Q. Zhang, Z. Shen, T. Nagai, Prolonged hypoglycemic effect of insulin-loaded polybutylcyanoacrylate nanoparticles after pulmonary administra-tion to normal rats, Int. J. Pharm. 218 (2001) 75–80.

[160] A. Grenha, B. Seijo, C. Remuñán-López, Microencapsulated chitosannanoparticles for lung protein delivery, Eur. J. Pharm. Sci. 25 (2005)427–437.

[161] R.A. DeFronzo, R.M. Bergenstal, W.T. Cefalu, J. Pullman, S. Lerman, B.W.Bode, L.S. Phillips, Efficacy of inhaled insulin in patients with type 2 diabetesnot controlled with diet and exercise: a 12 week, randomized, comparativetrial, Diabetes Care 28 (2005) 1922–1928.

[162] J. Rosenstock, B. Zinman, L.J. Murphy, S.C. Clement, P. Moore, C.K.Bowering, R. Hendler, S.P. Lan, W.T. Cefalu, Inhaled insulin improvesglycemic control when substituted for or added to oral combination therapyin type 2 diabetes: a randomized, controlled trial, Ann. Intern. Med.143 (2005) 549–558.

[163] J.S. Skyler, R.S. Weinstock, P. Raskin, J.F. Yale, E. Barrett, J.E. Gerich,H.C. Gerstein, Use of inhaled insulin in a basal/bolus insulin regimen intype 1 diabetic subjects: a 6-month, randomized, comparative trial,Diabetes Care 28 (2005) 1630–1635.

[164] A.H. Barnett, S.A. Amiel, D. Hopkins, Six month efficacy and 2-yearssafety of inhaled insulin as adjunctive therapy in combination with oralagent, Diabet. Med. 22 (suppl. 2) (2005) P82.

[165] A. Himmelmann, J. Jendle, A. Mellén, A.H. Petersen, U.L. Dahl, P.Wollmer, The impact of smoking on inhaled insulin, Diabetes Care26 (2003) 677–682.

[166] R.R. Henry, S.R.D. Mudaliar, W.C. Howland, N. Chu, D. Kim, B. An, R.R.Reinhardt, Inhaled insulin using the AREx insulin diabetes managementsystem in healthy and asthmatic subjects, Diabetes Care 26 (2003) 764–769.

[167] E.A.M. Gale, Two cheers for inhaled insulin, Lancet 375 (2001) 324–325.[168] J.A. Stoever, J.P. Palmer, Inhaled insulin and insulin antibodies: a mew

twist to an old debate, Diabetes Technol. Ther. 4 (2002) 157–161.[169] R.A. Gerber, J.C. Cappelleri, I.A. Kourides, R.A. Gelfand, Treatment

satisfaction with inhaled insulin in patients with type 1 diabetes, DiabetesCare 24 (2001) 1556–1559.

[170] G.C. Chiou, Systemic delivery of polypeptide drugs through ocular route,J. Ocul. Pharmacol. 10 (1994) 93–99.

[171] Y.C. Lee, P. Simamora, S. Pinsuwan, S.H. Yalkowsky, Review on thesystemic delivery of insulin via the ocular route, Int. J. Pharm. 233 (2002)1–18.

[172] S.B. Koevary, Pharmacokinetics of topical ocular drug delivery: potentialuses for the treatment of diseases of the posterior segment and beyond,Curr. Drug Metab. 4 (2003) 213–222.

[173] J.D. Bartlett, A. Turner-Henson, J.A. Atchison, T.W. Woolley, D.J. Pillion,Insulin administration to the eyes of normoglycemic human volunteers,J. Ocul. Pharmacol. 10 (1994) 683–690.

[174] A. Yamamoto, A.M. Luo, S. Dodda-Kashi, V.H. Lee, The ocular routefor systemic insulin delivery in the albino rabbit, J. Pharmacol. Exp. Ther.249 (1989) 249–255.

[175] G.C. Chiou, B.H. Li, Chronic systemic delivery of insulin through theocular route, J. Ocul. Pharmacol. 9 (1993) 85–90.

[176] D.J. Pillion, J.A. Atchison, R.X. Wang, E. Meezan, Alkylglycosidesenhance systemic absorption of insulin applied topically to the rat eye,J. Pharmacol. Exp. Ther. 271 (1994) 1274–1280.

[177] R.V. Morgan, M.A. Huntzicker, Delivery of systemic regular insulin via theocular route in dogs, J. Ocul. Pharmacol. Ther. 12 (1996) 515–526.

[178] F. Ahsan, J.J. Arnold, E. Meezan, D.J. Pillion, Sucrose cocoate, acomponent of cosmetic preparations, enhances nasal and ocular peptideabsorption, Int. J. Pharm. 251 (2003) 195–203.

[179] V. Soni, R. Singh, R. Srinivasan, S.K. Jain, Pulsatile insulin deliverythrough the ocular route, Drug Deliv. 5 (1998) 47–51.


[180] R. Srinivasan, S.K. Jain, Insulin delivery through the ocular route, DrugDeliv. 5 (1998) 53–57.

[181] Y.C. Lee, S.H. Yalkowsky, Ocular devices for the controlled systemicdelivery of insulin: in vitro and in vivo dissolution, Int. J. Pharm.181 (1999) 71–77.

[182] Y.C. Lee, P. Simamora, S.H. Yalkowsky, Effect of Brij-78 on systemicdelivery of insulin from an ocular device, J. Pharm. Sci. 86 (1997)430–433.

[183] Y.C. Lee, P. Simamora, S.H. Yalkowsky, Systemic delivery of insulin viaan enhancer-free ocular device, J. Pharm. Sci. 86 (1997) 1361–1364.

[184] Y.C. Lee, S.H. Yalkowsky, Effect of formulation on the systemic absorptionof insulin from enhancer-free ocular devices, Int. J. Pharm. 185 (1999)199–204.

[185] Y.C. Lee, S.H. Yalkowsky, Systemic absorption of insulin from a Gelfoam®ocular device, Int. J. Pharm. 190 (1999) 35–40.

[186] E.A. Hosny, Relative hypoglycemia of rectal insulin suppositoriescontaining deoxycholic acid, sodium taurocholate, polycarbophil, andtheir combinations in diabetic rabbits, Drug Dev. Ind. Pharm. 25 (1999)745–752.

[187] E.A. Hosny, H.I. Al-Shora, M.M.A. Elmazar, Effect of different bile saltson the relative hypoglycemia of Witepsol W35 suppositories containinginsulin in diabetic beagle dogs, Drug Dev. Ind. Pharm. 27 (2001) 837–845.

[188] E. Hosny, H.I. Al-Shora, M.MA. Elmazar, Relative hypoglycemic effect ofinsulin suppositories in diabetic beagle dogs: optimization of variousconcentrations of sodium salicylate and polyoxyethylene-9-lauryl ether,Biol. Pharm. Bull. 24 (2001) 1294–1297.

[189] M.O. Yun, H.G. Choi, J.H. Jung, C.K. Kim, Development of a thermo-reversible insulin liquid suppository with bioavailability enhancement, Int.J. Pharm. 189 (1999) 137–145.

[190] M.U. Adikwu, Evaluation of snail mucin motifs as rectal absorptionenhancer for insulin in non-diabetic rat models, Biol. Pharm. Bull.28 (2005) 1801–1804.

[191] M.R. Prausnitz, S. Mitragotri, R. Langer, Current status and future potentialof transdermal drug delivery, Nat. Rev., Drug Discov. 3 (2004) 115–124.

[192] B.J. Thomas, B.C. Finnin, The transdermal revolution, Drug Discov. Today9 (2004) 697–703.

[193] M.R. Prausnitz, Overcoming skin's barrier: the search for effective anduser-friendly drug delivery, Diabetes Technol. Ther. 3 (2001) 233–236.

[194] A.C. Williams, B.W. Barry, Penetration enhancers, Adv. Drug Deliv. Rev.56 (2004) 603–618.

[195] Y.N. Kalia, A. Naik, J. Garrison, R.H. Guy, Iontophoretic drug delivery,Adv. Drug Deliv. Rev. 56 (2004) 619–658.

[196] I. Lavon, J. Kost, Ultrasound and transdermal drug delivery, Drug Discov.Today 9 (2004) 670–676.

[197] M.R. Prausnitz, Microneedles for transdermal drug delivery, Adv. DrugDeliv. Rev. 56 (2004) 581–587.

[198] C.H. Purdon, C.G. Azzi, J. Zhang, E.W. Smith, H.I. Maibach, Penetrationenhancement of transdermal delivery—current permutations and limita-tions, Crit. Rev. Ther. Drug Carr. Syst. 21 (2004) 97–132.

[199] J.D. Bos, M.M. Meinardi, The 500 Dalton rule for the skin penetration ofchemical compounds and drugs, Exp. Dermatol. 9 (2000) 165–169.

[200] P. Karande, A. Jain, S. Mitragotri, Discovery of transdermal penetrationenhancers by high-throughput screening, Nat. Biotechnol. 22 (2004)192–197.

[201] P. Karande, A. Jain, K. Ergun, V. Kispersky, S. Mitragotri, Designprinciples of chemical penetration enhancers for transdermal drug delivery,Proc. Natl. Acad. Sci. 102 (2005) 4688–4693.

[202] Y. Chen, Y. Shen, X. Guo, C. Zhang, W. Yang, M. Ma, S. Liu, M. Zhang,L.P. Wen, Transdermal protein delivery by a coadministered peptideidentified via phage display, Nat. Biotechnol. 24 (2006) 455–460.

[203] J. Guo, Q. Ping, L. Zhang, Transdermal delivery of insulin in mice by usinglecithin vesicles as a carrier, Drug Deliv. 7 (2000) 113–116.

[204] M. Higaki, M. Higaki, M. Kameyama, M. Udagawa, Y. Ueno, Y.Yamaguchi, R. Igarashi, T. Ishihara, Y. Mizushima, Transdermal delivery ofCaCO3-nanoparticles containing insulin, Diabetes Technol. Ther. 8 (2006)369–374.

[205] B. Priya, T. Rashmi, M. Bozena, Transdermal iontophoresis, Expert Opin.Drug Deliv. 3 (2006) 127–138.

[206] V. Nair, O. Pillai, P. Ramarao, R. Panchagnula, Transdermal iontophoresis.Part I: Basic principles and considerations, Methods Find Exp. Clin.Pharmacol. 21 (1999) 139–151.

[207] O. Pillai, V. Nair, P. Ramarao, R. Panchagnula, Transdermal iontophoresis.Part II: Peptide and protein delivery, Methods Find Exp. Clin. Pharmacol.21 (1999) 229–240.

[208] R. Panchagnula, O. Pillai, V.B. Nair, P. Ramarao, Transdermal iontopho-resis revisited, Curr. Opin. Chem. Biol. 4 (2000) 468–473.

[209] X.M. Mao, B.W. Liang, S.Z. Fang, Q. Li, Y.P. Yao, M.W. Zhou, Facilitatedtransdermal delivery of insulin by pulse current iontophoresis, Yao XueXue Bao 30 (1995) 302–306.

[210] C.A. Zakzewski, J. Wasilewski, P. Cawley, W. Ford, Transdermal deliveryof regular insulin to chronic diabetic rats: effect of skin preparation andelectrical enhancement, J. Control. Release 50 (1998) 267–272.

[211] N. Kanikkannan, J. Singh, P. Ramarao, Transdermal iontophoretic deliveryof bovine insulin and monomeric human insulin analogue, J. Control.Release 59 (1999) 99–105.

[212] O. Pillai, S.D. Borkute, N. Sivaprasad, R. Panchagnula, Transdermaliontophoresis of insulin. II. Physicochemical considerations, Int. J. Pharm.254 (2003) 271–280.

[213] O. Pillai, N. Kumar, C.S. Dey, Borkute, N. Sivaprasad, R. Panchagnula,Transdermal iontophoresis of insulin: III. Influence of electronicparameters, Methods Find Exp. Clin. Pharmacol. 26 (2004) 399–408.

[214] O. Pillai, V. Nair, R. Panchagnula, Transdermal iontophoresis of insulin: IV.Influence of chemical enhancers, Int. J. Pharm. 269 (2004) 109–120.

[215] E.H. Choi, S.H. Lee, S.K. Ahn, S.M. Hwang, The pretreatment effectof chemical skin penetration enhancers in transdermal drug deliveryusing iontophoresis, Skin Pharmacol. Appl. Skin Physiol. 12 (1999)326–335.

[216] O. Pillai, R. Panchagnula, Transdermal iontophoresis of insulin V. Effect ofterpenes, J. Control. Release 88 (2003) 287–296.

[217] O. Pillai, R. Panchagnula, Transdermal iontophoresis of insulin. VI.Influence of pretreatment with fatty acids on permeation across rat skin,Skin Pharmacol. Physiol. 17 (2004) 289–297.

[218] S.N. Murthy, Y.L. Zhao, K. Marlan, S.W. Hui, A.L. Kazim, A. Sen, Lipidand electroosmosis enhanced transdermal delivery of insulin by electro-poration, J. Pharm. Sci. 95 (2006) 2041–2050.

[219] O. Pillai, R. Panchagnula, Transdermal delivery of insulin from poloxamergel: ex vivo and in vivo skin permeation studies in rat using iontophoresisand chemical enhancers, J. Control. Release 89 (2003) 127–140.

[220] W.G. Pitt, G.A. Husseini, B.J. Staples, Ultrasonic drug delivery — ageneral review, Expert Opin. Drug Deliv. 1 (2004) 37–56.

[221] S. Mitragotri, J. Kost, Low-frequency sonophoresis. A review, Adv. DrugDeliv. Rev. 56 (2004) 589–601.

[222] B.W. Barry, Novel mechanisms and devices to enable successfultransdermal drug delivery, Eur. J. Pharm. Sci. 14 (2001) 101–114.

[223] M.R. Prausnitz, Reversible skin permeabilization for transdermal deliveryof macromolecules, Crit. Rev. Ther. Drug Carr. Syst. 14 (1997) 455–483.

[224] R.H. Guy, Current status and future prospects of transdermal drug delivery,Pharm. Res. 13 (1996) 1765–1769.

[225] D.G. Kassan, A.M. Lynch, M.J. Stiller, Physical enhancement ofdermatologic drug delivery ― iontophoresis and phonophoresis, J. Am.Acad. Dermatol. 34 (1996) 657–666.

[226] N.N. Byl, The use of ultrasound as an enhancer for transcutaneous drugdelivery: phonophoresis, Phys. Ther. 75 (1995) 539–553.

[227] P. Tyle, P. Agrawala, Drug delivery by phonophoresis, Pharm. Res.6 (1989) 355–361.

[228] D.M. Skauen, G.M. Zentner, Phonophoresis, Int. J. Pharm. 20 (1984)235–245.

[229] D. Bommannan, H. Okuyama, P. Stauffer, R.H. Guy, Sonophoresis. I. Theuse of high-frequency ultrasound to enhance transdermal drug delivery,Pharm. Res. 9 (1992) 559–564.

[230] D. Bommannan, G.K. Menon, H. Okuyama, P.M. Elias, R.H. Guy,Sonophoresis. II. Examination of the mechanism(s) of ultrasound-enhancedtransdermal drug delivery, Pharm. Res. 9 (1992) 1043–1047.

[231] G.K. Menon, D.B. Bommannan, P.M. Elias, High-frequency sonophoresis:permeation pathways and structural basis for enhanced permeability, SkinPharmacol. 7 (1994) 130–139.


[232] S.P. Vyas, R. Singh, R.K. Asati, Liposomally encapsulated diclofenac forsonophoresis induced systemic delivery, J. Microencapsul. 12 (1995)149–154.

[233] S. Mitragotri, Synergistic effect of enhancers for transdermal drug delivery,Pharm. Res. 17 (2000) 1354–1357.

[234] M.E. Johnson, S. Mitragotri, A. Patel, D. Blankschtein, R. Langer,Synergistic effects of chemical enhancers and therapeutic ultrasound ontransdermal drug delivery, J. Pharm. Sci. 85 (1996) 670–677.

[235] A. Tezel, A. Sens, J. Tuchscherer, S. Mitragotri, Synergistic effect of low-frequency ultrasound and surfactants on skin permeability, J. Pharm. Sci.91 (2002) 91–100.

[236] S. Mitragotri, D. Blankschtein, R. Langer, Ultrasound-mediated transder-mal protein delivery, Science 269 (1995) 850–853.

[237] S. Mitragotri, D.A. Edwards, D. Blankschtein, R. Langer, A mechanisticstudy of ultrasonically-enhanced transdermal drug delivery, J. Pharm. Sci.84 (1995) 697–706.

[238] S. Mitragotri, D. Blankschtein, R. Langer, Transdermal drug delivery usinglow-frequency sonophoresis, Pharm. Res. 13 (1996) 411–420.

[239] J. Kost, U. Pliquett, S. Mitragotri, A. Yamamoto, R. Langer, J. Weaver,Synergistic effect of electric field and ultrasound on transdermal transport,Pharm. Res. 13 (1996) 633–638.

[240] S. Mitragotri, D. Blankschtein, R. Langer, An explanation for the variationof the sonophoretic transdermal transport enhancement from drug to drug,J. Pharm. Sci. 86 (1997) 1190–1192.

[241] S. Mitragotri, J. Kost, Low-frequency sonophoresis: a non-invasivemethod of drug delivery and diagnostics, Biotechnol. Prog. 16 (2000)488–492.

[242] A. Tezel, A. Sens, J. Tuchscherer, S. Mitragotri, Frequency dependence ofsonophoresis, Pharm. Res. 18 (2001) 1694–1700.

[243] H. Tang, S. Mitragotri, D. Blankschtein, R. Langer, Theoretical descriptionof transdermal transport of hydrophilic permeants: application to low-frequency Sonophoresis, J. Pharm. Sci. 90 (2001) 545–568.

[244] T. Terahara, S. Mitragotri, J. Kost, R. Langer, Dependence of low-frequency sonophoresis on ultrasound parameters; distance of the horn andintensity, Int. J. Pharm. 235 (2002) 35–42.

[245] A. Tezel, A. Sens, S. Mitragotri, Incorporation of lipophilic pathways intothe porous pathway model for describing skin permeabilization during low-frequency sonophoresis, J. Control. Release 83 (2002) 183–188.

[246] R. Langer, Biomaterials in drug delivery and tissue engineering: onelaboratory's experience, Acc. Chem. Res. 33 (2000) 94–101.

[247] J. Wu, J. Chappelow, J. Yang, L. Weimann, Defects generated in humanstratum corneum specimens by ultrasound, Ultrasound Med. Biol.24 (1998) 705–710.

[248] N. Yamashita, K. Tachibana, K. Ogawa, N. Tsujita, A. Tomita, Scanningelectron microscopic evaluation of the skin surface after ultrasoundexposure, Anat. Rec. 247 (1998) 455–461.

[249] A. Tezel, S. Mitragotri, On the origin of size-dependent tortuosity forpermeation of hydrophilic solutes across the stratum corneum, J. Control.Release 86 (2003) 183–186.

[250] A. Tezel, A. Sens, S. Mitragotri, Description of transdermal transport ofhydrophilic solutes during low-frequency sonophoresis based on amodified porous pathway model, J. Pharm. Sci. 92 (2003) 381–393.

[251] A. Tezel, S. Mitragotri, Interactions of inertial cavitation bubbles withstratum corneum lipid bilayers during low-frequency sonophoresis,Biophys. J. 85 (2003) 3502–3512.

[252] A. Boucaud, M.A. Garrigue, L. Machet, L. Vaillant, F. Patat, Effect ofsonication parameters on transdermal delivery of insulin to hairless rats,J. Control. Release 81 (2002) 113–119.

[253] S. Lee, B. Snyder, R.E. Newnham, N.B. Smith, Non-invasive ultrasonictransdermal insulin delivery in rabbits using the light-weight cymbal array,Diabetes Technol. Ther. 6 (2004) 808–815.

[254] M. Hippius, C. Uhlemann, U. Smolenski, U. Schreiber, S. Reissig, A.Hoffmann, In vitro investigations of drug release and penetration-enhancingeffect of ultrasound on transmembrane transport of flufenamic acid, Int. J.Clin. Pharmacol. Ther. 36 (1998) 107–111.

[255] J. Asano, F. Suisha, M. Takada, N. Kawasaki, S. Miyazaki, Effects ofpulsed output ultrasound on the transdermal absorption of indomethacinfrom an ointment in rats, Biol. Pharm. Bull. 20 (1997) 288–291.

[256] J.Y. Fang, T.L. Hwang, Y.B. Huang, Y.H. Tsai, Transdermal iontophoresisof sodium non-ivamide acetate. V. Combined effect of physicalenhancement methods, Int. J. Pharm. 235 (2002) 95–105.

[257] D. Levy, J. Kost, Y. Meshulam, R. Langer, Effect of ultrasound ontransdermal drug delivery to rats and guinea pigs, J. Clin. Invest. 83 (2002)2074–2078.

[258] S. Miyazaki, H. Mizuoka, Y. Kohata, M. Takada, External control of drugrelease and penetration. VI. Enhancing effect of ultrasound on thetransdermal absorption of indomethacin from an ointment in rats, Chem.Pharm. Bull. 40 (1992) 2826–2830.

[259] L.K. Pershing, S. Bakhtian, C.E. Poncelet, J.L. Corlett, V.P. Shah,Hydrogels with enhanced mass transfer for transdermal drug delivery,J. Pharm. Sci. 85 (2002) 1312–1316.

[260] J. Chen, K.D. Wise, J.F. Hetke, S.C. Bledsoe, A multichannel neural probefor selective chemical delivery at the cellular level, IEEE Trans. Biomed.Eng. 44 (1997) 760–769.

[261] J. Brazzle, I. Papautsky, A.B. Frazier, Micromachined needle arrays fordrug delivery or fluid extraction, IEEE Eng. Med. Biol. Mag. 18 (1999)53–58.

[262] L. Lin, A.P. Pisano, Silicon-processed microneedles, J. Microelectromech.Syst. 8 (1999) 78–84.

[263] P. Griss, P. Enoksson, H.K. Tolvanen-Laakso, P. Merilainen, S. Ollmar, G.Stemme, Micromachined electrodes for biopotential measurements,J. Microelectromech. Syst. 10 (2001) 10–16.

[264] J.A. Matriano, M. Cormier, J. Johnson, W.A. Young, M. Buttery, K. Nyam,P.E. Daddona, Macroflux microprojection array patch technology: a newand efficient approach for intracutaneous immunization, Pharm. Res.19 (2002) 63–70.

[265] J.A. Mikszta, J.B. Alarcon, J.M. Brittingham, D.E. Sutter, R.J. Pettis, N.G.Harvey, Improved genetic immunization viamicromechanical disruption ofskin-barrier function and targeted epidermal delivery, Nat. Med. 8 (2002)415–419.

[266] D.V. McAllister, P.M. Wang, S.P. Davis, J.H. Park, P.J. Canatella, M.G.Allen, M.R. Prausnitz, Microfabricated needles for transdermal delivery ofmacromolecules and nanoparticles: fabrication methods and transportstudies, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 13755–13760.

[267] W. Martanto, S.P. Davis, N.R. Holiday, J. Wang, H.S. Gill, M.R. Prausnitz,Transdermal delivery of insulin using microneedles in vivo, Pharm. Res.21 (2004) 947–952.

[268] http://www.emisphere.com.[269] http://www.phx.corporate-ir.net/phoenix.zhtml.[270] A.R. Sircar, Alternate routes of insulin delivery, Int. J. Diabet. Dev. Ctries.

22 (2002) 119–121.[271] http://www.highbeam.com.[272] http://www.autoimmuneinc.com.[273] The Diabetes Prevention Trial-Type 1 Study Group, Effect of oral insulin in

relatives of patients with type 1 diabetes, Diabetes Care 28 (2005)1068–1076.

[274] http://www.generex.com.[275] http://www.in-pharmatechnologist.com.[276] http://www.bentleypharm.com.[277] http://www.vectura.com.[278] http://sec.edgar-online.com.[279] http://pfizer.com.[280] http://nektar.com.[281] http://exubra.com.[282] T. Quattrin, A. Bélanger, N.J.V. Bohannon, S.L. Schwartz, Efficacy and

safety of inhaled insulin (Exubera) compared with subcutaneous insulintherapy in patients with type 1 diabetes: results of a 6-month, randomized,comparative trial, Diabetes Care 27 (2004) 2622–2627.

[283] P.A. Hollander, L. Blonde, R. Rowe, A.E. Mehta, J.L. Milburn, K.S.Hershon, J.L. Chiasson, S.R. Levin, Efficacy and safety of inhaled insulin(Exubera) compared with subcutaneous insulin therapy in patients withtype 2 diabetes: results of a 6-month, randomized, comparative trial,Diabetes Care 27 (2004) 2356–2362.

[284] S. White, D.B. Bennett, S. Cheu, P.W. Conley, D.B. Guzek, S. Gray, J.Howard, R. Malcolmson, J.M. Parker, P. Roberts, N. Sadrzadeh, J.D.Schumacher, S. Seshadri, G.W. Sluggett, C.L. Stevenson, N.J. Harper,


Exubera: pharmaceutical development of a novel product for pulmonarydelivery of insulin, Diabetes Technol. Ther. 7 (2005) 896–906.

[285] S. Bindra, W.T. Cefalu, Exubera. Inhaled therapeutic systems, Curr. Opin.Investig. Drugs 3 (2002) 758–762.

[286] R.A. Defronzo, for the Exubera® phase III study group, Efficacy andsafety of inhaled insulin (Exubera®) compared with rosiglitazone in type2 diabetes patients not optimally controlled on diet and exercise: results ofa 3-month, randomized, comparative trial, Diabetes 52 (suppl. 1) (2003)A38.

[287] http://aradigm.com.[288] K. Hermansen, T. Rönnemaa, A.H. Petersen, S. Bellaire, U. Adamson,

Intensive therapy with inhaled insulin via the AREx insulin diabetesmanagement system: a 12-week proof-of-concept trial in patients with type2 diabetes, Diabetes Care 27 (2004) 162–167.

[289] A. McElduff, L.E. Mather, P.C. Kam, P. Clauson, Influence of acute upperrespiratory tract infection on the absorption of inhaled insulin using theAERx® insulin diabetes management system, Br. J. Clin. Pharmacol.59 (2005) 546–551.

[290] J. Cramer, J. Okikawa, P. Clauson, Compliance with inhaled insulintreatment using AERx® iDMS insulin diabetes management system,Diabetes 52 (suppl. 1) (2003) A103.

[291] G.A. Brunner, B. Balent, M. Ellmerer, L. Schaupp, A. Siebenhofer, J.H.Jendle, J. Okikawa, T.R. Pieber, Dose–response relation of liquid aerosolinhaled insulin in type 1 diabetic patents, Diabetologia 44 (2001)305–308.

[292] R.R. Henry, S.Mudaliar, N. Chu, D. Kim, D. Armstrong, T.T. Davis, B. An,R.R. Reinhardt, Young and elderly type 2 diabetic patients inhaling insulinwith the AERx® insulin diabetes management system: a pharmacokineticand pharmacodynamic comparison, J. Clin. Pharmacol. 43 (2003)1228–1234.

[293] C. Kapitza, M. Hompesch, L.K. Thomsen, T. Heise, Intra-subjectvariability of pulmonary insulin via AERx® insulin diabetes managementsystem versus subcutaneous insulin, Diabetes 51 (suppl. 2) (2002) A103.

[294] C. Kapitza, M. Hompesch, B. Scharling, T. Heise, Intra-subject variabilityof inhaled insulin in type 1 diabetes: a comparison with subcutaneousinsulin, Diabetes Technol. Ther. 6 (2004) 466–472.

[295] K. Hermansen, T. Rönnemaa, A.H. Petersen, U. Adamson, Intensivetreatment with pulmonary insulin using the AREx® insulin diabetesmanagement system: a proof of concept trial in patients with type 2 diabeticpatients, Diabetes 51 (suppl. 2) (2002) A48.

[296] M. Kipnes, B. Otulana, J. Okikawa, S. Farr, J. Jendle, J. Thipphawong, S.Schwartz, Pharmacokinetics and pharmacodynamics of pulmonary insulindelivered via AERx® diabetes management system in type 1 diabetics,Diabetologia 43 (suppl. 1) (2000) A201.

[297] M. Hompesch, C. Kapitza, B. Scharling, L.K. Thomsen, T. Heise, Intra-subject variability of pulmonary insulin via AERx® insulin diabetesmanagement systemversus subcutaneous insulin,Diabetologia 44 (suppl. 1)(2001) A212.

[298] http://alkermes.com.[299] http://lilly.com.[300] K.M. Rave, L. Nosek, A. de la Peña, M. Seger, C.S. Ernest, L. Heinemann,

R.P. Batycky, D.B. Muchmore, Dose response of inhaled dry powderinsulin and dose equivalence to subcutaneous insulin lispro, Diabetes Care28 (2005) 2400–2405.

[301] http://www.mannkindcorp.com.[302] http://www.aerogen.com.[303] A.D. Perera, C. Kapitza, L. Nosek, R.S. Fishman, D.A. Shapiro, T. Heise,

L. Heinemann, Absorption and metabolic effect of inhaled insulin: intra-patient variability after inhalation via the Aerodose insulin inhaler inpatients with type 2 diabetes, Diabetes Care 25 (2002) 2276–2281.

[304] http://www.prnewswire.com.[305] http://www.abbott.com.[306] http://www.microdose-tech.com.[307] http://www.baxterbiopharmasolutions.com.[308] http://www.alteatherapeutics.com.[309] http://www.openpr.com.[310] G. Cevc, Transferosomes, liposomes and other liquid suspension on the

skin: permeation enhancement vesicle penetration and transdermal drugdelivery, Crit. Rev. Ther. Drug Carr. Syst. 13 (1996) 257–388.

[311] http://www.idea-ag.de.[312] http://www.encsys.com.[313] http://www.alza.com.

Documents

Current Challenges in Non-Invasive Insulin Delivery Systems a Comparative Review