challenges in delivery of therapeutic genomics and proteomics || other routes of protein and peptide...

Challenges in Delivery of Therapeutic Genomics and Proteomics. DOI:© 2010 Elsevier Inc. All rights reserved.

10.1016/B978-0-12-384964-9.00012-82011

Other Routes of Protein and Peptide Delivery: Transdermal, Topical, Uterine, and RectalBhavik Shah1, Naazneen Surti2, Ambikanandan Misra1

1Pharmacy Department, TIFAC – Centre of Relevance and Excellence in New Drug Delivery Systems, The Maharaja Sayajirao University of Baroda, PO Box 51, Kalabhavan, Vadodara 390 001, Gujarat, India2Baroda College of Pharmacy, Parul Arogya Seva Mandal, Limda, Waghodia 391 760, Gujarat, India

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12.1 Transdermal Delivery of Proteins and Peptides

12.1.1 Introduction

The delivery of peptides or proteins is extremely difficult. When administered orally in the form of solution, peptides and proteins are degraded in the gastrointestinal (GI) tract. When consumed in a formulation that prevents their degradation, their uptake in the gut remains more difficult and highly inefficient; even with the best currently avail-able formulations, the total uptake efficiency does not exceed a few fractions of the administered dose. In addition to this, hepatic first-pass metabolism always has a con-siderable effect. These are the reasons why nearly all therapeutic peptides still have to be introduced into the body through an injection needle, in spite of the inconvenience of this method. Numerous attempts have therefore been made to find acceptable alter-natives. In particular, the delivery of drugs across the skin or mucosa has attracted a great deal of attention in recent years [1,2]. But due to the skin permeability barrier, normally only a fraction of the applied drug crosses the intact mammalian skin, except for selected low-molecular-weight molecules. The use of supporting methods such as iontophoresis, ultrasound, or electroporation improves the situation to some extent.

12.1.2 Structure of the Skin

Skin is our outermost protective shield against detrimental environmental agents. Consequently, it is a good permeability barrier. Human skin is about 0.5 mm thick (ranging from 0.05 to 2 mm in different parts of body) and is composed of four main layers: the (1) stratum corneum (SC), (2) viable epidermis, (3) dermis, and (4) sub-cutaneous tissue. A schematic cross-section of the skin is presented in Fig. 12.1, with

Challenges in Delivery of Therapeutic Genomics and Proteomics624

different layers identified according to their role in influencing skin permeability through various pathways.

12.1.2.1 Stratum Corneum

The thick (approximately10–20 m) surface layer, the SC, is highly hydrophobic and contains nearby 10–15 layers of dead or dying keratinized cells, corneocytes, which are constantly shredded and renewed. Its organization can be described by the “brick-and-mortar” mode, in which extracellular lipid accounts for approximately 10% of the dry weight of this layer, and 90% is intracellular protein (mainly keratin). Such “brick-and-mortar” architecture of the outer skin layers effectively prevents transder-mal drug permeation. The SC lacks phospholipids but is enriched in ceramides and neutral lipids (cholesterol, fatty acids, cholesteryl esters) that are arranged in a bilayer format called lipid channels. Interdigitated long-chain -hydroxyceramides provide cohesion between corneocytes by forming tight lipid envelopes around the corneo-cyte protein component. The barrier function of the skin is created by lamellar gran-ules, which are synthesized in the granular layer and later become organized into the intercellular lipid bilayer domain of the SC. Barrier lipids are tightly packed, and any impairment to the skin results in active synthetic processes to restore them. The skin’s barrier function appears to depend on the specific ratio of various lipids; among polar

Pore

Dermal papillae

Free nerveending

Sebaceousgland

Arrectorpili muscle

Sensorynerve fiber

Hair follicle

Hair root

VeinArtery

Root hair plexus

Pacinian corpuscle

Adipose tissue

Hypodermis(superficial fascia)

DermisPapillary layer

Stratumbasale

Stratumspinosum

Stratumgranulosum

Epidermis

Stratumlucidum

Stratumcorneum

Hair

Meissner’scorpuscle

Reticular layer

Eccrine sweat gland

Figure 12.1 Schematic cross-section of the skin.

Other Routes of Protein and Peptide Delivery: Transdermal, Topical, Uterine, and Rectal 625

and nonpolar lipids, polar lipids are more crucial to skin barrier integrity. Because of its highly organized structure, the SC is the major permeability barrier to any external material and is regarded as the rate-limiting factor in penetration of therapeutic agents utilizing skin as a route. The ability of various agents to interact with the intercellular lipid therefore dictates the degree to which absorption is enhanced.

12.1.2.2 Viable Epidermis

The viable epidermis contains multiple layers of keratinocytes at various stages of differentiation. The basal layer contains actively dividing cells, which travel upward successively to form the spinous, granular, and clear layers. As part of this process, the cells steadily lose their nuclei and undertake changes in composition. The role of the viable epidermis in skin barrier function is mainly related to the intercellular lipid channels and continuous partitioning phenomena. Normally, when applied, drugs can partition from layer to layer, depending on their solubility after the drug starts to dif-fuse through the SC. Several other cells (e.g., melanocytes, Langerhans cells, dendritic T cells, epidermotropic lymphocytes, and merkel cells) are available throughout the viable epidermis, which also contains a variety of active catabolic enzymes, for exam-ple, esterases, phosphatases, proteases, nucleotidases, and lipases. Lipid catabolic enzymes like acid lipase, phospholipase, sphingomyelinase, and steroid sulfatase, although mainly concentrated in the SC and granulosum, have been demonstrated throughout the epidermal layers. Lipase and sphingomyelinase were localized primar-ily to intercellular domains in the SC [3]. Although the basal and spinous layers are rich in phospholipids, as the cells differentiate during their journey to the surface, the phospholipid content decreases and the sphingolipid (glucosylceramide and ceramide) and cholesterol content simultaneously increases.

12.1.2.3 Dermis and Hypodermis

The dermis is largely acellular and is rich in blood vessels, lymphatic vessels, and nerve endings. A widespread network of dermal capillaries connects to the systemic circulation, with considerable horizontal branching from the arterioles and venules in the papillary dermis, to form plexuses and to supply capillaries to hair follicles and glands. Dermal lymphatic vessels help to drain surplus extracellular fluid and clear antigenic materials. The elasticity of the dermis is attributed to a network of protein fibers, including collagen (type I and III) and elastin, which are surrounded in an amorphous glycosaminoglycan ground substance. The dermis also contains scattered fibroblasts, macrophages, mast cells, and leukocytes. Hair follicles, sebaceous glands, and sweat glands found in the dermis and subcutis might serve as additional, albeit fairly limited, pathways for drug absorption.

In some cases, for example, hair follicles might act as target sites for drug delivery.

12.1.3 Penetration Pathways Through the Skin

A compound may use two different ways to penetrate normal intact human skin: the transappendageal pathway and the transepidermal pathway. The transappendageal


pathway involves transport via the sweat glands or the pilosebaceous units (hair fol-licles with their associated sebaceous glands). This route avoids penetration through the SC and is therefore known as the shunt route. The transappendageal route is considered to be of less significance than the transepidermal route for the reason of its relatively very small area, approximately 0.1% of the total skin area. The rate of success for transfer largely depends on the lipophilicity and the composition of the entrant. Figure 12.2 shows pathways through the SC. The transcellular (intracellular) route is important during electrically enhanced transport methods such as iontophore-sis. Compounds that penetrate the SC via the transepidermal route may follow a trans-cellular (intracellular) or intercellular pathway. Because of the highly impermeable character of the cornified cells, the intercellular pathway is suggested as the route of preference for most drug molecules [4]. Moreover, it was demonstrated that drug per-meation across the SC increases many folds once the lipids are extracted [5]. Hence, knowledge of the structure and physical properties of the intercellular lipids is vital to broaden our insight into the skin’s barrier role in permeation of therapeutics.

12.1.4 Approaches to Enhance Transdermal Peptide Delivery

12.1.4.1 Physical Approaches

12.1.4.1.1 Electrical Methods: Electroporation and IontophoresisAs already discussed, proteins and peptides are poorly bioavailable when deliv-ered orally and therefore are normally administered invasively by intravenous or

Paracellular route Transcellular route

Mucosal side

Serosal side

Passive diffusion Carrier-mediated transport

Epithelial cell

Blood capillary

Cell junction

Lymph capillary

Endocytosis

Figure 12.2 Pathways through SC.


subcutaneous injections. The requirement for higher frequency of administration due to short plasma half-life and poor patient compliance stress the need for an alter-native drug-delivery mode. Transdermal protein and peptide delivery is a unique technique because of its ease of administration, noninvasiveness, better patient com-pliance, and avoidance of GI degradation and the first-pass effect of active molecules in the liver. But the highly lipophilic nature of the SC impedes the passive trans-port of charged macromolecules across the skin into systemic circulation [6]. Several investigations explored this aspect, studying different methods, including chemical and physical methods, to overcome this most important barrier in transdermal protein and peptide delivery [7]. Electrical methods like iontophoresis and electroporation have been studied extensively to enhance the transport of macromolecules across the skin by overcoming the barrier of the SC.

Electroporation As the human skin is the largest single organ of the body, it may at first sight be attractive to formulators as an accessible means of drug input. Electroporation holds many advantages that include avoidance of GI and liver first-pass effects, controlled and continuous drug delivery, easy removal of the dosage form in case of an accidental release, and good patient compliance. However, the skin’s func-tion as a barrier to macromolecules ensures a difficult passage for most drugs both into and through the skin. The main reasons for the good barrier properties of the organ skin lie within the highly organized lipid matrix within the SC, the outermost layer of the epidermis. It is therefore desirable to devise strategies both to enhance the penetration of molecules, which can break the skin barricade passively and reversibly, and also to widen the spectrum of drug molecules that can penetrate the skin at therapeutically ben-eficial doses. Many strategies have been utilized to help conquer this barrier function.

Electroporation of the cell membrane has been studied extensively and used since the 1970s for deoxyribonucleic acid transfection of the cells by reversibly permeabi-lizing the cell membranes with the application of brief electric pulses. Electroporation is an electrical technique that involves the application of high-voltage electric pulses for very short duration (microsecond or millisecond) to enhance the skin permeability reversibly, for macromolecules. Unlike iontophoresis, which employs small currents (0.5 mA/cm2) for relatively long periods of time (many minutes to hours), electro-poration involves exposure of the skin to relatively high voltages (on the order of 30–100 V imposed across the skin) for rather short times, typically one to several hun-dred milliseconds. The voltage-induced permeability change is consistent with the formation of pores in the membrane. Electroporation involves the creation of new, low- resistance pathways through the SC. Depending on the applied electrical field, the electrical force produces partial rupture of cell membrane [8,9]. Several publications of the application of electroporation to increase transdermal delivery have been pub-lished within the last few years [10–14]. The use of electroporation has been shown recently to reversibly permeabilize skin for enhancing the transdermal delivery of several molecules such as calcein [10], oligonucleotides [11], and calcium-regulating hormones [12]. Skin electroporation is believed to produce new transport pathways, in addition to expanding existing pathways, although these new pathways shows clearly persistent structural changes [13].


Electroporation is still in the experimental stage, and many researchers have employed model nontherapeutic molecules to demonstrate the feasibility of its use. More biotechnologic and therapeutically derived molecules have now been investigated for transdermal delivery, including heparin [14], oligonucleotides [15], and genes [16]. The main hurdle to electroporation is its further development for practical applications.

Mechanism of Electrical Methods Electroporation involves the application of high-voltage pulses for a very short duration. The pulses are thought to produce transient aqueous pores in the lipid bilayers of the SC, which may be seen as a localized area capable of drug transport called the localized transport region (LTR). These openings provide momentary pathways for drug diffusion all the way through the horny layer [17–19]. The technique of electroporation is more typically applied to the unilamellar phospholipid bilayers of cell membranes. The feasibility of electorporation for trans-dermal drug delivery was first demonstrated by Prausnitz et al. [14,18]. The electri-cal behavior of the human epidermal membrane (HEM) as a function of the scale and period of the applied voltage mimics closely the breakdown and recovery of bilayer membranes seen during electroporation. In contrast to iontophoresis, electroporation acts mainly on the skin, with less effect by electromigration due to the short pulse “on” time. The SC requires approximately 1 V pulse per bilayer, and 100 multilamel-lar bilayers require 100 V pulses for electroporation [18,19]. As with iontophoretic and electroporative transport, some studies have indicated that high-voltage pulse-induced transdermal delivery of charged or even neutral drugs could be controlled by the proper use of electric considerations, that is, pulse voltage, width, and number [20–22]. Although it is generally believed that electroporation involves the creation of aqueous pathways (pores) in the SC, to some extent this theory is controversial [23]. These proposed channels have not yet been identified in any microscopic study, which may be due to their small size (about 10 nm), sparse distribution (0.1% of the total skin area), and ephemeral nature (millisecond to second). Electroporation results in the breakdown of lipid bilayers, hydration of skin, and decreased resis-tance of skin. Existing successful in vitro studies should be supplemented with in vivo studies for a step toward the development of the electroporation system [24,25]. Notably, during electroporation highly localized pockets of molecular transport are observed, revealed by real-time video imaging for fluorescent molecules called as LTR [17]. The mechanism that explains the perturbation of the lipid barrier of the horny layer during electroporation is a heat dissipation phenomenon. Considering three compartment models in skin, it was observed that localized areas with large micropore is generated [26]. Although it was stated to be a nonthermal process, the temperature rose after electric pulses of 100 V (transmembrane voltage) applied for 1 ms. A “pore” is supposed to form very rapidly, preceding any potential tempera-ture rise. However, localized heating may also occur at sites of large current den-sity, especially with extended pulses. Even though convection spreads the heat front across the skin, the Joule heating could be sufficient for the melting of skin lipids with phase transitions around 70°C, which suggests that the temperature augmenta-tion plays an important role. The transport of water-soluble molecules was observed to be facilitated by the electric field due to the electrophoretic impetus in conjunction


with high permeability due to the breakdown of the multilamellar system of the SC lipids and the formation of LTRs [27,28]. The area involved in drug transport during electroporation is subdivided into two distinct domains: a number of LTRs with radii ranging from 10 to 100 m depending on pulse voltage, and surrounding local dis-sipation regions (LDRs), larger than LTRs with their size depending on the length of the applied pulse [28]. Experimental and theoretical investigations of localized heat-ing by skin electroporation predict that LTRs formation can be partly explained by Joule heating during pulsing and that temperature rise is relatively small in the LDRs that surround the LTRs. This is further supported by field-driven transport, recov-ery of the skin barrier function, and the creation of aqueous pores [23,28,29]. The mechanisms of molecular transport during electroporation likely involve passive dif-fusion and electrically driven transport during the brief pulsing time, but unlike ion-tophoresis, electroosmosis (EO) is thought to be unimportant and safe. The safety of applying high voltage was assessed in vivo [21,22,30]. The augmentation in the horny layer water content, and consequent disorganization of the SC, is also believed to occur but seemed to be less important than during iontophoresis [24]. The bar-rier effects of the horny layer are also reduced for an additional period after pulsing, which further enhances molecular penetration.

A major safety concern is associated with the use of electrical methods like elec-troporation, even though several reports indicated the injury to the skin to be gen-tle, reversible, and safe [22,24,30]. Electrochemotherapy has been used effectively in preclinical and clinical studies. The only skin alteration seen with electropora-tion was slight erythema that diminishes within a few hours. [31]. Patients subjected to electrochemotherapy seemed to tolerate well the application of 10,000 V/cm for 100 ms2/wave pulses. It is also observed that 10 pulses of 400 V–10 ms were more efficient than 10 low voltage, long duration pulses [31,32]. However during electro-poration, milder conditions achieved by shortening the pulses, lowering voltage, or improving the electrode design could be used [32] that may give less pain.

Iontophoresis Iontophoretic delivery is a system that has been successfully engaged to introduce proteins and peptide drugs into the systemic circulation [33–37]. Iontophoresis uses a mild electric current that facilitates the transdermal delivery of a variety of agents. The current progress in technological advancement is due to huge patient acceptance, and the commercial success of passive transdermal patches such as fentanyl, nicotine, estradiol, and nitroglycerin has piqued curiosity within the industry for additional transdermal agents. Technological breakthroughs in the micro-electronics industry have enabled minimization of the size of electronic components that can be programmed at lower cost. Finally, rational drug design and advancement in recombinant DNA technology yields a growing number of therapeutically impor-tant peptides and proteins. Peptides typically contain acidic and basic functional groups and are usually charged at physiological conditions. Consequently, passive transdermal diffusion of these ionic large-molecular-weight agents across the hydro-phobic outermost layers of the skin is difficult. In recent years, a large number of published articles, issued patents, and patent applications demonstrate the potential of this technology for peptide and protein delivery.


The movement of charges due to electromigration is illustrated in Fig. 12.3. In this diagram, the positively charged drug (D) and its counter ion (B–) are formu-lated from the anodic donor reservoir for drug delivery. The cathodic counter reser-voir contains biologically acceptable cations (C) and anions (B–). When an electric field is applied, drug ions transfer into the skin, and endogenous anions, mostly chlo-ride, transfer from the body into the donor reservoir. Simultaneously, at the cathodic counter electrode, anions transfer from the counter reservoir into the skin, whereas endogenous cations, mostly potassium and sodium, migrate from the body into the counter reservoir. The movement of ions maintains a neutral environment for the skin throughout the process [34,35].

The increased flux during iontophoresis includes flux due to the electrochemical potential gradient across the skin and change in the skin permeability due to the elec-tric field applied by electroosmotic water flow and the resultant solvent drag. Hence, Eq. (12.1):

J J J Jionto electric passive convective (12.1)

where:

Jelectric flux due to electric current applicationJpassive flux due to passive delivery through the skin

Jconvective flux due to convective transport due to electroosmosis

Iontophoretic delivery is advantageous in many ways. It considerably reduces the inter- and intraindividual variability because in iontophoretic delivery the rate of drug delivery is more dependent on applied current than on characteristics of the SC. It can deliver drugs both in ionized and unionized form. It enables continuous or pulsatile delivery of the drug, depending on the applied current. Iontophoresis offers enhanced control on the amount of drug delivered, as this depends on the area

Source of current

Donorelectrode

D+Drug repulsion

C+ B–

Blood vessel

Blood vessel

B–

Anode CathodeReceptorelectrode

Skin

Movement tocathod via anode

+ –

Figure 12.3 Schematic diagram of an iontophoresis system showing flow of ions when voltage is applied.


of skin exposed to the current, the duration of applied current, and the module of applied current. It permits fast and easy cessation of drug delivery in case of emer-gency. Iontophoresis improves the delivery of high-molecular-weight compounds as well as polar molecules. Iontophoresis is safe as it allows restoration of the skin bar-rier function without producing severe skin irritation and has the ability to be used for systemic delivery as well as local (topical) delivery of drugs.

Factors Affecting Iontophoretic DeliveryMolecular Weight and Size Molecular weight and size is the most significant factor affecting iontophoretic drug delivery.

Figure 12.4 shows the effect of molecular size on the relative importance of elec-trorepulsion (ER) and EO to the overall iontophoretic transport of ions.

Small, highly mobile cations are principally moved across the skin by ER. But with an increase in molecular size, the fraction of charge carried by a cationic drug decreases, and the principal mechanism of transport becomes EO. As molecular size increases for cations, there will therefore be a transition in the dominant mechanism from ER to EO [35]. For anions, EO is a negative contribution to the total flux, and once the molecular size reaches a critical value, it completely cancels out the ER con-tribution to electrotransport (resulting in no net flux). Highly mobile ions (e.g., Na, Cl–, and small charged amino acids) are principally moved across the skin by ER, whereas large, bulky species carrying only a part of the charge passing across the skin can only be transported by EO. As molecular size increases for cations, there will be a transition in the dominant mechanism from ER to EO. For anions, on the other

Anionic drug

ER EO Total

Molecular size mc

0

Flu

x Cationic drug

Molecular size

0

Flu

x

Figure 12.4 Effect of molecular size on the relative importance of ER and EO to the overall iontophoretic transport of ions.


hand, as molecular size increases, it is clear that, at the transition, the electrorepulsive contribution will be cancelled by electroosmotic convective flow going in the opposite direction. The movement of an ion across skin under the influence of an electric field increases as molecular size decreases (in normal conditions it is inversely proportional to size/molecular weight). This has been confirmed for a sequence of amino acids, acetyl amino acids, and tripeptides [36], and for alkanoic acids for a homologous series of oligonucleotides [37]. Because the mobility of ions is related to the molecu-lar weight, or probably more importantly molecular volume and shape, it is likely that secondary, tertiary, and quaternary structures of the peptide are important in deter-mining the degree of iontophoretic delivery. Because a pore pathway is most likely involved, the flux is size reliant with a cut-off at some stage. Although an upper size limit is not known, the largest polypeptide with respect to molecular weight investi-gated extensively is insulin, although delivery is expected for a molecular weight of up to about 10 KDa, provided the polypeptide has other desirable attributes such as a high isoelectric point (pI) value.

Drug Charge and pH The pH of the buffer used, which is related to the isoelectric point of the peptide, controls the charge on the peptide. The peptide will be posi-tively charged at a pH below its isoelectric point and should be delivered under anode, whereas the peptide will have a negative charge if the pH is above the isoelectric point, in which case it should be delivered under cathode. The techniques of isoelectric focusing and capillary zone electrophoresis have also been used as tools to predict the ability of a peptide to be iontophoresed. Based on such studies, native luteinizing hor-mone–releasing hormone (LHRH) was predicted to be better suited for iontophoretic delivery than its free-acid analogue [38]. To stay charged in the skin environment, the isoelectric point of the protein should be away from the isoelectric point of the skin, that is, 3–4. Typically, polypeptides with high isoelectric point values, such as vaso-pressin or calcitonin, are good candidates for transdermal iontophoretic delivery from a delivery efficiency point of view. Another phenomenon that needs to be considered is electroosmotic flow.

It is suggested that the skin is a permselective membrane and exists with an “appar-ent” net negative charge at the free solution pH of 7.4 due to its isoelectric point. This makes the skin selectively permeable to cations (positively charged ions) and selec-tively restricts the entry of anions (negatively charged ions) [34]. As cations move into the skin, a solvent molecules move along with it, resulting in a mass flow of water or other solvent. This phenomenon, called EO, enhances the transport of neutral species across the skin. Also, the anodal (anode-to-cathode direction) flux is typically higher because it is aided by EO. At extremely low pH, the skin will be below its isoelectric point and will attain a positive charge, causing a change in the direction of electroos-motic flow to support cathodic flux. Also, adsorption of a positively charged drug on the negatively charged skin may lead to a change in the net charge of the skin, which may affect electroosmotic flow observed for polypeptide [39], leuprolide [40], and nafarelin [41]. At the pH used, these cationic peptides strongly associate with the skin and neutralize its charge, which in turn leads to a gradual reduction and eventually a reversal of the convective (electroosmotic) flow [42,43].


Current Density Normally, an increase in current will increase drug delivery, but once a certain transport number is achieved, further increase in current does not increase delivery flux. Actually, the plateau may be achieved at a certain current density, depending on the formulation used and the physicochemical properties of the drug molecule. Once the limiting transport number is achieved, further increase in current does not increase delivery flux. It was also shown that due to electroosmotic flow, transdermal delivery of a large anion (or negatively charged protein) from the anode compartment is more effective than delivery from the cathode compartment [43,44].

Electrode Material Proper selection of electrodes is an aspect that is vital to success-ful iontophoretic delivery of a drug. The electrode material in an iontophoretic device is very important as it decides the type of electrochemical reaction taking place at the electrodes. The possibility of introducing metallic ions into the skin must be care-fully considered. In the past, stainless steel, nickel, or other iron alloys have been used, but these are known to produce allergic reaction in the skin and are not being used nowadays. Electrodes silver–silver chloride are the most preferred as they resist the changes in pH which are generally seen during the use of platinum or zinc–zinc chloride electrode. Platinum offers another inert material for electrode construction. However, platinum causes electrolysis of water, resulting in pH drift. The oxidation reaction at the anode and reduction at the cathode may be described as follows:

H O H on the surface of the anode)2 22 2→ ∫ Ο e (

(12.2)

2 2 22 2H O H OH on the surface of the cathode) e → ( (12.3)

The electron is released in the circuit, and insoluble AgCl precipitates at the anode surface. In the case of other metals like platinum, the chloride ion at the anode will be converted to Cl2, which will in turn react with water to generate hydronium ions. As a function of iontophoresis time, the solution under the cathode becomes progres-sively more basic, and the solution under the anode becomes gradually more acidic. Because the type and magnitude of charge on peptides and proteins is directly depen-dent on the background pH or solution, such pH shifts must be prevented. The small mobile hydrogen and hydroxyl ions produced will carry a significant fraction of the current. They will thus compete with the peptide for electric current, thereby reducing the efficiency of iontophoretic transport. These pH changes may be avoided by using reversible electrodes such as silver–silver chloride electrodes. Reversible electrodes are consumed by the electrochemistry, and thus they do not force electrolysis of water to be a fuel for the electrochemistry. The use of a silver wire for the anode and chlori-dized silver for the cathode in a buffer containing chloride ion provides an ideal elec-trochemical system [45]. At the anode, the silver will react with chloride ions to form insoluble silver chloride. Simultaneously, the silver chloride cathode is reduced to sil-ver metal, and these reactions prevent the electrolysis of water:

Ag Cl AgCl at anode → e ( ) (12.4)


AgCl Ag Cl at cathode e → + ( ) (12.5)

Therefore, the use of silver wire as an anode and silver–silver chloride wire as a cathode is not accompanied by pH drifts. However, the system must provide some chloride ions to drive the electrochemistry, as is evident from the above equations. There could be situations when silver–silver chloride electrodes may be unsuitable as they may react with some protein drugs or with hydrogel. In these conditions, the use of platinum electrodes may be suggested, although other methods that control pH can be chosen. Using mannitol as an appropriate marker molecule, electroosmotic flow during reverse iontophoresis has been examined as a function of the pH and ionic strength of the electrolyte solutions located on the skin surface, which were contained within the electrode compartments [45].

Formulation The basic principles that apply to iontophoresis are relevant to peptides and proteins due to the charge. If the drug carries a measurable fraction of the charge being passed, then it is important to minimize the presence of competing ions in the formulation. Generally, lower electrolyte levels also mean that EO is slightly higher [45–47]. Furthermore, due to stability, it may demand at least some level of back-ground electrolyte and/or buffer; thus, polymeric buffers, for example, which are not necessarily competitive for charge carrying, have been used to improve drug delivery [46,48]. Because iontophoretic formulations employ aqueous-based gels, there are, in particular, potential problems of hydrolysis that are tackled by making a dry res-ervoir disk for iontophoresis (via compression of a mixture of freeze-dried peptide and gelatin), which is hydrated during use [49]. Proof of concept of the idea has been achieved via the observation of a hypocalcemic response in animal [49]. The formu-lation ingredients in the drug and counter reservoirs typically consist of a solvent, a drug salt or a biocompatible salt, and a matrix-forming material. A formulation may also include additives such as buffers, antimicrobial agents, antioxidants, and addi-tional electrolyte salts or permeation enhancers. All of these interact in a complex fashion to affect rate of delivery, biocompatibility, and product shelf life.

Examples of Iontophoretic DeliveryAmino Acids and Small Peptides Peptide delivery seems to be one of the most promising applications of iontophoretic transdermal delivery. Iontophoresis of amino acids, which are building blocks of peptides, may give some information useful for predicting the delivery of small peptides. Amino acids may also have a direct benefit of moisturizing the skin [49,50]. It has been showed that the binding of a series of amino acids in the excised abdominal skin of hairless rat decreased with an increase in the alkyl side chain [50]. This suggests that binding is likely to be polar or electrostatic in nature. A series of amino acids across excised hairless mouse skin was delivered to investigate the effects of permeant charge (neutral, 1, or –1), lipophilicity, and vehicle pH [50]. As usual, the positively charged amino acids (i.e., positively charged at the vehicle pH used) had maximum flux under the anode, and the negatively charged amino acids had maximum flux under the cathode. For zwitterions (i.e., essentially neutral), the iontophoretic flux did not reach steady state under the experimental


conditions used and was inversely proportional to the permeant octanol/pH 7.4 buffer distribution coefficient. Iontophoretic transport of a tripeptide, thyrotropin-releasing hormone (TRH), across excised dorsal nude mouse skin was directly proportional to the applied current density [51]. Even uncharged TRH was transported, presumably by the electroosmotic or convective flow that accompanies iontophoresis. However, in the absence of current, the flux of TRH across skin was undetectable [52,53]. Another study showed that penetration enhancers also enable transdermal delivery of TRH at physiologically relevant concentrations [52]. Another tripeptide, threonine-lysine-proline (Thr-Lys-Pro), was successfully delivered across nude rat skin by iontophoresis under both in vitro and in vivo conditions. The delivery of Thr-Lys-Pro was directly proportional to the applied current density over the range 0.18–0.36 mA/cm2. Following 6 h of iontophoresis, 98.4% of the radioactivity showed intact peptide in the donor, and 94.0% of the radioactivity penetrated showed parent Thr-Lys-Pro in the receptor phase [53]. Therefore, it can be stated that metabolism in the skin is not likely to be a significant problem for several peptides. Iontophoretic delivery of another tripeptide, enalaprilat, has also been investigated across hairless guinea pig skin [54]. For the tetrapeptide hisetal, iontophoresis increased its permeation rate across human skin by a factor of 30 [55]. The iontophoretic treatment was much more effective than the use of penetration enhancers. The transport through skin under iontophoresis was shown to take place mainly through water-filled pores.

Oligopeptides LHRH, a decapeptide, and its analogues have been successfully delivered by iontophoresis [56]. Meyer et al. delivered therapeutic doses of leupro-lide, LHRH analogue in 13 normal men using a double-blind, randomized, cross-over study conducted under an investigational new drug (IND) process granted by the Food and Drug Administration (FDA). Data analysis by analysis of variance (ANOVA) showed significant differences between the active and passive patches. The magnitude of elevation of luteinizing hormone (LH) produced by the active patches was in the therapeutic range and comparable to that achieved by subcutane-ous administration. The only adverse effect reported was mild erythema at the site of the active patch in 6 of the 13 subjects. The erythema resolved rapidly without sequelae [57]. Vasopressin, a nonapeptide antidiuretic hormone; its analogues; and arginine vasopressin have been investigated for transdermal iontophoretic delivery across skin [58–61]. In contrast to insulin, the flux of vasopressin under iontopho-resis was observed to be reversible. Due to its high isoelectric point (pI 10.9), the vasopressin molecule will stay highly charged at the pH environment of the skin. Permeation of buserelin through isolated human SC by iontophoresis has also been reported [62]. In this study, it was found that passive permeation of buserelin through human SC is not feasible.

Polypeptides Iontophoretic delivery of calcitonin, salmon calcitonin (sCT), and human calcitonin has been investigated [63–65]. sCT, when applied to the abdominal skin of rats, did not produce any hypocalcemic effect. Conversely, when delivered under anode as a cation, the drug produced a small hypocalcemic effect. The hypo-calcemic effect was enhanced when aprotinin or camostat mesilate was used as a proteolytic enzyme inhibitor, but not when soybean trypsin inhibitor was used. That


is because aprotinin (MW 6500; pI 10.5) existed as a cation at pH 4.0 and was probably delivered into the skin during iontophoresis. In contrast, soybean trypsin inhibitor (MW 8000; pI 4.0–4.2) was anionic, or neutral, at the pH used and had a higher molecular weight. Other polypeptides that have been delivered iontopho-retically include an analogue of growth hormone-releasing factor with a molecular weight of 3929 Da [66].

Insulin Insulin, despite being a large molecule (MW ~6 kDa), has generated substan-tial interest and has been widely investigated for iontophoretic delivery in different animals [67–76]. The momentum for research is obviously the importance of insu-lin for control of diabetes and the lack of any breakthrough drug-delivery systems yet. In the first reported study on transdermal iontophoretic delivery of insulin for systemic effect, Stephen et al. were able to deliver a highly ionized and monomeric form of insulin to a pig and observed a turndown in blood glucose levels and a boost in serum insulin levels [67]. Increased penetration of insulin with application of a depilatory lotion, cream, or absolute alcohol, prior or in combination with iontopho-resis, has been reported [76,77]. Another group of scientists reported the successful transdermal delivery of a therapeutic dose of human insulin to diabetic albino rabbits across the skin with intact SC [69]. Although iontophoresis overcomes the physical barrier to some extent, the question remains whether enough insulin can be delivered to be of therapeutic benefit to diabetic patients. A drug flux of 2–4 mg/cm2 an hour is necessary to meet the basal insulin needs for a diabetic patient. Although the pro-teolytic activity of skin is low, skin contains both exo- and endopeptidases, which causes degradation of insulin during delivery through skin.

Another problem is the formation of a depot in the skin. It was observed that blood glucose levels were declining even after current was turned off, suggesting that insulin forms a reservoir and releases gradually from the skin [70]. Similar observa-tions on skin accumulation of insulin have been made by other researchers. Thus, it would seem that iontophoresis serves to load up the skin tissue with insulin to form a reservoir from which insulin molecules continue to creep slowly into the blood circu-lation until several hours after the current application. This could be a potential dis-advantage. In addition, most commercially available insulin products actually exist in hexameric form, so attempts are being made to deliver a protein with a molecular weight of about 36 kDa, which is most likely too high to be within the scope of ion-tophoretic delivery. Iontophoretic delivery of monomeric insulin analogues has been investigated with better success than regular hexameric insulin. Still, the isoelectric point of insulin (5.3) falls in the region of skin pI (4.0–6.0). This poses a major hur-dle to its delivery because insulin will lose its charge in the skin environment, and the predominant impetus for delivery (electrical repulsion) would end. Because some insulin then diffuses toward the blood or physiological interior (pH 7.4), it acquires a negative charge and may be drawn back toward the anode. Therefore, despite the considerable interest in iontophoretic delivery of insulin, it is not a model protein for delivery. Its isoelectric point and tendency toward aggregation make successful iontophoretic delivery of regular insulin unlikely. However, other enhancement tech-niques discussed in this chapter may be more promising for insulin delivery.


Commercial Aspects of Iontophoretic Delivery FDA requirements, regulatory issues in combination products, and patient compliance are some of the important market fac-tors. Ease of use, device adhesion, dosing and rate factors, electrode design, power requirements and battery technology, and prefilled disposable device designs are fac-tors which depend upon design of iontophoretic delivery device. Initial products (from Iomed, Empi, Life-Tech, Vyteris, and ALZA) have not achieved market success, per-haps because of drawbacks in some medical devices or unoptimized formulations for iontophoresis.

Several pharmaceutical companies are trying to commercialize iontophoretic skin delivery. Major companies that are involved in the development of iontopho-resis equipment include Iomed Inc. (Salt Lake City, UT), Empi Inc. (St Paul, MN), Life-Tech Inc. (Houston, TX), Alza Corporation (Pala Alto, CA), and Beckton and Dickinson (Franklin Lakes, NJ). Iontophoresis makes a positive contribution to the transport of cations and a negative contribution to the transport of anions under nor-mal physiological conditions. Iontophoresis is a major mode of transport for neutral molecules design but requires further research today in order to build convenient, efficient, and cost-effective devices. Many devices are already on the market and typ-ically include a current-controlling mechanism primarily incorporating a micropro-cessor, a pulse controller with timer, and electrodes.

An New Drug Application has been filed for a prefilled iontophoretic fentanyl patch. The iontophoretic patch from Alza is expected to be an integrated device. The lidocaine patch from Vyteris has a prefilled patch, a wearable battery-powered controller, and an interconnect module, and has been approved for marketing by the FDA. Elan Corporation (Ireland) investigated a device (Panoderm) that was designed like a watch and contained a disposable drug cartridge. Empi Inc. has announced FDA approval of a lidocaine HCl 2% and epinephrine 1:100,000 solution for use with the company’s Action Patch Smart Iontophoresis System for the iontophoretic induction of local analgesia during superficial dermatologic procedures. Although an iontophoretic patch is expected to be more costly than a passive transdermal patch, this may not be a disadvantage for a proteinous or peptide drug due to their higher initial cost. Also, passive transdermal or other alternative delivery systems may not be possible, so it is important to verify the efficiency of delivery.

As shown earlier, iontophoresis has been approved for the topical delivery of local anesthetics and corticosteroids. Gel drug-delivery systems containing proteins and peptides have been fabricated by preparing a formulation of the peptide or protein drug with the monomer and inducing polymerization by the application of electro-magnetic radiation. Use of hydrogel formulations in units available in the market will allow dosage replacement by the removal and replacement of a drug-loaded hydrogel patch that reuses the same iontophoretic device. Studies have shown that polypep-tide drugs are reliably loaded into the aqueous environment of a hydrogel, and their release from the hydrogel is controlled and modulated by controlling the iontopho-resis parameters, such as the current density. Feasibility of the commercialization of iontophoretic delivery and any long-term effects of current on skin will need to be carefully evaluated. The only short-term effects experienced following iontopho-retic delivery are a feeling of tingling or warmth and possibly general irritation and


erythema. Iontophoresis will be contraindicated for patients sensitive to the current levels used and to patients carrying electrically sensitive devices such as cardiac pacemakers. Based on differential scanning calorimetry (DSC) and attenuated total reflection–Fourier transform infrared (ATR–FTIR) studies, it has emerged that the effects of electrical treatment are less disruptive to the SC compared to the effects of penetration enhancers [78]. At present, several drug-delivery companies are in the process of developing prefilled wearable, battery-powered patches for clinical trials and eventual market launch.

Electroporation in Combination with Iontophoresis Iontophoresis and electropor-ation are both methods of electrically assisted transdermal drug delivery. Figure 12.5 shows drug penetration pathway in low-voltage iontophoresis and high-voltage elec-troporation. Iontophoresis is more usually used to deliver hydrophobic low-molecu-lar-weight drugs, whereas electroporation appears more effective for the delivery of some macromolecules such as antisense oligonucleotides, peptides, and proteins. Drug delivery with iontophoresis and electroporation are thought to utilize different penetration pathways (Fig. 12.5). Electroporation has the advantages of delivery of macromolecules, quick drug effect onset, and resultant insignificant or minor skin damage. There is also evidence showing greater drug uptake by skin cells during elec-troporation. A combination of iontophoresis and electroporation could possibly further enhance drug transport and allow rapid delivery of a bolus dose and precise control of drug-delivery rate and programmability. Electrically assisted delivery of sCT (MW 3600) was conducted by Chang et al. [12]. Electroporation pulses (six pulses of 120 V, 10 ms each) followed by iontophoresis (0.5 mA/cm2) gave a flux about 4 times higher than with iontophoresis alone. Lag time of the iontophoretic delivery was shortened significantly as well. However, pulsing at lower voltages (60 and 100 V) followed by iontophoresis did not result in sCT transport increase over iontophoresis alone. Pulsatile transdermal delivery of LHRH using electroporation followed by iontopho-resis was studied [79]. The application of a single pulse (500 V, 5 ms as exponential) to initiate the experiment resulted in a nearly twofold increase in LHRH concentration

New pathways(high-voltage)

Hair follicle

Transappendageal(low-voltage iontophoresis)

Sweat gland

Figure 12.5 Drug penetration pathways in low-voltage iontophoresis and high-voltage electroporation.


at the end of 30 min of iontophoresis (0.4 mA/cm2). LHRH transport in a pulsatile manner was achieved by repeated processes of one pulse, immediately followed by 30 min iontophoresis. Skin toxicity of electroporation together with iontophore-sis evaluated. Pulses of 0, 250, 500, and 1000 V were applied, followed by constant current anodal iontophoresis of 0, 0.2, and 2.0 mA/cm2 for 30 min or 10 mA/cm2 for 10 min. At the gross microscopic level, immediately after or 4 h after treatment, ery-thema increased with increasing pulse voltage. Erythema, edema, and petechiae all increase significantly with increased current in the absence of a pulse. The application of an electroporation pulse did not increase the iontophoretic-induced irritation with any current tested. All skin changes tended to decrease within 4 h after the treatments.

Nevertheless, at times lowered combined effects in contrast to the effects achieved with each individual treatment were also reported. Denet et al. reported lowered transdermal delivery of the hydrophobic drug timolol with iontophoresis and elec-troporation combination than with iontophoresis alone [80]. The decreased transport was explained as being due to an accumulation of positively charged timolol in the SC, which was amplified by electroporation, and a resulting decrease of electroos-motic flux during iontophoresis [44,80]. The practical application of combining electroporation with iontophoresis is still in its initial trial stage, similar to the com-mercial development of electroporation devices for transdermal delivery of drugs. Iontophoretic studies have resulted in a few marketed medical device products, some containing drugs, that are close to FDA approval.

12.1.4.1.2 Skin MicroporationSkin microporation involves the creation of micron-sized micropores or microchan-nels in the skin that allow the transport of macromolecules and soluble drugs, as shown in Fig. 12.6. Technologies that create these microchannels in the skin include mechanical microneedles [81,82], thermal or radiofrequency ablation, and laser abla-tion. The approach looks very promising and is likely to revolutionize transdermal drug delivery of proteins and peptides.

Microneedle approaches are designed to circumvent the primary skin barrier without intruding on the underlying pain receptors and blood vessels. Microneedles

Stratumcorneum

Epidermis

Microneedles

Drug

Dermis

Pain receptor

Blood vessel

Figure 12.6 Schematic representation of delivery of a drug through microneedle-punctured skin.


commonly range from 100 to 1000 m in length are designed to pierce the SC, and provide a direct and controlled route of access to the underlying tissue layers. When inserted into the skin, microneedles create microscopic punctures through the SC and into the viable epidermis. The length of the microneedle is controlled to ensure that the depth of penetration does not encroach on the nerve fibers and blood vessels that reside primarily in the dermal layer. The micron-sized channels therefore facilitate the delivery of both small- and large-molecular-weight therapeutics into skin without causing pain [82] and bleeding at the site of application.

Microneedles are used to augment the impact of concomitant delivery meth-ods such as transdermal patches. Use of microneedles is a robust delivery for any formulation—solution, suspension, emulsion, dry powder, and gel. This method exposes large surface areas of the epidermis to the delivery agents rapidly (micronee-dle arrays can contain over 1000 microneedles) and controls direct delivery of the medicament. It does not need extra effort and is a convenient and painless delivery for the patient. It is a minimally invasive methodology and suited to patient self-administration, without the need for medical supervision. The materials, types of structure, and dimensions of the needle can be adapted to facilitate the delivery of macromolecules, nanoparticles, and vaccines [83].

Solid microneedle arrays present an opportunity to create conduits through the restrictive skin barrier layer. The formulation is applied into the channels, through dry coating of the microneedle array or solution, suspension, emulsion, or gel con-taining the medicament. The formulation is administered simultaneously and relies on passive delivery mechanisms. The capacity to fabricate small hollow micronee-dles allows a controlled quantity of the medicament to be actively delivered from the tip of the inserted microneedle at a predefined rate and also provides the opportunity to withdraw material from the skin for analysis and monitoring of response.

12.1.4.1.3 Phonophoresis/SonophoresisThe use of ultrasound to enhance percutaneous absorption of a drug molecule is called sonophoresis or phonophoresis [84–87]. The proposed mechanisms that enhance skin penetration include cavitation, thermal effects, and mechanical per-turbation of the SC [84,88]. It can be inferred that ultrasound acts on the barrier function of the membrane. Sonophoresis has employed three distinct categories of ultrasound:

1. High-frequency, or diagnostic, ultrasound (2–10 MHz)2. Mid-frequency, or therapeutic, ultrasound (0.7–3 MHz)3. Low-frequency ultrasound (5–100 kHz).

Ultrasound is defined as sound with a frequency longer than 16 kHz, although a frequency of about 1 MHz is often used for diagnostic purpose. To attain biologic effects from ultrasound, the energy must be captivated by the tissues. The depth of penetration in tissue is controlled by the attenuation of sound, which is inversely related to frequency. A contact medium or coupling agent is required to transfer ultrasonic energy from the ultrasonic device to the skin and ultimately to the body. A gel, emulsion, or ointment is used for purpose. The coupling agent may also be


used as a drug carrier, but the amount of coupling agent to be applied must be opti-mized. Phonophoresis, a feasible technique for skin permeation enhancement, offers a potential for delivery of peptides and polypeptides. Any possible effect of ultra-sound on the stability of peptide drugs is still to be evaluated.

The efficiency of ultrasound-mediated drug delivery depends on several factors, including ultrasound frequency, intensity, continuous versus pulsed exposure, duty cycle, duration, coupling medium, and other considerations. The ultrasound beam is made up of two components: the field closest to the transducer, and the field fur-ther away (the final, diverging conical part). The relative size of these two zones, and their separation, is a function of the ultrasound wavelength (i.e., frequency) and the transducer radius. The mechanistic aspects of the effects of low-frequency ultra-sound, cavitations, and thermal effects have been hypothesized, but it is not clear how ultrasound interacts with the skin barrier to increase its permeability. Very few publications have presented data to support that higher frequency (1 MHz) ultrasound can be used to improve peptide delivery across the skin.

Collective application of ultrasound and iontophoresis also has practical implica-tions. The combination of ultrasound and electric current offers a double enhance-ment greater than that offered by each of them independently under the same circumstances. Because ultrasonic pretreatment reduces skin resistivity, a lower volt-age is required to deliver a given current during iontophoresis compared to that in controls. This may result in lower power needs as well as possibly lessening skin irritation [89].

12.1.4.1.4 Electrical Methods in Conjunction with Other TechniquesThe combinations of iontophoresis with electroporation [90], enhancers [91], and poloxamer gels [92] were also found to enhance the penetration ability of insulin through skin more than iontophoresis alone.

Iontophoresis in Conjunction with Microneedles A combination of microneedle with iontophoresis technologies can be studied for delivery. This combination may provide the possibility of macromolecule transdermal delivery with precise electronic control. The Macrofluxw array, 2 cm2, had a microprojection density of 240/cm2 and a needle length of 430 mm. The Macrofluxw and iontophoresis combined system is made by assembling the Macrofluxw array, a drug reservoir, a membrane, a conductive gel, and the iontophoretic electrode. Macrofluxw and iontophoresis combined trans-dermal delivery system for the delivery of an antisense oligonucleotide ISIS 2302 is already designed [93]. The system may be capable of delivering therapeutically signif-icant amounts of drug through the SC. The rate of delivery is controlled by duration of the patch application, donor drug concentration, current density, and active patch area.

Iontophoresis in Conjunction with Ion-Exchange Materials The ion-exchange process is a stoichiometric and reversible process wherein an ion from the solution is replaced with a similarly charged ion attached to an immobile solid phase (e.g., ion-exchange device) in order to fulfill the electroneutrality requirement. Ion-exchange devices will exhibit greater preference for a particular ion. The higher the preference an ion exchanger exhibits for a particular ion, the greater the exchange efficiency in


terms of ion-exchanger capacity for removal of that particular ion from a solution. Novel applications of ion-exchange materials include enhancement of drug release and increasing the dissolution rate of the poorly soluble drugs. Ion-exchange materi-als used for medical and pharmaceutical applications are usually in a form of fibers [94–99], membranes [100], and resins [101]. The equilibrium reaction between the ion-exchange device and a particular organic ion of significant molecular weight of typical drugs is controlled by the environment in which the drug and the ion-exchange material are found.

Factors controlling the equilibrium constant include molecular weight, pKa of drug and resin, pH of the solvent, ionic strength, hydrophobicity and hydrophilicity, and concentration of competing ions. Drug ions, which are attached to the ion-exchange materials via electrostatic interactions, provide a more accurate and homogeneous control of the ion-exchange process so that drug release rate can be easily adjusted. Ion-exchange fibers could be a good material to successfully store an easily degrad-able drug. Drug stability was greatly enhanced by attaching drug to ion-exchange fibers in an acidic or basic environment. Drug ions attached to the ion-exchange mate-rials through electrostatic interactions provide a more precise and uniform control of the ion-exchange process, so the drug release rate can be simply adjusted [97,98].

A combination of iontophoresis with electroporation, chemical enhancers, sono-phoresis, microneedle, and ion-exchange material may provide easier and more accurate delivery of macromolecules and poorly water-soluble compounds. The skin irritation associated with iontophoresis has been addressed by several studies and is an issue preventing wide application of the technology [12]. Nevertheless, the blend with other enhancement techniques may result in the need for less strong current to reach therapeutically effective delivery amounts, and hence will considerably reduce the skin irritation problem.

12.1.4.2 Chemical Approach

Application of peptides and proteins as clinically useful drugs is a major challenge. This is due to their poor delivery characteristics caused by their metabolic instability and general hydrophilic character, resulting in poor transport across biomembrane. This typically leads to bioavailability less than 1–2%. Once within the systemic cir-culation, a short biological half-life is normally observed due to rapid metabolism and clearance from the body. A possible approach to solve these delivery problems could be chemical amendment or derivatization.

Two approaches may be possible:

1. Permanent chemical change in the drug molecule (also referred as the analogue approach).2. Bioreversible derivatization of the bioactive peptide or protein (also referred as the prodrug

approach).

Normally, analogues are not bioreversible. Both the prodrug and the analogue should have better absorption and/or stability characteristics over the parent drug molecule. The analogue should also have high receptor selectivity and affinity. In the literature, different terms such as analogues and peptidomimetics find use to describe


permanent chemical changes in the parent molecule [102–107]. There seems to be no consistency in the usage, but in general the chemically modified drug molecule is defined by the degree of peptide backbone structure left after derivatization. Thus, analogues have more peptide-like structure left after chemical change than pepti-domimetics [106,107]. In recent past several types of analogues of various biologi-cally active peptides and proteins have been explored. Possible strategies used in the development of analogue include N- and C-terminal modifications (e.g., conversion of the C-terminal carboxylic acid residue to an amide); amino acid manipulations (e.g., systematic replacement of l-amino acids with d-amino acids); peptide back-bone modifications, where the use of amide isosters is common (e.g., N-methylation of the peptide amide bond); and replacement of larger structural moieties in a com-pound with dipeptide or tripeptide analogue structures or analogues of the secondary structure [108–112].

12.1.4.2.1 Prodrug ApproachA prodrug is by definition a pharmacological inactive derivative of a drug molecule that is capable of releasing the parent molecule quantitatively, due to enzymatic or spontaneous reaction in the body. The chemical group used for derivatization of the parent drug molecule, called the progroup, should be nontoxic. The use of prodrug suggests a promising biochemical approach for improving the skin permeation of drug molecules [108]. As we all know that the low skin permeability of peptides is partly because of their hydrophilic nature. The synthesis of lipophilic prodrugs of such peptides is expected to improve transdermal transport. After diffusing into or through the skin, the prodrug undergoes conversion into the original active drug molecule. Such an approach was found feasible for TRH. The prodrugs studied are N-isobutyloxycarbonyl and N-octyloxycarbonyl derivatives of TRH. Results of dif-fusion experiments using excised human skin indicate that the N-octyloxycarbonyl derivative demonstrated better permeability. The prodrug penetrated into the receptor phase was found to exist primarily as TRH. The authors showed that the quantities equivalent to those given by infusion or injection of TRH can be delivered in this manner [103]. The prodrug approach was introduced by Albert in 1958, but focus was not directed to this specialized area until later. Research in the design of pro-drugs of various chemical functional groups and of well-known drug substances, as well as types of usable progroups, intensified, and today several drugs are used clini-cally as prodrug derivatives of a parent drug molecule [104–109]. In the late 1980s, attention to the use of the prodrug approach to improve the delivery of peptides and proteins increased [107,108,112–114].

12.1.4.2.2 Permeation EnhancersSeveral chemical compounds have the capacity to increase the permeation of drugs across biomembranes, mostly by altering the barrier properties of skin [115–118]. These are the substances that speed up the transfer across skin by altering the intercel-lular lipid or intracellular protein domains of the SC. Their interaction with intercellular lipids may disorder the highly ordered lamellar structure, thus increasing the diffu-sivity of drugs across skin. In addition, they may solubilize or plasticize skin–tissue


components. Examples of commonly investigated chemical permeation enhancers include phospholipids, surfactants, dimethylsulfoxide (DMSO), and 1-odecylazacy-cloheptan-2-one (azone). Ideal characteristics of a permeation enhancer are that it must be pharmacologically inert, nonirritating, nontoxic, compatible with most drugs and excipients, and nonallergenic. However, no single agent meets all the required qualities of an ideal enhancer. A blend of enhancers may thus be necessary [117]. In addition to these chemical enhancers, many of the generally regarded as safe (GRAS) parenteral vehicles that are GRAS also enhance percutaneous drug absorption. The addition of cosolvents in transdermal and dermal delivery products is another good approach. These cosolvents include polyethylene glycol 400, propylene glycol, iso-propyl myristate (IPM) and palmitate (IPP), ethanol, water, and mineral oil. Because an enhancer is delivered to the skin, its pharmacokinetic properties, such as its mech-anism of elimination, half-life in skin, degree of absorption, and metabolism, must be known. Also, the reversibility of skin barrier properties should be determined as any permanent breakdown in the barrier properties of SC could result in infection [119]. Use of a transdermal patch creates occlusive conditions on the skin, leading to increased hydration and irritation of skin beneath the patch. Increased skin hydration may increase the permeation of the enhancer itself, which may result in even more irritation or toxicity. Further, the enhancer may increase the permeation of the formu-lation ingredients along with the drug. Therefore, careful evaluation of the long-term local and systemic toxicity of the chemical enhancers in the final transdermal dosage form is important [114,115].

12.1.4.2.3 Protease InhibitorsTherapeutic proteins and peptide are becoming increasingly important. These com-pounds are degraded by the luminally secreted and brush border membrane-bound proteolytic enzymes. Because of their nonhydrophobic character and comparatively large size, they are frequently taken up via the paracellular route after oral admin-istration. Coadministration of inhibitors of proteolytic enzymes provide a practical means to avoid the enzymatic barrier in achieving the delivery of peptide and protein drugs. A number of inhibitors, like misleading aprotinin (trypsin/chymotrypsin inhib-itor), amastatin, bestatin, boroleucine, and puromycin (aminopeptidase inhibitors), have been reported for this purpose. Interestingly, these inhibitors were not effective in skin diffusion experiments [116,117]. The drawback of using protease inhibitors is that the inhibition may also affect the absorption of other peptides or proteins that normally would be degraded, thereby decreasing any effect. Moreover, high doses of inhibitor are needed, and the bioavailability of the drug is still limited by the physical barrier of the cells.

This section of the chapter deals with the transdermal route as a delivery route. The transdermal route has less proteolytic activity compared to the mucosal route. The transdermal route has the potential to hydrolyze peptides. Protease inhibitors are used to avoid or reduce the enzymatic barrier of the skin. Other potentially useful protease inhibitors are pepstatin, leupeptin, p-chloromercuribenzoate, and phenylmethylsulfo-nyl fluoride. A metalloprotease inhibitor, o-phenanthroline, has been shown to inhibit degradation of delta sleep-inducing peptide and increase its transdermal iontophoretic


delivery eightfold at pH 7.4 [120]. Sodium glycocholate, a penetration enhancer, may also act as a protease inhibitor. A dual role may be played by a variety of mechanisms, such as by tightly binding to or covalent modification of the active sites of proteases or by chelating metal ions essential for proteolytic activity.

12.1.4.3 Other Technologies

Delivery of peptides and proteins across the skin is carried out by physically circum-venting the SC barrier in ways designed to be less invasive than the use of a classic injection. Among the approaches used are ablation of the SC by laser radiation, heat, or erosion, and particle bombardment. All these approaches can be used in combination with electrical methods [121]. The mechanism by which this laser beam disrupts the SC is complex, yet quite reproducible and controllable, in that the number of laser pulses is correlated to the degree of SC damage. Lasers are physical devices that have been used for medical diagnosis and therapeutic purposes. The erbium:yttrium–aluminum– garnet (Er:YAG) laser, a tool used to provide overall skin rejuvenation, from simple lentigines to deeper rhytides, causes minimal residual thermal damage [122]. The dura-tion of the skin’s exposure to the laser is in the range of nano- to microseconds. To obtain pure photomechanical waves (PWs) generated by the laser and exclude the SC ablation effect, a polystyrene lens is used to filter the laser light for comparison. The Micro PorsTM technology consists of directing tightly focused thermal energy into the SC to create micropores. The skin is contacted by a wire mesh through which a current is passed, causing local heating sufficient to create small holes in the barrier. Delivery of insulin by this approach has been tried [122]. A more macroscopic method to cre-ate an erosion in the SC is via suction deepithelialization [123]. Using a vacuum, a small blister (6 mm diameter) is formed on the skin; the tissue separates at the dermal–epidermal junction. The roof of the blister is then removed, or “guillotined,” expos-ing a small area of dermis to which a drug solution is directly applied. The dermal microcirculation remains intact and functional following creation of the erosion. Mild inflammation is observed and the erosion self-heals over time with, apparently, mini-mal scarring. But practical applications of this approach, especially for chronic disease treatment, look unlikely, and issues related to local infections must be addressed. High-velocity particle delivery across the skin is the technology of a major drug-delivery operation designated in the UK and the USA (PowderJect Inc.) [124]. Once a drug has been formulated as an appropriate and well-characterized powder, it is then introduced into a compact handheld device in which a supersonic flow of gas accelerates the par-ticles to a speed high enough that they collide with the skin, creating enough energy to penetrate the skin’s outer layers and effect drug delivery. The depth and extent of deliv-ery depends on the speed, diameter, and density of the drug particles.

12.2 Topical Delivery of Proteins and Peptides

A great deal of research for delivery of peptide drugs is currently centered on trans-dermal delivery, that is, percutaneous absorption for systemic results. Nevertheless,


topical delivery of peptide and protein drugs is also significant; one example is the use of the growth factor for wound healing. Proteins are also given as antigens for topical delivery for vaccination. Biotechnology products in development for skin dis-orders include the recombinant proteins onercept and interleukin (IL) 18 bp, the fusion protein denileukin diftitox, and the monoclonal antibodies eculizumab, adalimumab, and antiangiogenesis monoclonal antibody [125,126]. For conventional drugs, topical products to treat dermatological ailments have been in continual use from the outset. Although transdermal delivery is moderately new, the principles involved are better understood than for topical delivery. This is mainly because with transdermal delivery the blood concentration needed to achieve therapeutic effects is recognized. For topical delivery, the skin is the target organ. In contrast to transdermal delivery, a non-steady-state transport generally characterizes a topical drug product. An optimal drug buildup in the skin, with little or no flux through the skin, is desirable, which does not occur in transdermal delivery. The topical delivery of small molecule drugs has been used to deliver therapeutics transdermally and transmucosally. Again, because of the size of the protein, it is often not feasible to deliver enough protein to achieve systemic levels of the drug. It is possible to deliver protein-based therapeutics locally by a topical for-mulation [125,127]. Regranex, a platelet-derived growth factor–based topical formula-tion, has been approved by the FDA for the treatment of diabetic ulcers [128,129].

12.2.1 Growth Factors

The wound-healing process in the body is always accompanied by the discharge of a number of protein-based growth factors that assist the healing [130,131]. These growth factors are now formed biotechnologically in a commercially feasible manner, therefore increasing the possibility of fast healing of wounds. The use of growth fac-tors can hasten the healing process after surgery. With respect to health care costs, this could result in enormous savings. This rationale is driving a number of companies and other academic and government organizations to develop growth factors. Although growth factors also have application in ophthalmology, a chief indication for their use is to accelerate wound healing in the skin. The most promising agent for such use is the epidermal growth factor (EGF), a polypeptide of 53 amino acid residues, which has shown of the capability to heal open and burn wounds. It was found that the existence of protease inhibitors is necessary in the formulation to stabilize EGF at the wound site [132,133]. Formulations of acidic fibroblast growth factor were also reported to accelerate wound healing in a diabetic mouse model [133]. In the same way, transforming growth factor- (TGF-) is used topically to accelerate wound healing. One patent involves a lyophilized protein formulation containing a cellulose polymer as a gelling agent. When reconstituted, the formulation forms a gel that can then be used topically [134].

12.2.2 Liposomes

The literature on the use of liposomes for topical or transdermal delivery appears to have reached an accord on the belief that liposomes cannot traverse the skin in intact


form due to their structure. Consequently, they will usually not improve percutaneous absorption of entrapped drugs, although they are likely to augment topical delivery into epidermis and perhaps to dermis as well [135–138]. It has been suggested that liposomes, being lipophilic, may enter the lipid-rich external skin layers and then are localized because of the hydrophilic environment of the dermis. Nevertheless, it may be difficult to ascertain how relatively large lipid vesicles cross densely packed epidermal tissue to reach within the intact dermal layers; the mechanism involved is not apparent. Analytical techniques must be developed that can distinguish skin and liposomes [137]. The reason for reduced epidermal and dermal clearance of liposome-entrapped drugs is recognized as being due to the unavailability of the metabolizing enzymes within the skin to the encapsulated drug. It has also been sug-gested that larger liposomes, unable to penetrate the underlying blood vessels, act as localized sustained release vesicles [139]. Additionally, liposomes improve solubil-ity of poorly soluble drugs, and their phospholipid constituents may act as penetra-tion enhancers to facilitate topical absorption [138]. Lecithin is commonly used in pharmaceutical, cosmetic, and food products and has GRAS status. Lipids as such and liposomes have been incorporated into many cosmetic formulations [140,141]. Unlike liposome-, lipid-containing preparations have been marketed as cosmetics. The liposomes-containing products in the market include the antifungal drugs econ-azole (Pevaryl®, Cilag, AG) and amphotericin B. Unlike water-soluble peptide and protein drugs, which are entrapped in the aqueous environment within the liposome, drugs that are associated with the lipid bilayer use liposomes for topical delivery. The method of liposomal preparation is the most important factor determining the effec-tiveness of the formulation. The dehydration–rehydration method was most effective, presumably because dehydration and subsequent rehydration of the liposomes facili-tates partitioning of the interferon into liposomal bilayers [142]. In continuing stud-ies using an in vitro diffusion setup, it was observed that liposomes made from “skin lipids” were twice as effective as those made from phospholipids, perhaps because they are better able to transfer the drug to the skin [142]. Unlike other biological membranes, the SC does not contain phospholipids and is made primarily of cerami-des, cholesterol, fatty acids, and cholesteryl sulfate.

12.2.3 Iontophoresis and Phonophoresis

Although investigations of the potential use of iontophoresis and phonophoresis tech-niques to achieve systemic delivery of drugs are relatively new, having only begun over the last one or two decades, these techniques have been used for topical delivery of drugs and in physical therapy for several years. The technique of iontophoresis is used routinely in clinics by physical therapists for the delivery of corticosteroids and local anesthetics to treat inflammatory conditions of muscles and tendons, such as tendonitis, bursitis, carpal tunnel syndrome, arthritis, temporomandibular joint dys-function, and others. Several devices and electrodes are commercially available for this purpose, and the most commonly used drug for iontophoretic delivery is dexa-methasone sodium phosphate. Similarly, phonophoresis is also routinely used in clin-ics by physical therapists, with hydrocortisone a commonly administered drug [143].


Use of iontophoresis and phonophoresis to achieve topical delivery may be extended to peptide and protein drugs. In fact, as discussed, insulin forms a depot or reser-voir in the skin on iontophoretic or phonophoretic delivery. Although this may not be a good scenario for drugs delivered for systemic effects, depot formation may be advantageous for certain topical drugs.

12.3 Intrauterine Delivery of Proteins and Peptides

12.3.1 Introduction

Generally, high-molecular-weight drugs, and peptides and proteins in particular, are usually delivered by parenteral route because they are either degraded in the GI tract or poorly absorbed. Repeated injections are often required because of the drug’s short half-life and the chronic nature of many diseases. To optimize therapy, it is essential to search for a nonparenteral route of drug administration. Intrauterine drug delivery has been proposed as a new route of drug administration relatively recently. Two polypep-tides, calcitonin and insulin, were reported to be absorbed from the uterus of the rat in a biologically active form [144,145]. The extent and duration of hypocalcemic and hypoglycemic effects induced by intrauterine delivery of calcitonin and insulin, respec-tively, were measured and were equivalent to those obtained after subcutaneous injec-tions. The role of cervical mucus in drug delivery via the vagina and cervix has been reviewed [146]. The viability and integrity of the mucosal tissue must be monitored during in vitro studies, such as those done in using chambers [147]. Only very few stud-ies have evaluated this route thus far, and more work is likely to appear in the future.

12.3.2 Drug Absorption Through the Intrauterine Route

A number of drug substances are known to act directly upon the uterus, including uterine relaxants (e.g., -agonists) and stimulants (e.g., prostanoids, oxytocin). The administration of drugs to the uterus is achieved by the application of a formulated product to the vagina or the cervix. But it has been demonstrated that the mechanism by which the drug is transported from the cervicovagina to the uterus is not limited to passive diffusion, but is facilitated by a preferential transport mechanism termed the first uterine pass effect.

As shown in Fig. 12.7, the uterus in a human is a pear-shaped, thick, muscular organ composed of three major anatomical divisions—corpus, isthmus, and cervix. The main uterine cavity comprises the upper two-thirds of the uterus and is known as the corpus. It is several centimeters in diameter and connected on both sides to the fallopian tubes. Below the corpus, the uterus narrows to form the isthmus, which ter-minates, through the cervical canal, in a constricted opening into the vagina known as the cervix. The main functions of the uterus include transportation of sperm from vagina to uterine tubes for fertilization; site for implantation, nourishment, and pro-tection of embryo and fetus; expulsion of the fetus at term, through the vagina to outside, by powerful contractions of its thick muscular walls.


From a histological perspective, the uterine tissue consists of three well-defined layers: the tunica serosa (perimetrium), the tunica muscularis (myometrium), and the tunica mucosa and submucosa (endometrium). The tunica mucosa is the muco-sal lining of the uterus, consisting of epithelial tissue, tubular glands, and connec-tive stroma tissue. Hormonal changes associated with the menstrual cycle cause the superficial two-thirds surface of the endometrium (also known as the functionalis or stratum functionale) to be sloughed in a process known as menstruation. However, the basalis (or stratum basale) component of the endometrial layer remains intact and permits regeneration of the sloughed funtionalis layer. The highly vascularized tunica muscularis consists of a thick inner circular layer and a thinner outer longitudinal layer of smooth muscle. The region in between the two layers of smooth muscle con-tains large blood vessels. The tunica serosa consists of loose connective tissue con-taining a large number of lymphatic vessels.

Clinically, the oral route is the most accepted and common way for delivering drugs having relatively low molecular mass of up to 400–600 Da. As high-molecular-weight drugs are poorly absorbed by the digestive tract [148], they must be delivered parenter-ally in order to obtain therapeutic effect. Due to enzymatic degradation in the intestinal lumen, enterocytes, or liver (first-pass effect), and/or a low permeability of the intesti-nal epithelia, many low-molecular-weight drugs must also be delivered parenterally. In these cases, alternative routes can be selected when even parenteral route is not feasible.

The design of delivery systems for peptides and proteins and their evaluation depends on physicochemical and biological properties, and the pharmacokinetics and pharmacodynamics of the substance itself. The desire to deliver protein and peptide biopharmaceuticals suitably and efficiently has led to extensive study of site-specific drug-delivery systems. Despite challenges, progress toward the convenient noninva-sive delivery of proteins and peptides has been achieved through specific routes of administration. In addition, the delivery of proteins and peptides to specific sites of action has been exploited to reduce the total dose to be delivered; gain access to spe-cific organs or body compartments; and concentrate a therapeutic dose at a specific site of pharmacological action. The delivery pattern is vital for the pharmacologi-cal result of diverse peptide and protein drugs. Pulsatile release rather than constant release may be needed for several drugs with regulatory functions. Dosing at steady state of several drugs could lead to tolerance, or “downregulation” of receptors due to

Lateral cornuCavityFundus

Body

IsthmusInternal OS

Cervical canal

Lateral fornix

External OS

Ovarian artery

Urinary artery

Ureter

Vagina

Figure 12.7 Frontal sections through the uterus and upper part of the vagina.


the continual presence of the agent at a receptor site [149]. Proteins have been applied directly to tissues such as the uterine horns with the aid of biodegradable hydrogel in an effort to stop postsurgical adhesions. This method demonstrates proteins being targeted by direct application to the uterine horns with the aid of a polymer or device designed to hold the protein at the site of action. Other examples of this principle include the application of TGF-1 to the surface of titanium hip implants coated with hydroxyapatite and tricalcium phosphate to enhance bone growth [150], and the appli-cation of growth factors such as acidic fibroblast growth factor (aFGF) directly into topical wounds with the aid of methylcellulose gels [151].

12.3.3 Intrauterine Drug Delivery Systems

Implantable delivery systems are used for long-term controlled delivery and site- specific activity with the use of medicated intrauterine devices (IUDs)

An IUD is a small T-shaped device, prepared from a metal or flexible polymer, which is fitted within the uterine cavity for the purpose of preventing conception. The most widely used IUDs are copper-bearing IUDs, although unmedicated (inert) and medicated (levonorgestrel or progesterone) progestin-releasing IUDs are also available. The IUD induces an intense local inflammatory response, especially by the copper-containing devices, which in turn leads to lysosomal activation and other inflammatory changes that are spermicidal. Whenever fertilization does occur, the same inflamma-tory actions are directed against the developing embryo. Inert devices, such as the Lippes loop, are more effective with increased size and extent of contact with the endometrium. The progesterone-carrying devices induce atrophic endometrial changes that make the endometrium a hostile site for implantation if fertilization and successful tubal transport have occurred. Since the 1970s, WHO has conducted 10 large trials to compare and evaluate the safety and efficacy of six different IUDs [154]. The new-est generations of copper IUDs combine high continuation rates with very low preg-nancy rates [155]. Because little can be done to increase the efficacy of these devices, recent research has focused on developing devices to address side effects, particularly bleeding and pain, which account for a significant number of removals. The levon-orgestrel-releasing IUD, a device with high effectiveness and acceptability, reduces menstrual blood loss compared to preinsertion levels. The levonorgestrel-releasing IUD, MirenaTM, has been available in Europe for 10 years and has been used by 2 mil-lion women; it was approved for sale in the USA in December 2000. Frameless IUDs, such as the GynefixTM [156–158], have been specifically designed to reduce cramp-ing and pain. This device consists of a surgical nylon thread that holds copper sleeves and is anchored to the uterine fundus during insertion. It recently became available in Europe, and is licensed for 5-year use. Studies suggest that the GynefixTM is as effec-tive as the Copper T380A, and expulsion rates are less than 1 per 100 women years. Very recently, clinical assessments of an IUD-releasing danazol for the treatment of endometrial hyperplasia and endometriosis-related pelvic pain have been reported.

Contraceptive-medicated IUDs (containing copper or progesterone) have been widely used in humans. Copper-bearing IUDs consist of a copper wire wound around the vertical leg of a T- or 7-shaped plastic device, having a surface area of 200 or


380 mm2. The in utero device releases the copper mainly in the uterine fluid, partly taken up by the endometrium, with insignificant levels in the blood. However, itching or allergic dermatitis, possibly due to the absorption of copper from the uterus into the circulation, was reported several months after insertion of the device. In contrast, a recent study clearly shows that a copper IUD has no effect on gonadal, hepatic, or renal function, or on blood copper concentrations in the Japanese monkey [152]. The increased number of migrating cells in the endometrium was attributed to the contra-ceptive action of the device. The risk factors for infection-related removal of an IUD have been identified as young age, first month postinsertion, multiple sex, and high sexual activity. Most of these risk factors are irrelevant to medicated IUD use in post-menopausal women. It was concluded recently that the Nova-T, being widely used all over the world, is a safe, effective, and acceptable device for contraception [153]. The concept of using a conventional IUD as a carrier for long-term, continued intrauterine administration of contraceptive steroids within the uterine cavity was carried over to humans, following the observation of localized, site-specific antifertility activity of hormones. It was shown that a progestin, released at a controlled rate from an intra-uterine silicone capsule, was able to prevent implantation in the experimental horn of a rabbit uterus, whereas the normal implantation proceeded in the contralateral con-trol horn. There was no difference in plasma profiles of progesterone, estradiol, LH, and follicle-stimulating hormone in women wearing a progesterone-releasing IUD as compared to those using only a placebo device, indicating that the intrauterinally administered progesterone does not have any systemic effect, but a local effect only. The ProgestasertTM IUD contains progesterone delivered at a slightly decreasing but continuous rate (about 65 pug/day) during the 1-year lifetime of the system.

12.4 Rectal Delivery of Proteins and Peptides

12.4.1 Introduction

The rectal route of drug administration has been used for several years because drugs are readily introduced and retained in the rectal cavity. Rectal administration may be a practical substitute to oral administration when patients are prone to nausea, vom-iting, convulsion, and, in particular, disturbances of consciousness. Therefore, rectal administration has been used to deliver many kinds of drugs such as anticonvulsants, analgesics (including narcotics), antiemetics, antibacterial agents, anesthetics for chil-dren, some anticancer agents, and proteins and peptides. On the one hand, rectal drug delivery is efficient because of the extensive rectal vasculature and the presence of lymphatic vessels in the rectal region. On the other hand, patient acceptability of rec-tal administration is much lower and drug absorption may be affected by defecation.

Peptides and proteins, a very important class of therapeutic agents, have poor oral bioavailability due to poor absorption and easy degradation by proteolytic enzymes in the GI tract; for therapeutic results, parenteral administration is necessary. However, these administration routes are very poorly accepted by patients and doctors due to allergic reaction. Thus, alternative routes such as the buccal, nasal, pulmonary, rectal,


and vaginal are used. Conjunctival and transdermal routes are being investigated. Rectal administration may potentially be an important route for peptide administra-tion, although among available options it is poorly accepted by patients. In contrast to the oral route of administration, the rectal delivery of peptide and protein drugs pro-vides the advantage of greater systemic bioavailability, especially with the coadmin-istration of adjuvant. An additional advantage is that it avoids first-pass elimination. However, the rectal absorption of peptide and protein drugs is still poor as compared with intravenous administration. Consequently, various approaches have been exam-ined to improve the absorption of these drugs from the intestine, including the rec-tum. However, it is generally agreed that absorption enhancers are required to achieve therapeutic plasma levels of rectally dosed peptides.

12.4.2 Rectal Absorption

The rectal route is extremely useful for delivery of drugs to newborn and young chil-dren. Disadvantages are limited surface area, dissolution problems, and interruption of drug absorption during defecation and inconsistent patient acceptability. However, the rectal route does offer a more convenient way to control drug release, using osmotic pumps and hydrogel cylinders, although to date they have only been tested with low-molecular-weight drugs, such as propranolol [159] and nifedipine [160,161].

Traditionally, the rectum has been an accepted site of drug delivery. Its principal applications have been for local therapy, for example, for hemorrhoids, and for sys-temic delivery of drugs to groups presenting practical problems for parenteral or oral dosing to patients such as infants and epileptics. Some populations are less willing to accept this route as a standard method for drug delivery, but it is the most easily accessible area of the lower GI tract. As such, it offers the potential for delivery of peptides without the need for targeted devices, and its efficiency is simpler to test in vivo in both animals and humans. However, it is generally agreed that absorption enhancers are needed to achieve therapeutic plasma levels of rectally dosed peptides. Many groups have looked at the bioavailability of a variety of peptides in the rat after rectal administration. Mikaye et al. [162] and Morimoto et al. [163] detected low levels of absorption of [Asu 1,7]-eel calcitonin (MW 3415) after rectal admin-istration to rats. Hypoglycemic response was detectable only in the presence of an enhancer. Rectal absorption of desglycinamide arginine vasopressin (dGAVP, MW 1100) has been investigated by van Hoogdalem et al. [164]. Negligible bioavailability with dGAVP was observed, but with sodium tauro-24,25-dihydrofusidate (STDHF), which is an absorption enhancer, a bioavailability of 27 6% was observed. After rectal administration of insulin (MW 5800), it was demonstrated a bioavailability of 0.2 0.2%, increasing to 4.2 3.2% and 6.7 2.1% following coadministration of different doses of STDHF [165]. The significance of absorption from the rectum into the lymphatic system has been investigated by Yoshikawa et al. [166]. They have demonstrated that negligible levels of human -interferon (MW 17,000) in serum and plasma were detected after rectal administration. Coadministered mixed micelles attained selective lymphatic uptake. These data indicate that rectal absorption of even large peptides is feasible. The generally low bioavailability by simple rectal absorption


resulted in more recent work concentrating on the optimization of enhancement strategies. A widespread array of enhancers has been used to overcome the difficul-ties associated with poor and erratic availability following rectal administration, with some limited success. Nonionic surfactants and glycerol esters have been used to deliver therapeutic amounts of insulin and nonionic surfactants in a polyacrylic acid gel. Bile salts have been used to deliver interferon-1 and insulin. However, given the damaging effects of bile salts on the mucosa and the possible carcinogenic effects of rectally administered bile salts, safer enhancers have been developed.

Yoshikawa et al. [166] have demonstrated the successful enhancement of recom-binant -interferon using suppositories containing lipid-surfactant mixed micelles composed of linoleic acid (0.5%) and HCO-60 (0.4%), a polyoxyethylene castor oil derivative. -Interferon entered the lymph circulation in superior proportion to the blood compartment. The same researchers investigated the enhancing effects of another mixed micelle formulation, monoolein/sodium taurocholate with dextran sulfate, on bleomy-cin absorption. Once again they demonstrated a preferential uptake into lymph and a significant degree of absorption enhancement. In anesthetized rats, Miyake et al. [162] showed that [Asu 1,7]-eel calcitonin (5 U kg) dosed rectally achieved a bioavailability of 0.6% relative to intramuscular administration. However, the presence of an enhancer, polyacrylic acid gel, increased absorption from the rectum, producing a significant hypocalcemic response. For some forms of treatment, the rectal route could provide a more acceptable alternative to multiple injections. It is evident from the studies cited that peptide and protein delivery is strongly influenced by numerous formulation factors and does require the careful use of enhancing agents. However, this mode of delivery shares many problems with the oral and nasal route. It is difficult to predict widespread use of peptide and protein formulations suitable for rectal delivery [163,167–170].

12.4.3 Advantages and Disadvantages of Peptide and Protein Drug Delivery Through the Rectal Route

The transition from columnar to stratified epithelium in the rectum permits rapid absorption of low-molecular-weight proteins and peptide. The rectal site provides potential for absorption into the lymphatic system, which may be due to large pore radii in the rectum. The rectum allows retaining a large volume of formulation at the site of application (10–25 ml). The potential for time-controlled release and repro-ducible absorption is higher at the rectum due to constant environment at the site of application.

Specifically, the advantages for rectal delivery of peptides and proteins are sum-marized here: the rectal route has low levels of pancreatic-originated protease activ-ity. The rectal route provides a large surface area for absorption that is potentiated by using spreading/foaming agents in the formulation. The rectal route avoids the first-pass metabolism of proteins and peptides because middle and inferior rectal veins drain into the inferior vena cava. The disadvantages for rectal delivery of drugs include poor or erratic absorption across the rectal mucosa of many drugs and a limiting absorbable surface area. Slow dissolution of the formulation is due to the small fluid content in the rectum.


12.4.4 Approaches to Improve Rectal Absorption of Proteins and Peptides

12.4.4.1 Absorption Enhancers

Extensive studies have been conducted related to rectal absorption of peptides and proteins. Nevertheless, in the absence of an absorption-promoting adjuvant, the rec-tal absorption of these drugs is much less than for intramuscular, intravenous, or sub-cutaneous administration. Incomplete absorption is probably due to a combination of poor membrane permeability and metabolism at the absorption site. Thus, a number of absorption enhancers have been utilized for improving rectal absorption of larger polypeptides and proteins. Examples of various peptides and proteins delivered through the rectal route along with their respective absorption enhancers are listed in Table 12.1. Many absorption enhancers were utilized to enhance the absorption of rebamipide [171], insulin [172,173], polypeptide [174], heparin [175], and interferon [166]. These absorption enhancers were adopted not only for the GI tract but also for other alternative routes such as nasal, buccal, ocular, pulmonary, vaginal, and rec-tal routes. There are many factors affecting the effectiveness of absorption enhanc-ers such as the physicochemical characteristics of the drugs, administration site, and species differences.

12.4.4.1.1 Absorption Enhancers and Their MechanismAmong the peptides and proteins, insulin is probably the most frequently studied pro-tein with respect to rectal absorption. Nishihata et al. examined the effect of salicy-lates on rectal absorption of insulin in dogs [172]. The absorption-promoting effect of sodium deoxycholic acid, sodium taurocholate, polycarbophil, and their combinations were also studied in diabetic rabbits with respect to rectal insulin suppositories [176]. It was found that sodium salicylate and 5-methoxysalicylate both increase the rectal absorption of insulin [174,177]. It was also found that bile salts and ethylenediamine

Table 12.1 Absorption Enhancer for Different Proteins and Peptides

Protein or Peptide Enhancer Reference

Human chorionic gonadotropin

-Cyclodextrin [170]

Insulin Nitric oxide donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP)

[167]

()-(E)-4-methyl-2-[(E)-hydroxyimino]-5-nitro-6-methoxy-3-hexenamide (NOR1)

[173]

()-N-[(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-hexen-1-yl]3-pyridine carboxamide (NOR4)Docosahexaenoic acid [168]Sodium laurate and acrylic hydrogel [169]

Polypeptides 5-Methoxysalicylate [174]Rebamipide Sodium laurate and taurine [171]


tetra-acetic acid (EDTA) may improve the in vitro rectal penetration of insulin in the albino rabbit. [178]. The absorption-promoting effect of sodium 5-methoxysalicylate was also observed in the rat with respect to rectal delivery of pentagastrin and gastrin [174]. Rectal bioavailability was quantified by direct comparison of pharmacological effect with intravenous dose response. Rectal absorption of des-enkephalin--endor-phin (DE--E) was enhanced by medium-chain glycerides and EDTA in conscious rats [179]. Coadministration of the absorption adjuvant greatly enhanced the rectal bioavailability of the model peptides [180]. Enamine derivatives of phenylglycine are also studied as an adjuvant for the rectal absorption of insulin [181]. Enamine derivatives have been shown to increase support absorption of insulin in the dog and rabbit [181–184]. Recently, the effect of various absorption enhancers was examined on the transport of insulin across the rectal membrane of albino rabbits by in vitro using the chamber method. Insulin could not cross the rectal mucosa without absorp-tion enhancers, but its transport was improved in the presence of various absorption enhancers. Among absorption enhancers, Na-glycocholate (Na-GC) was more effec-tive than Na-taurocholate (Na-TC), but less effective than Na-deoxycholate (Na-DC) and polyoxyethylene-9-lauryl ether (BL-9) in enhancing rectal transport of insu-lin [185]. The transport of YAGFM (d-Ala2 Met enkephalinamide) [119] was also enhanced by the addition of 1% Na-GC. Increasing the Na-GC concentrations further increased rectal insulin transport. Although EDTA at 0.01% and 0.1% did not affect rectal transport of insulin, it augmented the penetration enhancement effect of 1% Na-GC. Uchiyama et al. examined the transport of insulin across the colonic membranes in vitro using the chamber method in the presence of various absorption enhancers [173]. Insulin transport was enhanced by the addition of Na-DC, EDTA, n-lauryl- P-d-maltopyranoside (LM), and Na-caprate (Na-Cap), although other enhancers did not improve its transport across the colonic membrane. Insulin suppositories in rabbits are also studied [186], and rectal insulin suppositories have been studied for hypogly-cemic effect [187]. Various nonsteroidal antiinflammatory drugs (NSAIDs) have been studied for effect of drug absorption through the rectal mucosa [188,189]. Salicylate and other enhancers were found to increase rectal absorption of erythropoietin in rats [190]. In the same way, rectal bioavailability was shown to be enhanced by sodium 5-ethoxysalicylate [174]. Enhanced absorption was also shown by nonsurfactant adju-vants in rats [191] and by salicylates [192] for insulin, heparin, and dextran in rats. Thus, some of the absorption enhancers were effective for improving the absorption of insulin [193,194] (Table 12.2).

12.4.4.1.2 Various Absorption Enhancers for the Rectal RouteIn this section, we describe those factors that regulate the effectiveness of various absorption enhancers.

Cyclodextrins Cyclodextrins are known to alter various properties of drugs, phar-maceutical formulations, and biomembranes, resulting in enhancement and modula-tion of rectal drug absorption. Cyclodextrins, cyclic oligosaccharides consisting of several glucopyranose units, are host molecules, which form inclusion complexes. Hydrophobic cyclodextrins derivatives may modulate the release of drugs from the


vehicles. Hydrophilic cyclodextrins increase the bioavailability of drugs in different administration routes by increasing the solubility, dissolution rate, and wettability of poorly water-soluble drugs and by preventing the degradation or disposition of chem-ically unstable drugs in GI tracts, as well as during storage. It perturbs the membrane fluidity to lower the barrier function, which consequently enhances the absorption of drugs, including peptide and protein drugs, through the rectal mucosa. It also releases the included drug by competitive inclusion complex formation with third components (bile acid, cholesterol, lipids, etc.) and inhibits P-glycoprotein–mediated efflux of the drug from intestinal epithelial cells.

Cyclodextrins were found to enhance the permeability of proteins such as insu-lin, recombinant human granulocyte colony-stimulating factor (rhG-CSF) [195], and human chorionic gonadotropin (hCG) [170] through the rectal epithelium cells. With respect to insulin suppository, the absorption of insulin from the rectum of rabbits after the administration of hollow-type suppositories containing insulin and cyclodextrins significantly increased, with a marked decrease in the glucose levels observed. To expand the practical use of other enhancers, vital issues such as their safety and local irritation as well as variability of the efficacy should be tackled.

Surfactants Surfactants are capable of improving various pharmaceutical proper-ties, such as wettability, solubility, dissolution rate, and miscibility, and are useful for improving the rectal bioavailability of impermeable drugs across biomembranes, including p-aminobenzoic acid (PABA), sulfaguanidine, and proteins [196,197]. The enhancing effects of surfactants on rectal absorption of proteins such as insulin and calcitonin [163] in various formulations have been reported: BL-9 enhanced the rec-tal insulin absorption in suppository (witepsol W35) and solution [185]. Recently, the use of Tween 60 as an absorption enhancer for insulin suppository in rabbits was demonstrated [198]. The mechanisms through which surfactants enhance rec-tal absorption of poorly membrane permeable drugs are likely to be solubilization of membrane lipids, sequestration of Ca2, and protein release from rectal mucosa. Solvent drag effect in drug intestinal absorption was studied on drug and D2O absorption clearances [199].

Table 12.2 Absorption-Enhancing Systems Used in Rectal Drug Administration of Proteins and Peptides

Enhancer Protein or Peptide

Bile acids Heparin [175,180], insulin [178]Ethylenediamine tetra-acetic acid Des-enkephalin--endorphin [179]Enamine derivatives Lysozyme [180], insulin [181–184]Nonionic surfactants Calcitonin [163]Nitric oxide donor Insulin [167]Nonsteroidal antiinflammatory drugs Insulin [188,189]Salicylate, 5-methoxysalicylate Erythropoietin [190], heparin [191], dextran

[192], insulin [193,194]


Bile Acids Bile acids are surface-active compounds that consist of a facially amphi-philic steroid nucleus with a hydrophobic- side and a -hydrophilic side, which are known to enhance impermeable drugs in the rectal routes. The enhancing mecha-nisms of bile acids for rectal absorption seem to involve sequestration of Ca2, an increase in solvent drag and water channel, dissociation of protein oligomers, and solubilization of membrane lipids.

Enamine Derivatives Amino acid enamines (phenylalanine and phenylglycine) of -diketones (ethylacetoacetate) are known to be novel absorption enhancers in the rectal routes. The supporting effects of enamines on the rectal absorption of other macromolecules such as lysozyme [180], calcitonin, and heparin are also reported. On the other hand, enamine derivatives are somewhat labile in aqueous solution. They may have an absorption-enhancing effect with a very short duration, and may be more useful in prodrug development and as absorption enhancers on insulin [181–184,200].

Salicylate and its Derivatives Salicylate and salicylic acid enhance the rectal absorption of various drugs including macromolecules [172,190–193]. Fluorescence spectroscopy was used to demonstrate that the salicylate ion increases plasma mem-brane permeability by effecting the plasma membrane proteins rather than directly interacting with the membrane lipids. The action of salicylate ion on these vesicles could possibly promote nonabsorbable drug absorption in vivo [201,202]. However, we must pay particular attention to pharmacological activity of salicylate derivatives. The enhancing mechanisms of sodium salicylate may relate to the membrane per-turbation by interaction with membrane proteins, but not membrane lipids, and a decrease in intracellular glutathione levels [177,185,193].

Nonsteroidal Antiinflammatory Drugs NSAIDs such as indomethacin, phenylbuta-zone, diclofenac sodium, and aspirin have been reported to act as absorption enhanc-ers in the rectal route [188,189]. The enhancing effect of NSAIDs could be attributed to a change in membrane permeability of poorly permeable drugs, owing to the accu-mulation of the drugs in rectal mucosa [203].

Caprate and Fatty Acids The enhancing mechanisms of Na-C10 are proposed due to an increase in the intracellular calcium level, Ca2 sequestration, increase in pore size and solvent drag, and interaction with membrane proteins and lipids of these enhancers. Na-C10 has been successfully included in commercially available sup-positories [204–207].

Mixed Micelles Mixed micelles are composed of lipids and bile acids or surfactants. These micelles are known to enhance poorly absorbable drugs in the small intestine as well as the large intestine. It is believed that the mixed micelles encapsulate mol-ecules with a high degree of efficiency (90% encapsulation). These mixed micelles are extremely small in size (1–10 nm) and are smaller than the pores of the mem-branes in the GI tract. It is therefore believed that the extremely small size of mixed micelles helps encapsulated molecules penetrate efficiently through the mucosal membranes. The absorption of proteins and peptides is thought to be enhanced by the diffusion of large molecules entrapped in the mixed micellar form through the


aqueous pores and the cell structure perturbation of the tight paracellular junctions. Actually, there are only a few reports available about the use of mixed micelles in the rectal route.

EDTA and Egtazic Acid Strong chelating agents such as EDTA and egtazic acid have been employed for the study of tight junctions between rectal epithelial cells. In fact, these chelating agents increased the rectal absorption of insulin and DE--E [179,208]. It was revealed that EDTA activates protein kinase C by depletion of extracellular calcium through chelation, resulting in expansion of the paracellular route in CaCo2 cell monolayers [209–211].

Other Absorption Enhancers The main issue in using other absorption enhancers is local irritation and safety; as well, the variability of the efficacy should be verified. Saponin is known to be surface-active, and may be involved in enhancing effects. Nitric oxide donors NOR1 and NOR4 increase insulin absorption. Utoguchi et al. reported that the nitric oxide donor S-nitroso-N-acetyl-d,l-penicillamine (SNAP) induced a significant increase in the absorption of insulin and FITC-dextran (MW 4000) from the rabbit’s rectum [167,170]. The absorption-enhancing effect of SNAP was inhibited by simultaneous administration of the nitric oxide scavenger car-boxy-PTIO. Thus, nitric oxide donors act as powerful absorption enhancers. Other absorption enhancers such as diethylethoxy methylene malonate (DEEMM) [212], phosphate derivative [213], and glycerin ester [214] have been reported as well. Thus, a number of absorption enhancers have been extensively developed for their potential use.

12.4.4.1.3 Efficacy and Safety of Absorption EnhancersA large number of absorption enhancers, including surfactants, bile salts, chelating agents, and fatty acids, are used to improve the absorption of macromolecules like proteins and peptides. For practical use, it is essential that these enhancers do not affect the membrane integrity of the epithelium. However, some of these excipients lead to membrane damage and irritate the rectal mucosal membrane. Thus, nontoxic and effective enhancers should be urbanized.

12.4.4.2 Protease Inhibitors

The oral administration of peptides often results in very low bioavailability due to extensive hydrolysis of peptides by digestive enzymes of the GI tract (enzymatic barrier) and poor membrane penetration characteristics (transport barrier). Of these two barriers, the former is of great importance for certain unstable small peptides, because these peptides can be transported across the intestinal membrane unless they are degraded by various proteases enzymes. Because the use of protease inhibitors could reduce the degradation of various peptides and proteins, this is a promising approach for overcoming the delivery problems of peptides and proteins. Many com-pounds have been used as protease inhibitors to improve the stability of various pep-tides and proteins, for example, aprotinin, trypsin inhibitors, bacitracin, puromycin, bestatin, and bile salts such as Na-glycholate.


The effect of protease inhibitors on the stability of peptidesIt was found that the protease inhibitors may reduce the degradation of insulin

in the large intestinal homogenate, thereby improving large intestinal absorption of insulin. These findings suggest that coadministration of protease inhibitors would be useful for improving large intestinal absorption of insulin.

The effect of protease inhibitors on the intestinal absorption of peptidesInformation on the relative potential of proteolytic activity at different regions

along the GI tract is required for the use of a protease inhibitor as an absorption enhancer. The activities of various proteases in the small intestine are generally higher than those in the large intestine. This tendency is more remarkable in the case of endopeptidases such as trypsin, chymotrypsin, and elastase, because little activity of these enzymes is in the large intestine. Of all proteases, aminopeptidase B had a more uniform distribution pattern than the other proteases used.

Absorption-enhancing mechanisms of protease inhibitorsProtease inhibitors are one of the most useful approaches to improve the stability

and absorption of peptides and proteins, although the mechanism by which peptides and proteins are stabilized by these inhibitors is not fully understood. Because some protease inhibitors may also have absorption-enhancing activities in addition to their protease inhibitory action, when they are applied in practical use, it is essential that these inhibitors do not affect the membrane integrity of the epithelium.

12.4.4.3 Chemical Modification of Protein and Peptide Drugs

Alternative methods are needed for peptide delivery via the GI tract because absorp-tion enhancers and protease inhibitors improve the absorption of usually nonabsorbed substances from the GI tract. Normally, peptides and proteins have been coadminis-tered with absorption enhancers and protease inhibitors in order to promote the passage through GI epithelial barriers and to reduce degradation in the gut [215]. But limita-tions, for instance, local irritation of the mucosa and nonselective absorption of other antigenic compounds, are considered a negative aspect in the use of absorption enhanc-ers. A potentially useful approach to solve these delivery problems may be chemical modification of peptides and proteins to produce analogues and prodrugs. So, it is likely that this chemical approach may protect peptides against degradation by the pres-ence of peptidases and other enzymes at the mucosal barrier, leaving the peptides and proteins more lipophilic, resulting in augmented permeability. From these standpoints, some novel lipophilic derivatives of TRH, tetragastrin, human calcitonin, insulin, and lysozyme can be synthesized through chemical modification by various fatty acids.

12.5 Conclusion

Various routes have been explored to achieve therapeutic concentration, such as trans-dermal, topical, uterine, and rectal. Low oral bioavailability is often recorded for pro-teins and peptides because of some limiting factors. Development of a transdermal


delivery technique for the controlled administration of proteins and peptides could potentially yield one or more biomedical benefits, such as less variation in absorp-tion and metabolism; avoidance of risks and inconveniences; continuous drug deliv-ery for proteins and peptides normally having a short biological half-life; bypassing hepatic first-pass metabolism; prolonged or preprogrammed delivery of the drug at the required therapeutic rate; rapid termination of the medication, if needed; and a simplified therapeutic regimen, leading to better patient compliance. For conven-tional drugs, topical products to treat dermatological ailments have been in continua-tion from the start. For topical delivery of proteins and peptides, the drug must reach local site skin ailments. Many topical drugs are proteins or peptides; one prominent example is growth hormone, which has been discussed. Delivery systems like lipo-somes are used for topical delivery of these drugs.

The vagina is a complex genital organ with multiple functions. First-pass effects with local and hepatic proteases are almost avoided by using the uterine route. However, bioavailability is less and fluctuates, so absorption promoters are required. In summation, the uterine is a possible site for the systemic administration of pep-tides and proteins. However, variations in absorption resulting from cyclic changes in the reproductive organs throughout the life cycle must be taken into account in developing new uterine delivery systems.

There are numerous examples of effective delivery of peptide and protein drugs using the rectal route of administration. Use of an adjuvant to enhance the absorption of peptides and proteins in order to obtain adequate absorption is needed for practical use. This is true for larger molecules having relatively high aqueous solubility. Overall, for a number of situations, the rectal route of administration has advantages over oral and parenteral routes of administration for protein and peptide delivery. Rectal drug delivery has emerged as a possible alternative for administering a number of protein and polypeptide drugs and the method of choice for some proteins and peptides.

Other routes like transdermal, topical, rectal, and uterine are discussed. These are used for possible protein and peptide delivery, taking into account various aspects like local biological environment, use of various formulation excipients like absorp-tion promoters, advantages and disadvantages of each route, and inherent properties of the proteins or peptide.

Acknowledgment

The authors acknowledge the financial assistance from TIFAC CORE in Novel Drug Delivery System (NDDS), Department of Science and Technology, Government of India, New Delhi, for providing research facilities to the team.

References

[1] Flynn GL. Developmental issues and research initiatives. In: Hadgraft J, Guy RH, editors. Transdermal drug delivery. New York, NY: Marcel Dekker; 1989. p. 59–81.


[2] Wearley LL. Recent progress in protein and peptide delivery by non invasive routes. Crit Rev Ther Drug Carrier Syst 1991;8:331–94.

[3] Menon GK, Grayson S, Elias PM. Cytochemical and biochemical localization of lipase and sphingomyelinase activity in mammalian epidermis. J Invest Dermatol Symp Proc 1986;86:591–7.

[4] Williams ML, Elias PM. The extracellular matrix of stratum corneum: role of lipids in normal and pathological function. Crit Rev Ther Drug Carrier Syst 1987;3:95–122.

[5] Rastogi SM, Singh J. Lipid extraction and transport of hydrophilic solutes through por-cine epidermis. Int J Pharm 2001;225:75–82.

[6] Flynn GL. Mechanism of percutaneous absorption from physicochemical evidence. In: Bronaugh RL, Maibach HI, editors. Percutaneous absorption: mechanisms–methodol-ogy–drug delivery. New York: Marcel Dekker; 1989. p. 27–51.

[7] Bronaugh RL. Determination of percutaneous absorption by in vitro techniques. In: Bronaugh RL, Maibach HI, editors. Percutaneous absorption: mechanisms–methodology– drug delivery. New York, NY: Marcel Dekker; 1989. p. 239–58.

[8] Auer D, Brandner G, Bodemer W. Dielectric breakdown of the red blood cell membrane and uptake of SV 40 DNA and mammalian cell RNA. Naturwissenschaften 1976;63:391.

[9] Kinosita K Jr, Tsong TY. Formation and resealing of pores of controlled sizes in human erythrocyte membrane. Nature 1977;268:438–41.

[10] Prausnitz MR, Bose VG, Langer R, Weaver JC. Electroporation of mammalian skin: a mechanism to enhance transdermal drug delivery. Proc Natl Acad Sci USA 1993;90: 10504–8.

[11] Zewert TE, Pliquett UF, Langer R, Weaver JC. Transdermal transport of DNA antisense oligonucleotides by elelctroporation. Biochem Biophys Res Commun 1995;212:286–92.

[12] Chang SL, Hofman GA, Zhang L, Deftos LJ, Banga AK. The effect of electroporation on iontophoretic transdermal delivery of calcium regulating hormones. J Control Release 2000;66:127–33.

[13] Pliquett U. Mechanistic studies of molecular transdermal transport due to skin electro-poration. Adv Drug Deliv Rev 1999;35:41–60.

[14] Prausnitz MR, Edelman ER, Gimm JA, Langer R, et al. Transdermal delivery of heparin by skin electroporation. Nat Biotechnol 1995;13:1205–9.

[15] Brand RM, Wahl A, Iverson PL. Effects of size and sequence on the iontophoretic deliv-ery of oligonucleotides. J Pharm Sci 1998;87:49–52.

[16] Zhang L, Hoffman GA. Electric pulse mediated transdermal drug delivery. Proc Int Symp Control Release Bioact Mater 1997;24:27–8.

[17] Pliquett UF, Zewert TE, Chen T, Langer R, Weaver JC. Imaging of fluorescent molecules and small ion transport through human stratum corneum during high voltage pulsing: localized transport regions are involved. Biophys Chem 1996;58:185–204.

[18] Prausnitz MR, Lee CS, Liu CH, Pang JC, Singh TP, Langer R, et al. Transdermal transport efficiency during skin electroporation and iontophoresis. J Control Release 1996;38:205–17.

[19] Higuchi WI, Li SK, Ghanem AH, Zhu H, Song Y. Mechanistic aspects of iontophoresis in human epidermal membrane. J Control Release 1999;62:13–23.

[20] Vanbever R, Lecoutturier N, Preat V. Transdermal delivery of metoprolol by electropora-tion. Pharm Res 1994;11:1657–62.

[21] Vanbever R, LE Boulengé E, Preat V. Transdermal delivery of fentanyl by electropora-tion. I. Influence of electrical factors. Pharm Res 1996;13:559–65.

[22] Vanbever R, Leroy M, Preat V. Transdermal penetration of neutral molecules by skin electroporation. J Control Release 1998;54:243–50.


[23] Weaver JC, Vaughan TE, Chizmadzhev Y. Theory of electrical creation of aqueous path-ways across skin transport barriers. Adv Drug Deliv Rev 1999;35:21–39.

[24] Jadoul A, Bouwstra J, Preat V. Effect of iontophoresis and electroporation on the stratum corneum. Adv Drug Deliv Rev 1999;35:89–105.

[25] Prausnitz MR. A practical assessment of transdermal drug delivery by skin electropora-tion. Adv Drug Deliv Rev 1999;35:61–7.

[26] Chizmadzhev YA, Zarnitsin VG, Weaver JC, Potts RO. Mechanism of electroinduced ionic species transport through a multilamellar lipid system. Biophys J 1995;68:749–65.

[27] Pliquett UF, Gusbeth CA. Perturbation of human skin due to application of high voltages. Bioelectrochemistry 2000;51:41–51.

[28] Pliquett UF, Vanbever R, Preat V, Weaver JC. Local transport regions (LTRs) in human stratum corneum due to long and short “high voltage” pulses. Bioelectrochem Bioenerg 1998;47:151–61.

[29] Pliquett UF, Weaver JC. Electroporation of human skin: simultaneous measurement of changes in the transport of two fluorescent molecules and in the passive electrical prop-erties. Bioelectrochem Bioenerg 1996;39:1–12.

[30] Preat V, Vanbever R. In vivo efficacy and safety of skin electroporation. Adv Drug Deliv Rev 1999;35:77–88.

[31] Heller R, Gilbert R, Jaroszeski MJ. Clinical applications of electrochemotherapy. Adv Drug Deliv Rev 1999;35:119–29.

[32] Denet A, Préat V. Transdermal delivery of timolol by electroporation through human skin. J Control Release 2003;88:253–62.

[33] Green PG. Iontophoretic delivery of peptide drugs. J Control Release 1996;41:33–48. [34] Burnette RR, Ongpipattanakul B. Characterization of the permselective properties of

excised human skin during iontophoresis. J Pharm Sci 1987;76:765–73. [35] Guy RH, Kalia YN, Delgado-Charro MB, Merino V, Lopez A, Marro D. Iontophoresis:

electrorepulsion and electroosmosis. J Control Release 2000;64:129–32. [36] Green PG, Hinz RS, Kim A, Szoka Jr FC, Guy RH. Iontophoretic delivery of a series of

tripeptides across the skin in-vitro. Pharm Res 1991;8:1121–7. [37] Oldenburg K, Vo KT, Smith GA, Selick HE. Iontophoretic delivery of oligonucleotides

across full thickness hairless mouse skin. J Pharm Sci 1995;84:915–21. [38] Heit MC, McFarland A, Bock R, Riviere JE. Isoelectric focusing and capillary zone elec-

trophoretic studies using luteinizing hormone releasing hormone and its analog. J Pharm Sci 1994;83:654–6.

[39] Hirvonen J, Guy RH. Transdermal iontophoresis: modulation of electroosmosis by poly-peptides. J Control Release 1998;50:283–89.

[40] Hoogstraate AJ, Srinivasan V, Sims SM, Higuchi WI. Iontophoretic enhancement of pep-tides: behaviour of leuprolide versus model permeants. J Control Release 1994;31:41–7.

[41] Delgadocharro MB, Rodriguezbayon AM, Guy RH. Iontophoresis of nafarelin: effects of current density and concentration on electrotransport in vitro. J Control Release 1995;35:35–40.

[42] Kalia YN, Naik A, Garrison J, Guy RH. Iontophoretic drug delivery. Adv Drug Deliv Rev 2004;56:619–58.

[43] Lattin GA, Padmanabhan RV, Phis JB. Electronic control of iontophoretic drug delivery. Ann N Y Acad Sci 1991;618:450–64.

[44] Pikal MJ. The role of electroosmotic flow in transdermal iontophoresis. Adv Drug Deliv Rev 1992;9:201–37.

[45] Santi P, Guy RH. Reverse iontophoresis—parameters determining electroosmotic flow. I. PH and ionic strength. J Control Release 1996;38:159–65.


[46] Sarpotdar PP, Daniels CR. Use of polymeric buffers to facilitate iontophoretic transport of drugs. Pharm Res 1990;7:(Suppl.)S185.

[47] Santi P, Colombo P, Bettini R, Catellani PL, Minutello A, Volpato NM. Drug reservoir composition and transport of salmon calcitonin in transdermal iontophoresis. Pharm Res 1997;14:63–6.

[48] Coderch L, Oliva M, Pons L, Parra JL. Percutaneous penetration in vivo of amino acids. Int J Pharm 1994;111:7–14.

[49] Wearley LL, Tojo K, Chien YW. A numerical approach to study the effect of binding on the iontophoretic transport of a series of amino acids. J Pharm Sci 1990;79:992–8.

[50] Green PG, Hinz RS, Cullander C, Yamane G, Guy RH. Iontophoretic delivery of amino acids and amino acid derivatives across the skin in vitro. Pharm Res 1991;8:1113–20.

[51] Burnette RR, Marrero D. Comparison between the iontophoretic and passive transport of thyrotropin releasing hormone across excised nude mouse skin. J Pharm Sci 1986;75: 738–43.

[52] Magnusson BM, Runn P, Karlsson K, Koskinen LOD. Terpenes and ethanol enhance the transdermal permeation of the tripeptide thyrotropin releasing hormone in human epider-mis. Int J Pharm 1997;157:113–21.

[53] Green P, Shroot B, Bernerd F, Pilgrim WR, Guy RH. In vitro and in vivo iontophoresis of a tripeptide across nude rat skin. J Control Release 1992;20:209–17.

[54] Gupta SK, Kumar S, Bolton S, Behl CR, Malick AW. Optimization of iontophoretic trans-dermal delivery of a peptide and a non-peptide drug. J Control Release 1994;30:253–61.

[55] Ruland A, Rohr U, Kreuter J. Transdermal delivery of the tetrapeptide hisetal (melanotro-pin (6–9) and amino acids: their contribution to the elucidation of the existence of an “aqueous pore” pathway. Int J Pharm 1994;107:23–8.

[56] Heit MC, Monteiroriviere NA, Jayes FL, Riviere JE. Transdermal iontophoretic deliv-ery of luteinizing hormone releasing hormone (LHRH): effect of repeated administration. Pharm Res 1994;11:1000–3.

[57] Meyer BR, Kreis W, Eschbach J. Successful transdermal administration of thera-peutic doses of a polypeptide to normal human volunteers. Clin Pharmacol Ther Ser 1988;44:607–12.

[58] Lelawongs P, Liu J, Chien YW. Transdermal iontophoretic delivery of arginine-vasopres-sin (II): evaluation of electrical and operational factors. Int J Pharm 1990;61:179–88.

[59] Craane-van-hinsberg WHM, Bax L, Flinterman NHM, Verhoef J, Junginger HE, et al. Iontophoresis of a model peptide across human skin in vitro: effects of iontophoresis pro-tocol, pH, and ionic strength on peptide flux and skin impedance. Pharm Res 1994;11: 1296–300.

[60] Banga AK, Katakam M, Mitra R. Transdermal iontophoretic delivery and degradation of vasopressin across human cadaver skin. Int J Pharm 1995;116:211–16.

[61] Lelawongs P, Liu J, Siddiqui O, Chien YW. Transdermal iontophoretic delivery of argi-nine-vasopressin (I): physicochemical considerations. Int J Pharm 1989;56:13–22.

[62] Knoblauch P, Moll F. In vitro pulsatile and continuous transdermal delivery of buserelin by iontophoresis. J Control Release 1993;26:203–12.

[63] Morimoto K, Iwakura Y, Nakatani E, Miyazaki M, Tojima H. Effects of proteolytic enzyme inhibitors as absorption enhancers on the transdermal iontophoretic delivery of calcitonin in rats. J Pharm Pharmacol 1992;44:216–18.

[64] Chang S, Hofmann GA, Zhang L, Deftos LJ, Banga AK. Transdermal iontophoretic delivery of salmon calcitonin. Int J Pharm 2000;200:107–13.

[65] Thysman S, Hanchard C, Preat V. Human calcitonin delivery in rats by iontophoresis. J Pharm Pharmacol 1994;46:725–30.


[66] Kumar S, Char H, Patel S, Piemontese D, Iqbal K, Malick AW, et al. Effect of iontopho-resis on in vitro skin permeation of an analogue of growth hormone releasing factor in the hairless guinea pig model. J Pharm Sci 1992;81:635–9.

[67] Stephen RL, Petelenz TJ, Jacobsen SC. Potential novel methods for insulin administra-tion: I. Iontophoresis. Biomed Biochim Acta 1984;43:553–8.

[68] Kari B. Control of blood glucose levels in alloxan-diabetic rabbits by iontophoresis of insulin. J Am Diabetes Assoc 1986;35:217–21.

[69] Meyer BR, Katzeff HL, Eschbach JC, Trimmer J, Zacharias SB, Rosen S, et al. Transdermal delivery of human insulin to albino rabbits using electrical current. Am J Med Sci 1989;297:321–5.

[70] Siddiqui O, Sun Y, Liu JC, Chien YW. Facilitated transdermal transport of insulin. J Pharm Sci 1987;76:341–5.

[71] Liu JC, Sun Y, Siddiqui O, Chien YW. Blood glucose control in diabetic rats by transder-mal iontophoretic delivery of insulin. Int J Pharm 1988;44:197–204.

[72] Srinivasan V, Higuchi WI, Sims SM, Ghanem AH, Behl CR. Transdermal iontophoretic drug delivery: mechanistic analysis and application to polypeptide delivery. J Pharm Sci 1989;78:370–5.

[73] Sun Y, Xue H. Transdermal delivery of insulin by iontophoresis. Ann N Y Acad Sci 1991;618:596–8.

[74] Zakzewski CA, Wasilewski J, Cawley P, Ford W. Transdermal delivery of regular insulin to chronic diabetic rats: effect of skin preparation and electrical enhancement. J Control Release 1998;50:267–72.

[75] Kanikkannan N, Singh J, Ramarao P. Transdermal iontophoretic delivery of bovine insu-lin and monomeric human insulin analogue. J Control Release 1999;59:99–105.

[76] Rastogi SK, Singh J. Passive and iontophoretic transport enhancement of insulin through porcine epidermis by depilatories: permeability and Fourier transform infrared spectros-copy studies. AAPS PharmSciTech 2003;4:29.

[77] Langkjaer L, Brange J, Grodsky GM, Guy RH. Iontophoresis of monomeric insulin analogues in vitro: effects of insulin charge and skin pretreatment. J Control Release 1998;51:47–56.

[78] Clancy MJ, Corish J, Corrigan OI. A comparison of the effects of electrical current and penetration enhancers on the properties of human skin using spectroscopic (FTIR) and calorimetric (DSC) methods. Int J Pharm 1994;105:47–56.

[79] Riviere JE, Moteiro-Riviere NA, Rogers RA, Bommannan D, Tamada JA, Potts RO. Pulsatile transdermal delivery of LHRH using electroporation: drug delivery and skin toxicology. J Control Release 1995;36:229–33.

[80] Denet AR, Ucakar B, Preat V. Transdermal delivery of timolol and atenolol using electro-poration and iontophoresis in combination: a mechanistic approach. Pharm Res 2003;20: 1946–51.

[81] Prausnitz MR. Overcoming skin’s barrier: the search for effective and user friendly drug delivery. Diabetes Technol Ther 2001;3:233–6.

[82] Prausnitz MR. Microneedles for transdermal drug delivery. Adv Drug Deliv Rev 2004;56:581–7.

[83] Bramson J, Dayball K, Evelegh C, Wan YH, Page D, Smith A. Enabling topical immuni-zation via microporation: a novel method for pain-free and needle-free delivery of adeno-virus-based vaccines. Gene Ther 2003;10:251–60.

[84] Levy D, Kost J, Meshulam Y, Langer R. Effect of ultrasound on transdermal drug deliv-ery to rats and guinea pigs. J Clin Invest 1989;83:2074–8.

[85] Tyle P, Agrawala P. Drug delivery by phonophoresis. Pharm Res 1989;6:355–61.


[86] Sanghvi P, Banakar UV. Ultrasonics: principles and biomedical applications. BioPharm Int 1991;4:32–40.

[87] Machluf M, Kost J. Ultrasonically enhanced transdermal drug delivery experimental approaches to elucidate the mechanism. J Biomater Sci Polym Ed 1993;5:147–56.

[88] Kendall M, Mitchell T, Wrighton-Smith P. Intradermal ballistic delivery of microparti-cles into excised human skin for pharmaceutical applications. J Appl Biomater Biomech 2004;37:1733–41.

[89] Le L, Kost J, Mitragotri S. Combined effect of low-frequency ultrasound and iontopho-resis: applications for transdermal heparin delivery. Pharm Res 2000;17:1151–4.

[90] Tokumoto S, Higo N, Sugibayashi K. Effect of electroporation and pH on the iontopho-retic transdermal delivery of human insulin. Int J Pharm 2006;326:13–19.

[91] Murthy SN, Zhao YL, Hui SW, Sen A. Synergistic effect of anionic lipid enhancer and electroosmosis for transcutaneous delivery of insulin. Int J Pharm 2006;326:1–6.

[92] Pillai O, Panchagnula R. Transdermal delivery of insulin from poloxamer gel: ex vivo and in vivo skin permeation studies in rat using iontophoresis and chemical enhancers. J Control Release 2003;89:127–40.

[93] Lin W, Cormier M, Samiee A, Griffin A, Johnson B, Teng CL, et al. Transdermal deliv-ery of antisense oligonucleotides with microprojection patches (Macroflux) technology. Pharm Res 2001;18:1789–93.

[94] Jaskari T, Vuorio M, Kontturi K, Urtti A, Manzanares JA, Hirvonen J. Controlled trans-dermal iontophoresis by ion-exchange fiber. J Control Release 2000;67:179–90.

[95] Jaskari T, Vuorio M, Kontturi K, Manzanares JA, Hirvonen J. Ion-exchange fibers and drugs: an equilibrium study. J Control Release 2001;70:219–29.

[96] Hanninen K, Kaukonen AM, Kankkunen T, Hirvonen J. Rate and extent of ion-exchange process: the effect of physico-chemical characteristics of salicylate anions. J Control Release 2003;91:449–63.

[97] Vuorio M, Manzanares JA, Murtomaki L, Hirvonen J, Kankkunen T, Kontturi K. Ion-exchange fibers and drugs: a transient study. J Control Release 2003;91:439–48.

[98] Kankkunen T, Huupponen I, Lahtinen K, Sundell M, Ekman K, Kontturi K, et al. Improved stability and release control of levodopa and metaraminol using ion-exchange fibers and transdermal iontophoresis. Eur J Pharm Sci 2002;16:273–80.

[99] Kankkunen T, Sulkava R, Vuorio M, Kontturi K, Hirvonen J. Transdermal iontophoresis of tacrine in vivo. Pharm Res 2002;19:704–7.

[100] Lira LM, Córdoba de Torresi SI. Conducting polymer–hydrogel composites for electro-chemical release devices: synthesis and characterization of semi-interpenetrating poly-aniline–polyacrylamide networks. Electrochem Commun 2005;7:717–23.

[101] Conaghey OM, Corish J, Corrigan OI. Iontophoretically assisted in vitro membrane transport of nicotine from a hydrogel containing ion exchange resins. Int J Pharm 1998;170:225–37.

[102] Ghosh TK, Banga AK. Methods of enhancement of transdermal drug delivery: part I, physical and biochemical approaches. Pharm Technol 1993;17:72–98.

[103] Moss J, Bundgaard H. Prodrugs of peptides. 7. Transdermal delivery of thyrotropin-releasing hormone (TRH) via prodrugs. Int J Pharm 1990;66:39–45.

[104] Kahn M. Peptide secondary structure mimetics: recent advantages and future chal-lenges. Synlett 1993;11:821–6.

[105] Hruby VJ. Conformational and topographical considerations in the design of biologi-cally active peptide ligands. Biopolymers 1993;33:1073–82.

[106] Luthman K, Hacksell U. Peptides and peptidomimetics. In: Krogsgaard-Larsen P, Liljefors T, Madsen U, editors. A textbook of drug design and development. Amsterdam: Harwood Academic Publishers; 1996. p. 386–406.


[107] Sawyer TK. Peptidomimetic design and chemical approaches to peptide metabolism. In: Taylor MD, Amidon GL, editors. Peptide based drug design: controlling transport and metabolism. Washington, DC: American Chemical Society; 1995. p. 387–422.

[108] Friis GJ, Bundgaard H. Design and application of prodrugs. In: Krogsgaard-Larsen P, Liljefors T, Madsen U, editors. A textbook of drug design and development. Amsterdam: Harwood Academic Publishers; 1996. p. 351–85.

[109] Taylor MD. Improved passive oral drug delivery via prodrugs. Adv Drug Deliv Rev 1996;19:131–48.

[110] Bundgaard H. Means to enhance penetration: prodrugs as a means to improve the deliv-ery of peptide drugs. Adv Drug Deliv Rev 1992;8:1–38.

[111] Oliyai R. Prodrugs of peptides and peptidomimetics for improved formulation and delivery. Adv Drug Deliv Rev 1996;19:275–86.

[112] Bommannan D, Potts RO, Guy RH. Examination of the effect of ethanol on human stra-tum corneum in vivo using infrared spectroscopy. J Control Release 1991;16:299–304.

[113] Spatola AF. Peptide backbone modifications: a structure-activity analysis of peptides containing amide bond surrogates, conformational constraints, and related backbone replacements. In: Weinstein B, editor. Chemistry and biochemistry of amino acids, pep-tides, and proteins, vol. III. New York, NY: Marcel Dekker; 1983. p. 287–300.

[114] Christrup LL, Møss J, Steffansen B. Prodrugs, preservation of pharmaceutical products to salt forms of drugs and absorption. In: Swarbrick J, Boylan JC, editors. Encyclopedia of pharmaceutical technology. New York, NY: Marcel Dekker; 1996. p. 39–70.

[115] Barry BW. Mode of action of penetration enhancers in human skin. J Control Release 1987;6:85–97.

[116] Barry BW. Lipid–protein-partitioning theory of skin penetration enhancement. J Control Release 1991;15:237–48.

[117] Walters KA. Penetration enhancers and their use in transdermal therapeutic systems. In: Hadgraft J, Guy RH, editors. Transdermal drug delivery: developmental issues and research initiatives. New York, NY: Marcel Dekker; 1989. p. 197–246.

[118] Pfister WR, Hsieh DST. Permeation enhancers compatible with transdermal drug deliv-ery systems: part II: system design considerations. Med Device Technol 1990;1:28–33.

[119] Choi H, Flynn GL, Amidon GL. Transdermal delivery of bioactive peptides: the effect of n-decylmethyl sulfoxide, pH, and inhibitors on enkephalin metabolism and transport. Pharm Res 1990;7:1099–106.

[120] Chiang CH, Shao CH, Chen JL. Effects of pH, electric current, and enzyme inhibitors on iontophoresis of delta sleep-inducing peptide. Drug Dev Ind Pharm 1998;24:431–8.

[121] Down JA, Harvey NG. Transdermal drug delivery. In: Guy RH, Hadgraft J, editors. Minimally invasive systems for transdermal drug delivery, vol. 123. New York, NY: Marcel Dekker; 2003. p. 327–60.

[122] Eppstein JA, Delcher HK, Hatch MR, McRae MS, Papp J, Woods TJ. Insulin infu-sion via thermal micropores. Proc Int Symp Control Release Bioact Mater 2000;27: 1010–11.

[123] Svedman P, Lundin S, Svedman C. Administration of antidiuretic peptide (DDAVP) by way of suction de-epithelialised skin. Lancet 1991;337:1506–9.

[124] Sarphie DF, Johnson B, Cormier M, Burkoth TL, Bellhouse BJ. Bioavailability follow-ing transdermal powdered delivery (TPD) of radio labelled insulin to hairless guinea pigs. J Control Release 1997;47:61–9.

[125] Banga A. Transdermal and topical delivery of therapeutic peptide and protein. In: Therapeutic peptides and proteins: formulation, processing and delivery systems. 2nd ed. Florida: Taylor & Francis; 2006 p. 259–81.


[126] Lin WW, Pulkkinen MT, Vitto L, Yoon KJ. The gene gun: current applications in cuta-neous gene therapy. Int J Dermatol 2000;39:161–70.

[127] Lee W, Pan T, Wang P, Zhuo R, Huang C, Fang J-Y. Erbium: YAG laser enhanced trans-dermal peptide delivery and skin vaccination. J Control Release 2008;128:200–8.

[128] Lenz GR, Mansson PE. Growth factors as pharmaceuticals. Pharm Technol 1991;15: 34–40.

[129] Papanas N, Maltezos E. Growth factors in the treatment of diabetic foot ulcers: new technologies, any promises? Int J Low Extrem Wounds 2007;6:37–53.

[130] Celebi N, Erden N, Gonul B, Koz M. Effects of epidermal growth factor dosage forms on dermal wound strength in mice. J Pharm Pharmacol 1994;46:386–7.

[131] Okumura K, Kiyohara Y, Komada F, Iwakawa S, Hirai M, Fuwa T. Improvement in wound healing by epidermal growth factor (EGF) ointment. I. Effect of nafamostat, gabexate, or gelatin on stabilization and efficacy of EGF. Pharm Res 1990;7:1289–93.

[132] Kiyohara Y, Komada F, Iwakawa S, Fuwa T, Okumura K. Systemic effects of epidermal growth factor (EGF) ointment containing protease inhibitor or gelatin in rats with burns or open wounds. Biol Pharm Bull 1993;16:73–6.

[133] Matuszewska B, Keogan M, Fisher DM, Soper KA, Hoe C, Huber AC, et al. Acidic fibroblast growth factor: evaluation of topical formulations in a diabetic mouse wound healing model. Pharm Res 1994;11:65–71.

[134] Hsu CC, Nguyen HM, Wu SS. Reconstitutable lyophilized protein formulation. US Patent, 5,192,743. 1993. March 9.

[135] Mezei M, Gulasekharam V. Liposomes—a selective drug delivery system for the topical route of administration: gel dosage form. J Pharm Pharmacol 1982;34:473–4.

[136] Ganesan MG, Weiner ND, Flynn GL, Ho NFH. Influence of liposomal drug entrapment on percutaneous absorption. Int J Pharm 1984;20:139–54.

[137] Mezei M. Liposomes as a skin drug delivery system. In: Breimer DD, Speiser P, editors. Topics in pharmaceutical sciences. Amsterdam: Elsevier Science Publishers; 1985. p. 345–58.

[138] Martin GP. Phospholipids as skin penetration enhancers. In: Walters KA, Hadgraft J, editors. Pharmaceutical skin penetration enhancement. New York, NY: Marcel Dekker; 1993. p. 57–60.

[139] Duplessis J, Ramachandran C, Weiner N, Muller DG. The influence of particle size of liposomes on the deposition of drug into skin. Int J Pharm 1994;103:277–82.

[140] Schreier H, Bouwstra J. Liposomes and niosomes as topical drug carriers dermal and transdermal drug delivery. J Control Release 1994;30:1–15.

[141] Siciliano AA. Topical liposomes—an update and review of uses and production meth-ods. Cosmetics Toiletries 1985;100:43–6.

[142] Egbaria K, Ramachandran C, Kittayanond D, Weiner N. Topical delivery of liposo-mally encapsulated interferon evaluated by in vitro diffusion studies. Antimicrob Agents Chemother 1990;34:107–10.

[143] Bogner RH, Banga AK. Iontophoresis and phonophoresis. US Pharm 1994;19:H10–26. [144] Golomb G, Avramoff A, Hoffman A. A new route of drug administration: intrauterine

administration of insulin and calcitonin for systemic effect. Pharm Res 1993;10:828–33. [145] Hoffman A, Nyska A, Avramoff A, Golomb G. Continues versus pulsatile administration

of erythropoietin (EPO) via the uterus in anemic rats. Int J Pharm 1994;111:197–202. [146] Katz DF, Dunmire EN. Cervical mucus—problems and opportunities for drug delivery

via the vagina and cervix. Adv Drug Deliv Rev 1993;11:385–401. [147] Reardon PM, Gochoco CH, Audus KL, Wilson G, Smith PL. In vitro nasal transport

across ovine mucosa: effects of ammonium glycyrrhizinate on electrical properties and


permeability of growth hormone releasing peptide, mannitol, and lucifer yellow. Pharm Res 1993;10:553–61.

[148] Golomb G, Shaked I, Hoffman A. Intrauterine administration of peptide drugs for sys-temic effect. Adv Drug Deliv Rev 1995;17:179–90.

[149] Hoffman A, Shaked I, Avramoff A, Golomb G. Pharmacokinetics and pharmacodynam-ics of trans-endometrial administered peptides and macromolecules. Adv Drug Deliv Rev 1995;17:191–203.

[150] Sumner DR, Turner TM, Purchio AF, Gombotz WR, Urban RM, Galante JO. Enhancement of bone in growth by transforming growth factor-beta. J Bone Joint Surg 1995;77: 1135–47.

[151] Matuszewska B, Keogan M, Fisher DM, Soper KA, Hoe C-MA, et al. Acidic fibroblast growth factor: evaluation of topical formulations in a diabetic mouse wound healing model. Pharm Res 1994;11:65–71.

[152] Shimizu K, Nishikawa T, Nozaki M, Oshima K. Effects of an intrauterine copper device on serum copper, endometrial histology and ovarian, hepatic, and renal functions in the Japanese monkey (Macaca fuscata fuscata). J Med Primatol 1991;20:277–83.

[153] Chi IC. The Nova-T IUD—a review of the literature. Contraception 1991;44:341–66. [154] World Health Organization (WHO). The IUD—worth singing about. WHO Progress in

Reproductive Health Research 2002;60:1–8. [155] Susan R, Chaudhari A, Peterson C. Mirena® (Levonorgestrel intrauterine system): a suc-

cessful novel drug delivery option in contraception. Adv Drug Deliv Rev 2009;61:808–12. [156] Smith B, Uhl K. Drug delivery in twenty-first century: a new paradigm. Clin Pharmacol

Ther 2009;85:451–5. [157] Van Os W, Edelman D. New directions in IUCD development. Adv Contracept

1998;14:41–4. [158] Wildemeersch D, Batár I, Webb A, Gbolade BA, Delbarge W, Temmerman M, et al.

GyneFIX. The frameless intrauterine contraceptive implant—an update for interval, emergency and postabortal contraception. Br J Fam Plann 1999;24:149–59.

[159] de Leede LGJ, Hug CC Jr, De Lange S, de Boer AG, Breimer DD. Rectal and intra-venous infusion of propranolo1 to steady-state; kinetics and -receptor blockade. Clin Pharmacol Ther Ser 1984;35:l48–55.

[160] Kleinbloesem CH, van Brummelen P, van Linde JA, de Voogd JA, Breimer DD. Nifedipine kinetic and hemodynamic during rectal infusion to steady-state with an osmotic system. Clin Pharmacol Ther Ser 1984;36:396–401.

[161] Kleinbloesem CH, van Brummelen P, van Linde JA, de Voogd JA, Breimer DD. Nifedipine kinetic and hemodynamic during rectal infusion to steady-state with an osmotic system. Clin Pharmacol Ther Ser 1984;35:742–9.

[162] Miyake M, Nishihata T, Nagantr A, Kyohahi Y, Kamada A. Rectal absorption of [Asu 1,7]-eel calcitonln in rat. Chem Pharm Bull 1985;33:740–5.

[163] Morimoto K, Ahatauchi H, Morisaka K., Karnada A. Effect of non-ionic surfactants in a polyacrylic acid gel base on the rectal absorption of [Asu 1,7 ]-eel calcitonln in rat. J Pharm Pharmacol 1985;37:759–60.

[164] van Hoogdalem EJ, Heijiliger-Feijien CD, Mathot RA, Wackwit AT, van Brec JB, et al. Rectal absorption enhancement of cefoxitin and desglycinamide arginine vasopressin by sodium tauro-24, 25-dihydrofusidate in conscious rat. J Pharmacol Exp Ther 1989;251: 741–4.

[165] van Hoogdalem EJ, Heijiliger-Feijien CD, Verhoef JC, de Boer AG, Breimer DD. Absorption enhancement of rectally infused insulin by sodium tauro-24, 25-dihydrofu-sidate (STDHF) in rats. Pharm Res 1990;7:180–3.


[166] Yoshikawa H, Takada K, Satoh Y, Naruse N, Muranishi S. Development of interferon suppositories. Enhanced rectal absorption of human fibroblast interferon by fusogenic lipid via lymphotropic delivery in rat. Pharm Res 1986;3:116–17.

[167] Utoguchi N, Watanabe Y, Shida T, Matsumoto M. Nitric oxide donors enhance rectal absorption of macromolecules in rabbits. Pharm Res 1998;15:870–6.

[168] Onuki Y, Morishita M, Takayama K, Tokiwa S, Chiba Y, Isowa K, et al. In vivo effects of highly purified docosahexaenoic acid on rectal insulin absorption. Int J Pharm 2000;198: 147–56.

[169] Uchida T, Toida Y, Sakakibara S, Miyanaga Y, Tanaka H, Nishikata M, et al. Preparation and characterization of insulin-loaded acrylic hydrogels containing absorption enhanc-ers. Chem Pharm Bull 2001;49:1261–6.

[170] Kouji K, Iori H, Hirono K, Naoki U, Makiko F, Yoshiteru W. Pharmacokinetics and pharmacodynamics of human chorionic gonadotropin (hCG) after rectal administration of hollow-type suppositories containing hCG. Biol Pharm Bull 2002;25:678–81.

[171] Miyake M, Kamada N, Oka Y, Mukai T, Minami T, Toguchi H, et al. Development of sup-pository formulation safely improving rectal absorption of rebamipide, a poorly absorb-able drug, by utilizing sodium laurate and taurine. J Control Release 2004;99:63–71.

[172] Nishihata T, Rytting JH, Kamada A, Higuchi T, Routh M, Caldwell L. Enhancement of rectal absorption of insulin using salicylates in dogs. J Pharm Pharmacol 1983;35:148–51.

[173] Uchiyama T, Sugiyama T, Quan Y-S, Yamamoto A, Muranishi S. Enhancement effects of various absorption enhancers on the transport of insulin across the intestinal mem-brane. The 12th Japan Society of DDS (Kyoto) 1996:7.

[174] Yoshioka S, Caldwell L, Higuchi T. Enhanced rectal bioavailability of polypeptides using sodium 5-methoxysalicylate as an absorption promoter. J Pharm Sci 1982;71:593–4.

[175] Ziv E, Eldor A, Kleinman Y, Bar-On H, Kidron M. Bile salts facilitate the absorption of heparin from the intestine. Biochem Pharmacol 1983;32:773–9.

[176] Hosny EA. Relative hypoglycemia of rectal insulin suppositories containing deoxycho-lic acid, sodium taurocholate, polycarbophil, and their combinations in diabetic rabbits. Drug Dev Ind Pharm 1999;25:745–52.

[177] Ehab HA, Zohair Al-MMH, Mohammed MES, Mamdouh SYS, Rhman AAAM. Evaluation of efficiency of insulin suppository formulations containing sodium salicylate or sodium cholate in insulin dependent diabetic patients. Boll Chim Farm 2003;142:361–6.

[178] Yamamoto A, Hayakawa E, Kato Y, Nishiura A, Lee VH. A mechanistic study on enhancement of rectal permeability to insulin in the albino rabbit. J Pharmacol Exp Ther 1992;263:25–30.

[179] Hoogdalem EJ, Heijliger-Feijien CD, Boer AG, Verhoef JC, Breimer DD. Rectal absorption enhancement of des-enkephalin--endorphin (DE--E) by medium-chain glycerides and EDTA in conscious rats. Pharm Res 1989;6:91–5.

[180] Miyake M, Nishihata T, Wada N, Takeshima E, Kamada A. Rectal absorption of lyso-zyme and heparin in rabbits in the presence of non-surfactant adjuvants. Chem Pharm Bull 1984;32:2020–5.

[181] Kamada A, Nishihata T, Kim S, Yamamoto M, Yata N. Study of enamine derivatives of phenylglycine as adjuvants for the rectal absorption of insulin. Chem Pharm Bull 1981;29:2012–19.

[182] Kim S, Kamada A, Higuchi T, Nishihata T. Effect of enamine derivatives on the rectal absorption of insulin in dogs and rabbits. J Pharm Pharmacol 1983;35:100–3.

[183] Yagi T, Hakui N, Yamasaki Y, Kawamori R, Shichiri M, Abe H, et al. Insulin suppository: enhanced rectal absorption of insulin using an enamine derivative as a new promoter. J Pharm Pharmacol 1983;35:177–8.


[184] Nishihata T, Okamura Y, Kamada A, Higuchi T, Yagi T, Kawamori R, et al. Enhanced bioavailability of insulin after rectal administration with enamine as adjuvant in depan-creatized dogs. J Pharm Pharmacol 1985;37:22–6.

[185] Hosny EA, Al-Shora HI, Elmazar MM. Relative hypoglycemic effect of insulin sup-positories in diabetic beagle dogs: optimization of various concentrations of sodium salicylate and polyoxyethylene-9-lauryl ether. Biol Pharm Bull 2001;24:1294–7.

[186] Ichikawa K, Ohata I, Mitomi M, Kawamura S, Maeno H, Kawata H. Rectal absorption of insulin suppositories in rabbits. J Pharm Pharmacol 1980;32:314–18.

[187] Kosior A. Investigation of physical and hypoglycaemic properties of rectal supposito-ries with chosen insulin. Acta Pol Pharm 2002;59:353–8.

[188] Nakanishi K, Ogata A, Masada M, Nadai T. Effect of nonsteroidal anti-inflammatory drugs on the permeability of the rectal mucosa. Chem Pharm Bull 1984;32:1956–66.

[189] Nakanishi K, Masada M, Nadai T. Effect of nonsteroidal anti-inflammatory drugs on the absorption of macromolecular drugs in rat rectum. Chem Pharm Bull 1986;34:2628–31.

[190] Mizuno A, Ueda M, Kawanishi G. Effects of salicylate and other enhancers on rectal absorption of erythropoietin in rats. J Pharm Pharmacol 1992;44:570–3.

[191] Nishihata T, Rytting JH, Higuchi T, Caldwell L. Enhanced rectal absorption of insu-lin and heparin in rats in the presence of non-surfactant adjuvants. J Pharm Pharmacol 1981;33:334–5.

[192] West GB. Rectal absorption of dextran in rats in the presence of salicylates. Int Arch Allergy Immunol 1982;68:283–5.

[193] Hosny E, el-Ahmady O, el-Shattawy H, Nabih Ael-M, el-Damacy H, Gamal- el-Deen S, et al. Effect of sodium salicylate on insulin rectal absorption in humans. Arzneimittelforschung Beih. 1994;44:611–13.

[194] Hauss DJ, Ando HY. The influence of concentration of two salicylate derivatives on rectal insulin absorption enhancement. J Pharm Pharmacol 1988;40:659–61.

[195] Watanabe Y, Kiriyama M, Ito R, Kikuchi R, Mizufune Y, Nomura H, et al. Pharmacodynamics and pharmacokinetics of recombinant human granulocyte colony-stimulating factor (rhG-CSF) after administration of a rectal dosage vehicle. Biol Pharm Bull 1996;19:1059–63.

[196] Nakanishi K, Masada M, Nadai T. Effect of pharmaceutical adjuvants on the rectal per-meability of drugs. II. Effect of Tween-type surfactants on the permeability of drugs in the rat rectum. Chem Pharm Bull 1983;31:3255–63.

[197] Kunio N, Shozo M, Mikio M, Tanekazu N. Effect of pharmaceutical adjuvants on the rectal permeability of drugs. Effect of pharmaceutical additives on the permeability of sulfaguanidine through the rat rectum. Yakugaku Zasshi 1982;102:1133–40.

[198] Muranishi S. Modification of intestinal absorption of drugs by lipoidal adjuvants. Pharm Res 1985;2:108–18.

[199] Akira K, Masahiro H, Toshiharu H, Shoji A, Haruo M, Manabu H. Solvent drag effect in drug intestinal absorption. I. Studies on drug and D2O absorption clearances. J Pharmacobio-Dyn 1982;5:410–17.

[200] Nishihata T, Rytting JH. Absorption-promoting adjuvants: enhancing action on rectal absorption. Adv Drug Deliv Rev 1997;28:205–28.

[201] Kajii H, Horie T, Hayashi M, Awazu S. Fluorescence study on the interaction of sali-cylate with rat small intestinal epithelial cells: possible mechanism for the promoting effects of salicylate on drug absorption in vivo. Life Sci 1985;37:523–30.

[202] Kajii H, Horie T, Hayashi M, Awazu S. Effects of salicylic acid on the permeability of the plasma membrane of the small intestine of the rat: a fluorescence spectroscopic approach to elucidate the mechanism of promoted drug absorption. J Pharm Sci 1986;75:475–578.


[203] Nakanishi K, Saitoh H, Masada M, Tatematsu A, Nadai T. Mechanism of the enhance-ment of rectal permeability of drugs by nonsteroidal anti-inflammatory drugs. Chem Pharm Bull 1984;32:3187–93.

[204] Hideyuki T, Tamayo S, Kenichi T, Fusao K, Masahiro H. The enhancing mechanism of capric acid (C10) from a suppository on rectal drug absorption through a paracellular pathway. Biol Pharm Bull 1997;20:446–8.

[205] van Hoogdalem EJ, Hardens MA, De Boer AG, Breimer DD. Absorption enhancement of rectally infused cefoxitin sodium by medium-chain fatty acids in conscious rats: con-centration–effect relationship. Pharm Res 1988;5:453–6.

[206] Lindmark T, Söderholm JD, Olaison G, Alván G, Ocklind G, Artursson P. Mechanism of absorption enhancement in humans after rectal administration of ampicillin in sup-positories containing sodium caprate. Pharm Res 1997;14:930–5.

[207] Lindmark T, Schipper N, Lazorová L, Boer AG, Artursson P. Absorption enhancement in intestinal epithelial CaCo2 monolayers by sodium caprate: assessment of molecular weight dependence and demonstration of transport routes. J Drug Targeting 1998;5: 215–23.

[208] Hayashi M, Tomita M, Awazu S. Transcellular and paracellular contribution to transport processes in the colorectal route. Adv Drug Deliv Rev 1997;28:191–204.

[209] Hayashi M, Sakai T, Hasegawa Y, Nishikawahara T, Tomioka H, Iida A, et al. Physiological mechanism for enhancement of paracellular drug transport. J Control Release 1999;62:141–8.

[210] Aungst BJ, Rogers NJ. Site dependence of absorption-promoting actions of laureth-9, Na salicylate, Na2EDTA, and aprotinin on rectal, nasal, and buccal insulin delivery. Pharm Res 1988;5:305–8.

[211] Tomita M, Hayashi M, Awazu S. Absorption-enhancing mechanism of EDTA, caprate, and decanoylcarnitine in CaCO2 cells. J Pharm Sci 1996;85:608–11.

[212] Nishihata T, Miyake M, Kamada A. Study on the mechanism behind adjuvant action of diethylethoxymethylene malonate enhancing the rectal absorption of cefmetazole and lysozyme. J Pharmacobio-Dyn 1984;7:607–13.

[213] Nishihata T, Lee C-S, Nghiem BT, Higuchi T. Possible mechanism behind the adju-vant action of phosphate derivatives on rectal absorption of cefoxitin in rats and dogs. J Pharm Sci 1984;73:1523–5.

[214] Nishihata T, Kim S, Morishita S, Kamada A, Yata N, Higuchi T. Adjuvant effects of glyceryl esters of acetoacetic acid on rectal absorption of insulin and insulin in rabbits. J Pharm Sci 1983;72:280–5.

[215] Akira Y, Muranishi S. Rectal drug delivery systems improvement of rectal peptide absorption by absorption enhancers, protease inhibitors and chemical modification. Adv Drug Deliv Rev 1997;28:275–99.

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