Keywords

1 Introduction

The human skin barrier hinders the widespread use of transdermal drug delivery (TDD) for drug administration. Despite the range of strategies devised and employed to reversibly overcome the skin barrier, TDD is restricted to potent and low molar mass therapeutic agents. Nonetheless, this noninvasive delivery mode offers advantages such as sustained and controlled drug release, reduced side effects, improved bioavailability, better patient acceptance and compliance, and easy termination of drug therapy. The use of chemical penetration enhancers (CPE) is the conventional approach to modify the skin structure and lower its resistance, so as to allow sufficient drug to reach desired therapeutic levels for systemic effects. Over the years, extensive screening and testing have identified different classes of chemicals as potential adjuvants. Among these, terpenes, the natural volatile oils extracted from plant sources, have been widely used as CPE by permeating into the human skin and reversibly decreasing its barrier resistance.

2 Structure of the Human Skin

The skin, with a surface area of ca. 1.72 m2, is the most accessible organ of the human body and continues to be the preferred site for the application of topical dosage forms [1]. It consists of three main histological layers: the epidermis, dermis, and subcutis as show in Fig. 124.1 [2]. The stratum corneum (SC) of the epidermis, the outermost part of the skin [3, 4] is a remarkable transport barrier which effectively retards the diffusion of exogenous and endogenous moieties into and out of the host and may be regarded as the rate-limiting layer [5]. Superficial SC is formed in the final stage of differentiation from several layers of dead cells embedded in a lipid matrix, and its morphology resembles a “brick and mortar” array [6].

Fig. 124.1
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Cross-sectional view of the uppermost layers of human skin (stratum corneum, viable epidermis, and dermis) (Adapted from Venkatraman and Gale [2])

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The protective quality of the skin is firmly attributed to the unique composition and structural configuration of the intercellular SC lipids. Lipid bilayers of ceramides, fatty acids, cholesterol, and cholesterol esters are arranged into a continuous semicrystalline and crystalline interconnecting domain [7–9]. As sweat glands and hair follicles occupy a fractional skin area of 0.1% [3], absorption across the SC layer can be geometrically subdivided into transcellular and intercellular routes. Tortuous intercellular pathway is supposedly the principal route [10–12].

3 Human Skin Permeation and Transdermal Drug Delivery

The continuous SC has been the usual target in TDD. In recent years, researchers have employed physical and chemical methods to temporarily disrupt its elegant molecular structure. Physical methods such as low-frequency ultrasound (sonophoresis) and electrical current (iontophoresis and electroporation) may facilitate absorption of drug molecules by physically altering the skin morphology [13]. Ultrasound causes cavitation, and the shock waves from the collapsing vacuum bubbles increase the free volume space within the lipid lamellae [14, 15]. Iontophoresis electrically drives or repels ionized drug molecules and peptides through current-induced defects [16, 17]. Skin electroporation creates transient aqueous pores in the lipid bilayers.

The conventional strategy of disrupting the skin barrier and improving the skin permeation of poorly absorbed drugs is to incorporate CPE into drug preparations [10, 18]. Lipid-protein-partitioning (LPP) model [19] theorizes that CPE intensifies TDD by increasing drug solubility in the donor formulation, increasing drug partitioning between the formulation and the skin, or disrupting the intercellular SC lipids [13, 20]. At clinically acceptable concentrations, most CPE interact with the lipoidal domains. These interactions include lipid fluidization, polarity alteration, phase separation, and lipid extraction as illustrated in Fig. 124.2 [10, 20]. Compounds such as sulfoxides, pyrrolidones, fatty acids, terpenes, and alcohols are potential enhancers. The ideal CPE would be pharmacologically inert, nontoxic, nonirritating, nonallergenic, and compatible with other components of the transdermal formulation and device as well as upon removal, the skin barrier properties would be restored rapidly and fully. In addition, the onset and duration of the action of a CPE should be predictable and reproducible [21].

Fig. 124.2
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Action of chemical penetration enhancers within the intercellular lipid domain (Adapted from Williams and Barry [20])

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4 Terpenes

4.1 Source, Nomenclature, Classification, and Uses of Terpenes

Terpenes are constituents of essential oils which are the volatile and fragrant substances present in flowers, fruits, and leaves of plants. They are named “terpenes” after “turpentine” as turpentine oil is a mixture of these compounds [22, 23]. They are usually named after the plants from which they were first isolated. Some terpenes share the same composition by percentage, and some have even the same molecular weights and similar boiling points. However, they smell different, have different optical properties, and behave differently in chemical reactions, they are not identical.

The term “terpene” is used to describe a compound, which is a constituent of an essential oil containing carbon and hydrogen or carbon atoms, hydrogen, and oxygen atoms, and is not aromatic in character [24, 25]. This definition is usually extended to include other compounds called terpenoids, which are not of natural occurrence but are very closely related to the natural terpenes. Most terpenes, which include terpenoids, are invariably hydrocarbons, alcohols, aldehydes, ketones, or oxides, and they may be solids or liquids. Terpene hydrocarbons are usually liquids, while terpenes of higher molecular weights, mostly obtained from the natural gums and resins of plants and trees, are not steam volatile.

Terpenes are defined and classified by the so-called isoprene rule, introduced by Wallach in 1887 [22]. Two isoprene units make one “terpene unit.” They are grouped according to the number of isoprene units, for example, monoterpenes [C10], sesquiterpenes [C15], diterpenes [C20], triterpenes [C30], and tetraterpenes [C40] contain 2, 3, 4, 6, and 8 isoprene units, respectively. A subsidiary classification is based on the number of carbon rings present in the terpene; monoterpenes, for example, may be acyclic, monocyclic, or bicyclic. Each group is further broken down into chemical divisions of hydrocarbons, alcohols, oxides (ethers), and ketones.

Terpenes have been utilized for a number of therapeutic purposes, such as in antispasmodics, carminatives, and perfumery. They have been found to be useful when incorporated into topical and transdermal pharmaceutical formulations as they act as chemical penetration enhancers facilitating the permeation of drugs through the skin barrier, both healthy and diseased in comprehensive reviews [26, 27].

4.2 Terpenes as Chemical Penetration Enhancers

Terpenes, in particular monoterpenes (Fig. 124.3), are generally recognized as safe (GRAS) adjuvants as indicated by high percutaneous enhancement [28–30], reversible effect on skin lipids, and mild cutaneous irritancy at low concentrations (1–5%w/v) [31, 32]. No skin irritation or sensitization was noted in humans for formulations containing hydrophobic limonene [31] and oxygen-containing anethole, linalool, carvacrol, thymol, and menthol [33, 34]. Sesquiterpenes (Fig. 124.4), probably due to a more bulky molecular structure, tend to be less effective and favorable. They are less active [21] and have a longer duration of action, implying poor reversibility, that is, they do wash out of the skin readily [29]. However, in a recent work by Nokhodchi et al. [35], sesquiterpenes (farnesol and nerolidol) were found to be more potent enhancers than monoterpenes (carvone, limonene oxide) for diclofenac sodium.

Fig. 124.3
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Molecular structures of the different chemical divisions of monoterpenes tested as skin penetration enhancers. Monoterpenes (C10) are made up of two isoprene units

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Fig. 124.4
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Molecular structures of sesquiterpenes tested as skin penetration enhancers. Sesquiterpenes (C15) are made up of three isoprene units

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The activity of terpenes is dependent on terpene concentration, but there is no clear activity-concentration relationship. Kunta et al. [32] examined the influence of terpene concentration on propranolol permeation. The terpenes investigated were carvacrol, linalool, menthol, and limonene. In general, propranolol transport initially increased and subsequently remained constant with terpene concentration. This phenomenon was also documented by Krishnaiah et al. [34], for menthol in the delivery of nicardipine hydrochloride. Probable reasons are limited terpene solubility in the vehicle and terpene saturation in the skin. In a study by Kararli et al. [33], the permeation flux of zidovudine dropped significantly as thymol and carvacrol concentrations increased from 5% to 10%w/w. Similarly, Babu and Pandit [36] noted a decrease in bupranolol permeation as menthol concentration increased from 2% to 5%w/v. No clear explanations were given for the reduced activities with terpene concentration. Many researchers have chosen a terpene concentration of around 5%w/v in their preparations, and at this level, significant improvements in drug permeation were obtained [28, 37–40]. The bottom line is that the enhancer concentration should be high enough to produce an adequate skin penetration but not high enough to elicit an irritating cutaneous response.

Oxygen-containing terpenes were more potent for hydrophilic drugs (Table 124.1) such as 5-fluorouracil [41], propranolol [32, 49], and zidovudine [33, 45]. Hydrocarbon terpenes apparently gave a better enhancement for lipophilic drugs (Table 124.1) such as ketoprofen [1] and indomethacin [20]. These collectively give rise to the general rule of thumb – the use of hydrophilic terpenes for polar drugs and the use of hydrocarbon terpenes for nonpolar drugs. Evidently, there are exceptions to the rule. Font et al. [37] and Ueda et al. [50] showed that hydrophobic limonene improved the deliveries of hydrophilic sumatriptan succinate (Table 124.1) and aminopyrine through excised animal skins the most as compared to oxygen-containing terpenes. The generic trend acts as a guideline for the screening of terpenes, but it does not provide any information on the enhancement level.

Table 124.1 Terpenes as skin penetration enhancers for hydrophilic and hydrophobic drugs
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The use of terpenes in conjunction with physical enhancers aims to further enhance drug permeation. Ueda and coworkers explored the coupled effect of ultrasound and several chemical enhancers on the percutaneous delivery of aminopyrine [50]. A greater enhancement in aminopyrine flux was obtained when ultrasound was used with monoterpenes (menthol, carvone, and limonene); the coupled effect was much greater than the sum of its individual effects. Rastogi and Singh [51] demonstrated a similar synergistic effect when iontophoresis was combined with limonene pretreatment in the percutaneous absorption of insulin through porcine epidermis.

4.3 Mechanisms of Skin Permeation of Terpenes

Penetration enhancers are applied onto the skin in an amount that is many times more than the skin lipid molecules beneath the area of administration. This means that there are possibly multiple mechanisms taking place at the same time, making it difficult to determine the relative importance and contribution of each mechanism [52, 53].

4.3.1 Techniques to Investigate the Mechanisms of Skin Permeation of Terpenes

Analytical techniques are used in elucidating changes in lipid conformation and composition caused by skin enhancers [54]. Differential scanning calorimetry (DSC), Fourier transform infrared (FTIR), and isothermal calorimetry (ITC) are techniques which have been utilized to examine macroscopic and molecular transitions of the skin.

DSC is sensitive to the thermal effects accompanying phase changes or transitions of the components of the SC layer. DSC thermograms of human SC, extracted lipids, and protein residue showed three endotherms at ca. 65 °C, 75 °C, and 95 °C [55]. The first is due to the melting of lipids. The second is believed to be the melting of lipid-protein complexes and the last represents protein denaturation. A decrease in lipid melting transition of the enhancer-treated skin denotes a plausible phase separation [39, 56, 57]. Phase separation involves the formation of interfacial defects in the SC lamellae, resulting in decreased diffusional path length or resistance. Based on the lowered lipid endotherms, Vaddi and our fellow researchers suggested that alcoholic terpenes in ethanol-water could exist as pools of fluid within the intercellular lipids [39]. The presence of fluid terpenes at physiological temperatures presumably aids molecular transport. Moghimi et al. reported some degree of phase transformation, the replacement of the lamellae by a reversed viscous isotropic phase, in limonene-treated matrices [57]. These macroscopic defects within the lipid structure may lead to enhanced drug permeation.

To better understand the effects of the terpenes on the permeability of a drug through the skin, the interactions of the terpenes with SC intercellular lipids were studied using the isothermal titration (ITC) method by our research group [58]. Cholesterol, palmitic acid, and stearic acid were found to be the most soluble among all the lipids in propylene glycol, and they were further significantly solubilized upon the addition of farnesol. The interactions between farnesol and four representative lipids, that is, cholesterol, behenic acid, ceramide 3, and ceramide 9 were studied using the ITC method. The binding ratios of farnesol to cholesterol, behenic acid, ceramide 3, and ceramide 9 were found to be 1, 2, 2, and 2, respectively. All were endothermic and entropy driven except for that between farnesol and behenic acid, which was exothermic and enthalpy driven. Hydrogen bonding may be the driving force of these interactions. The results suggested that the skin permeation enhancement mechanism of farnesol, the terpene of interest, could be due to lipid extraction and/or triggering lipid phase transition of the SC lamella. This finding was also consistent with the permeation study results, which showed the permeability coefficients of the drug increased as the lipophilicities of monoterpene and sesquiterpenes increased. It is perceivable that terpenes with high lipophilicities will have more interactions with skin lipids.

Infrared spectra bands of the SC can be attributed to the lipid or protein molecular vibrations. The hydrocarbon chains of SC lipids give rise to asymmetric and symmetric CH2 stretching vibrations at 2,920 and 2,850 cm−1, respectively [39]. The shift of these bands to a higher frequency occurs when the methylene groups change from a trans to a more energetic gauche conformation, and this shift is associated with lipid fluidization. The absorbance of the CH2 stretching bands is proportional to the amount of SC lipids, and thus, the extraction of lipids by a skin enhancer results in a decrease in absorbance [5, 39, 53, 59]. SC proteins give rise to CN stretching and NH bending vibrations at 1,550 cm−1, and C = O stretching vibration at 1,650 cm−1. The shift of these bands to either a higher or lower frequency signifies a change in the protein conformation [45, 53]. FTIR findings of several research groups identified partial lipid extraction as a dominant mechanism for alcoholic, oxide, and hydrocarbon terpenes [39, 40, 60, 61] and showed that delipidization was consistent with enhanced drug permeation.

After the permeation behaviors of the model drug and the enhancers have been investigated by studying the interactions between skin lipids and terpene enhancers, the modeling of drug permeation process through excised skin using both Franz cell and flow-through cell was used to compare the effects of terpenes on drug permeation [62]. A mathematical solution based on finite outflow volume was derived from Fick’s law. It can serve as a statistical model to estimate the permeability coefficient from in vitro skin permeation study with the accumulation of penetrants in the receptor compartment of the static diffusion cells. The model is suitable to describe the in vitro drug or chemical permeation studies using Franz cells. However, the flow-though cells have infinite outflow volume, so a different model that could enable the parameter estimation without impairing the integrity and quality of the original permeation data was proposed. The nonlinear regression model derived from Fick’s law is appropriate. Bootstrap sampling is useful for checking the precision of parameter inference based on the large-sample theory. For the in vitro permeation study that we conducted with flow-through cells, the method proved to be robust. The estimates of permeated drug/chemical are important in that, unlike in vivo environment where stratum corneum is replenished by the adjacent live stratum granulosum through keratinization, the excised stratum corneum, though composed of dead corneocytes, will deteriorate after days in contact with solvents, which will cause overhydration of stratum corneum that can destroy the lamella and decomposition that will leave highly permeable passages in the stratum corneum. The predictions are relevant for transdermal drug delivery, the cosmetic industry, and regulatory risk assessment on dermal exposure to toxic substances.

4.3.2 Comparison of the Skin Permeation Effects of Terpenes

The effects of a terpene as a CPE are very much dependent on its physicochemical properties and molecular structure. Increases in the skin transports of propranolol [32, 45], haloperidol [39], and zidovudine [33] were partially attributed to the hydrogen-bonding ability of oxygen-containing monoterpenes. The ether and hydroxyl groups of terpenes interact with the polar head groups of skin ceramides and fatty acids, thereby disrupting the lateral/traverse hydrogen bonding of the lipid bilayers [45, 49]. On the contrary, hydrocarbon terpenes would reside preferentially and cause structural perturbation in the alkyl tail regions of the lipid bilayers [61].

Cornwell and Barry [29] obtained a positive, linear relation between skin conductivity ratio and 5-fluorouracil enhancement ratio for eight terpenes (limonene, terpineol, carvone, pulegone, nerolidol, menthone, ascaridole, and cineole) with oxygen-containing cineole and hydrocarbon limonene positioned at the higher and lower ends of the curve, respectively. Increases in both skin conductivity and permeability after treatment with terpenes suggest the creation of new polar channels through which both ions and polar molecules traverse. These polar routes would be confined in the aqueous regions near the head groups of SC lipids [45], and their formation is possibly linked to the hydrogen-bonding potential of hydrophilic terpenes.

The enhancing effects of 49 terpenes were compared by in vitro drug permeation studies of haloperidol through excised human epidermis by our research group [62]. The derived multiple linear regression models which provided estimations of the permeability coefficients of a drug or chemical through the human skin were found to be useful for the preliminary screening of CPE. The authors reported that for monoterpenes and sesquiterpenes, the permeability coefficients of haloperidol increased as the lipophilicities of terpenes increased. For all terpenes studied, their enhancing abilities decreased as their molecular weights increased. Melting points and boiling points of terpenes were negatively correlated with the permeability coefficients of haloperidol. Sesquiterpenes were better than monoterpenes when only their enhancing effects were considered. The effects of the skin permeation enhancement by terpenes were ranked as follows: ester > aldehyde > oxide > hydrocarbon > alcohol > ketone > phenol > acid (Table 124.2).

Table 124.2 The second column is the name of each terpene, its CAS entry, and purity. The third column T indicates the terpene category (key: 1 monoterpene, 2 sesquiterpene, 3 diterpene, 4 triterpene, 5 tetraterpene). The fourth to seventh columns are molecular weight, melting point, boiling point, and logP of each terpene. The boiling point of (−)-isolongifolol is not available and is estimated at 300 °C to be similar to the boiling points of other sesquiterpenes. The eighth column, Sol, is the solubility of HP in PG at 37 °C with or without 5 % (w/v) enhancer. The last column LogKp is the in vitro permeability coefficient of HP though human stratum corneum. Data are given as mean ± SD. For columns 8 and 9, the data were determined experimentally in our lab. The other data were obtained from SciFinder Scholar® and original product information
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4.4 Reversible Effects of Terpenes

In addition to the enhancing effects, the reversibility of the effects of terpenes on the skin is also an important characteristic of an ideal CPE. The permeability of the pretreated epidermis was comparable to that of the control, so the insult to the barrier function of the skin caused by the enhancers was restored as reported by our research group [43]. As an in vitro study was performed, the recovery of the epidermal barrier function could not be due to the physical barrier being restored via cellular regeneration of the horny layer. The mechanism for this reversible enhancement would be attributed to the insertion of these enhancers within the SC intercellular lipid lamella. The disruptions in the lipid lamella eased the permeation of the lipophilic drug through the tortuous pathway, hence resulting in enhancement of drug permeation. Likewise, once the enhancers were removed, bonds between the lipids could start to re-form, and the depletion of the enhancers could allow the packing of the lipids to revert back to its original alignment. Of the terpenes studied, (R) – (−) carvone had a much faster elution profile out of the epidermis than eucarvone. The results also showed that (R) – (−) carvone, rather than eucarvone, retained more drug, haloperidol, within the epidermis, which suggests that (R) – (−) carvone could be useful as an enhancer for depot HP therapy. Both (R) – (−) carvone and eucarvone were shown to be effective and reversible enhancers for the in vitro permeation of haloperidol through the human epidermis.

4.5 Terpenes in Transdermal Formulations

The transdermal drug formulations could be in the form of a suspension, solution, gel, ointment, or multilayer transdermal patch. A controlled drug release from the patch is usually achieved by changing the properties of either the rate-controlling membrane or the drug matrix.

Lim et al. [42] demonstrated a controlled drug release by varying the gelator content of an organogel vehicle which presented different degrees of resistance to drug diffusion. He also went on to show the effect of CPE, such as terpenes, on the physical, rheological, and chemical characteristics of a model pharmaceutical formulation for topical and transdermal drug delivery [63] by examining the effects of three terpenes (linalool, cineole, limonene) on the rheology and chemical stability of an organogel composed of dibutyllauroylglutamide (GP1) and propylene glycol (PG). At a given GP1 concentration, oxygen-containing linalool and cineole decreased gel moduli (elastic and viscous) and brittleness, and the reverse was obtained for hydrocarbon limonene. Probably, linalool and cineole interfered with hydrogen bonding between GP1 molecules, while limonene could have initiated a phase separation-mediated gelation, changing the gel morphology. Microcalorimetry detected minute heat endotherms for gels (with and without terpenes) subjected to accelerated heat testing. These heat changes could arise from a small degree of structural disruption of the gel network. Heat endotherms normalized with respect to GP1 content were used to assess gel chemical stability. Although the terpenes altered rheology, they did not significantly affect the chemical stability of the gels. This is the first literature report on the effect of penetration enhancers, such as terpenes, on the physical, rheological, and chemical characteristics of a model pharmaceutical formulation for topical and transdermal drug delivery.

The enhancing effect of a selected enhancer, farnesol, incorporated into gels containing small molecule gelling agents (SMGA), was reported by our research group [38]. The SMGA gels developed for application on the skin retained their characteristic aesthetic and rheological properties with the incorporation of the drug and enhancer. These in vitro human skin permeation studies showed that the gels possessed desirable properties for both topical and transdermal delivery. The translucent lipophilic gels with ISA were stable and the permeation of the drug reached the pseudo steady state in less time compared to the PG-based gel. The latter, opaque white in color, delivered the drug at a faster rate with the addition of the enhancer. The gelator, GP1, did not influence the drug permeation rate but increased its permeation lag-time.

Experimental transdermal patches have also been fabricated by flanking a solution or gel containing the test drug and other excipients between an impermeable backing laminate and a rate-controlling membrane. A pressure-sensitive adhesive coated on the membrane ensures an intimate patch-skin contact. The patch is kept in a sealed aluminum pouch to minimize solvent loss [31, 62, 64].

Krishnaiah et al. [31] designed a transdermal therapeutic system (TTS) for nimodipine. The drug reservoir was an ethanolic gel with 4%w/w limonene as the penetration enhancer. A copolymer film (rate-controlling membrane) was coated with a pressure-sensitive adhesive. In vivo study performed on human volunteers showed that the TTS provided a steady-state nimodipine plasma level with minimal fluctuations for around 20 h and a much-improved bioavailability relative to a tablet dosage form of nimodipine. The intersubject variation in the drug plasma level was observed to be significantly lower for the transdermal route than for the oral route. Hepatic first-pass metabolism, differences in gastric emptying, and gastrointestinal absorption among the subjects are the key causes of a low bioavailability and a less reproducible pharmacokinetics, commonly associated with oral administration. Also, the absence of any local irritation at the application sites of the volunteers demonstrated that the components of the TTS patch (drug, terpene, vehicle, and adhesive) were well tolerated by the skin. Similar findings and conclusions were obtained for the TTS for nicardipine hydrochloride and nicorandil with menthol and nerolidol as the penetration enhancer, respectively [34, 65, 66].

5 Conclusions

Terpenes, as constituents present in plant essential oils, have been investigated as chemical enhancers in transdermal drug delivery. In particular, monoterpenes were generally more efficacious probably due to their small molecular sizes. The main mechanisms of action of terpenes on the skin as determined by several analytical techniques were lipid extraction and phase separation. Terpenes such as limonene and menthol in experimental patch formulations were demonstrated to be safe on the skin and effective in improving drug delivery across the skin.