High Throughput Screening of Transdermal Penetration Enhancers: Opportunities, Methods, and Applications

Fig. 8.1

Schematic of the INSIGHT screening apparatus. The INSIGHT screen is made up of a donor array (top) and a symmetric receiver array (bottom). A single screen can screen 100 formulations at one time. The skin is sandwiched between the donor (Teflon) and receiver (Polycarbonate) and the formulations contact the stratum corneum from the donor array. Conductivity measurements are made with one electrode inserted in the dermis and a second electrode moved sequentially in the donor wells. (a) and (b) is top and side view of the INSIGHT apparatus, respectively

Skin impedance has been previously used: (i) to assess the skin integrity for in vitro dermal testing (Lawrence 1997; Fasano et al. 2002; Davies et al. 2004), (ii) to evaluate the irritation potential of chemicals in a test known as skin integrity function test (SIFT) (Heylings et al. 2001), and (iii) to monitor skin barrier recovery in vivo following the application of current during iontophoresis (Turner et al. 1997; Curdy et al. 2002). Since it is evident from the literature that skin impedance can be used to confirm skin integrity, it is logical to hypothesize that alteration in skin barrier due to chemical enhancers can be used as an in vitro surrogate marker for permeability. Scattered literature data support this hypothesis. A study by Yamamoto and Yamamoto (1976a, b) showed that total skin impedance reduces gradually with tape stripping and after 15 stripping skin impedance approaches the impedance value of deep tissues (Yamamoto and Yamamoto 1976a, b). However, quantitative relationships between skin impedance and permeability in the presence of chemical enhancers and their validity for a wide range of markers have not been previously documented.

8.2.1 Skin Impedance–Skin Permeability Correlation

Stratum corneum is a composite of proteins and lipids in which protein-rich corneocytes are surrounded by lipid bilayers (Madison et al. 1987). Approximately 70–100 bilayers are stacked between two corneocytes (Elias et al. 1977; Elias 1983). Because of its architecture the SC is relatively nonconductive and possesses high electrical impedance (Lackermeier et al. 1999). Skin impedance (AC) can be measured either by applying a constant current and measuring the potential across the skin or by measuring transepidermal current following the application of a constant AC potential. Data reported in this chapter are based on measurement of transepidermal current following the application of a constant potential (100 mV rms). Frequency of the applied potential is also an important parameter. Due to the capacitive components of the skin, the measured electrical impedance of the skin decreases with increasing frequency (Yamamoto and Yamamoto 1976a, b). While the use of higher frequencies facilitates measurements due to decreased impedance, the correlation between electrical impedance and solute permeability is stronger at lower frequencies. Thus, an optimal frequency must be chosen. All experiments reported in chapter were performed at a frequency of 100 Hz.

INSIGHT screening is founded on the relationship between skin’s electrical impedance (reciprocal of skin conductance) and solute permeability. There is a dearth of literature on skin impedance (conductivity) and permeability relationship, and moreover in most of the studies this relationship was used to elucidate the mechanism of transport of hydrophilic molecules across the skin under the influence of temperature (Peck et al. 1995), hydration (Tang et al. 2002), electric current (Sims et al. 1991; Li et al. 1998), or ultrasonic waves (Tang et al. 2001; Tezel et al. 2003). Therefore existing data cannot be used to generalize the relationship between skin impedance and permeability. Accordingly, a large dataset was first generated to assess the correlation between skin impedance and permeability to small (mannitol) and macromolecule (inulin) hydrophilic solutes in the presence of different chemical enhancer formulations. A set of 22 enhancer formulations, chosen from the candidate pool was used to validate the relationship between skin conductivity and skin permeability. The candidate pool comprised of 15 single enhancer formulations and seven binary enhancer formulations. In order to establish the correlation between skin impedance and permeability for wide range of chemical enhancers, formulations were made from different classes of chemicals including cationic surfactants (CTAB, cetyl trimethyl ammonium bromide; BDAC, benzyl dodecyl ammonium chloride), anionic surfactants (NLS, N-lauorylsarcosine sodium; SLA, sodium laureth sulfate; SLS, sodium lauryl sulfate), zwitterionic surfactants (HPS, N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), nonionic surfactants (PEGE, polyethylene dodecyl glycol ether; S20, sorbitan monolaurate; T20, polyoxyethylene sorbitan monolaurate), fatty acids and their sodium salts (LA, lauric acid; OL, oleic acid; SOS, sodium octyl sulfate; SO, sodium oleate), fatty acid esters (TET, tetracaine HCl; IPM, isopropyl myristate), and others (DMP, N-dodecyl 2-pyrrolidone; MEN, menthol). Skin impedance and permeability to two model solutes, mannitol and inulin, were measured. Inulin (MW 5 kDa) was selected as a model solute as it satisfactorily represents a macromolecular hydrophilic drug. Mannitol (MW 182.2 Da, logK_o/w −3.1) was used as a representative of small hydrophilic drugs.

A strong correlation was observed between skin impedance and permeability of mannitol and inulin for different enhancer formulations (Figs. 8.2, 8.3, and 8.4). The measurements reported in Fig. 8.2a, b were performed in FDCs. There is a reasonable scatter in these data, which is inherent to biological systems such as skin that exhibits high variability. Also, measurements reported in Fig. 8.2a, b represent an aggregate of experiments performed over several different animals and anatomical regions. The correlation between skin permeability and impedance was improved when data for individual enhancers were plotted separately; example for inulin (with DMP enhancer r ² = 0.85) and mannitol (with OL enhancer r ² = 0.86) is given in (Fig. 8.3a, b). The correlation between skin permeability and impedance can be clearly seen in Fig. 8.4a, b where data in Fig. 8.2a, b are replotted after averaging over ~5 kΩ-cm² intervals (inulin r ² = 0.86 and for mannitol r ² = 0.90). Permeability data of mannitol and inulin with a variety of chemical enhancer formulations showed that skin impedance is inversely related to permeability of hydrophilic solutes, which is in agreement with existing data in literature. Correlation coefficient (inulin r ² = 0.86 and mannitol r ² = 0.90) of average data for all enhancer formulations indicates that a remarkable correlation exists between skin permeability and impedance for single and binary enhancers formulations irrespective of the nature of the formulation. These results indicate that skin impedance can be used as a parameter to measure the extent of barrier alteration by chemicals irrespective of their mode of action (which, in most cases, is not precisely known). Specifically, good correlations were observed between permeability and skin impedance for enhancers, which act by lipid extraction (NLS, MEN, BDAC) or by lipid bilayer fluidization (OL, LA, IPM). Note, however, that the nature of these correlations is an integral function of the physicochemical properties of the drug or permeant. Educated discretion must therefore be exercised when selecting a delivery formulation for a particular model permeant or drug of interest.

Fig. 8.2

Skin impedance–permeability correlation for (a) inulin and (b) mannitol. Test formulations used in this study (in parenthesis total concentration of chemical enhancer w/v, weight fraction used): (a)

-MEN (1.5 %w/v);

-SO (1.5 %w/v);

-PEGE (1.5 %w/v);

-OL (1.5 %w/v);

-S20 (1.0 %w/v);

-DMP (1.5 %w/v);

-OL:MEN (1.5 %w/v, 0.4:0.6);

-IPM (1.5 %w/v);

-TET (2.0-%w/v);

-LA (1.5 %w/v);

-NLS (1.0 %w/v);

-SOS (2.0 %w/v);

-NLS:S20 (1.0 %w/v, 0.6:0.4);

-TET:SLS (1.0 %w/v, 0.6:0.4);

-TET:HPS (2.0 %w/v, 0.1:0.9);

-MEN:T20 (2.0 %w/v, 0.5:0.5);

-DMP:TET (2.0 %w/v, 0.4:0.6);

-CTAB (1.0 %w/v). (b)

-OL (1.5 %w/v);

-DMP (2.0 %w/v);

-DMP:TET (2.0 %w/v, 0.4:0.6);

-PEGE (1.5 %w/v);

-TET (2.0 %w/v);

-LA (1.5 %w/v);

-S20 (1.0 %w/v);

-HPS (1.5 %w/v);

-NLS (1.0 %w/v);

-BDAC (1.5 %w/v);

-MEN (1.5 %w/v);

-DMP:HPS (1.5 %w/v, 0.6:0.4);

-NLS:S20 (1.0 %w/v, 0.6:0.4);

-DMP (1.5 %w/v)

Fig. 8.3

Skin impedance-permeability correlation for single enhancer. (a) Plot of skin permeability to inulin vs. skin impedance in the presence of DMP (1.5%w/v in 1:1 EtOH:PBS); (b) Plot of skin permeability to mannitol vs. skin impedance in the presence of NLS (1.5%w/v in 1:1 EtOH:PBS). A much tighter correlation can be observed compared to Fig. 8.2

Fig. 8.4

Skin impedance – permeability correlation for (a) inulin and (b) mannitol. Modified plot of permeability-impedance data shown in Fig. 8.2. Permeability data for different enhancers are grouped in the bins of 5 kΩ-cm² along the x-axis representing skin impedance. The correlation is much tighter as compared to the one in Fig. 8.2

8.3 Validation of INSIGHT with FDC

Conductivity enhancement ratio (ER), that is, the ratio of skin impedances at time zero and 24 h following the application of enhancer formulation, measurements in INSIGHT were plotted against conductivity enhancement and permeability enhancements in FDCs (Fig. 8.5a). Inulin was used as a model permeant in these studies. Results shown in Fig. 8.5a reflect that the predictions obtained from INSIGHT on the potency of enhancer formulations are essentially the same as those obtained from FDCs. However, INSIGHT allows collection of information at a much greater speed (~1000 per day) and less skin utilization (about 0.07 cm² per experiment as compared to 2 cm² in a 16 mm diameter FDC, >25-fold reduction in skin utilization).

Fig. 8.5

Validation of INSIGHT predictions with FDC. Plot of conductivity enhancement ratios in INSIGHT at 24 h vs. conductivity and permeability enhancement ratios in FDC at 96 h for 19 enhancer formulations. A strong linear correlation indicates the validity of observations in INSIGHT when compared with those from traditional tools like FDC. The closed circles indicate conductivity enhancement numbers, and the filled circles indicate permeability enhancement numbers in FDC. (a) Plot of 24 h predictions in INSIGHT vs. 4 h predictions in INSIGHT on the potency of enhancer formulations. A strong correlation indicates that predictions on potency of formulations can be obtained at significantly lower incubation periods of 4 h

Further improvements in INSIGHT screening speed can be obtained by reducing the formulation incubation period. Capabilities of INSIGHT in assessing formulation potency after a 4-h incubation are demonstrated in Fig. 8.5b where potency rankings of 438 single and binary formulations randomly prepared from the enhancer library based on 4-h screening are compared to those based on 24-h screening. Rank 1 corresponds to most potent formulation in the library and rank 438 to the weakest formulation. The predictions of the potency made in 4 h were consistent with those made after a contact time of 24 h, thus indicating that the efficiency of INSIGHT screening can be further improved.

8.4 Applications of INSIGHT Screening

8.4.1 Discovery of Rare Formulations

INSIGHT screening can be used to screen huge libraries of chemicals within a short span of time and without the fear of failure that exists with traditional tools. Many current single enhancers are also potent irritants to the skin at concentrations necessary to induce meaningful penetration enhancement. Attempts have been made to synthesize novel chemical enhancers such as laurocapram (Azone); however, achieving sufficient potency without irritancy has proved challenging, especially for macromolecules. A number of studies have shown that formulations made up of combination of chemical enhancers are more potent than its individual components (Karande et al. 2004; Tezel et al. 2002; Mitragotri 2000). The addition of components increases the number of formulations exponentially. However, the use of INSIGHT screening allows one to tackle this challenge in a more cost-effective way compared to FDCs. In addition, synergies between CPEs not only lead to new transdermal formulations but also potentially offer insight into mechanisms by which CPEs enhance skin permeability. Prediction of synergies from the first principles is challenging. INSIGHT screening offers an effective tool for identifying synergies (positive or negative) between the CPEs.

To identify synergistic combinations of penetration enhancers (SCOPE) formulations, a library of chemical enhancers was first generated from 32 chemicals chosen from a list of >250 chemical enhancers belonging to various categories. Random pairing of CPEs from various categories led to 496 binary chemical enhancers pairs. For each pair, 44 distinct chemical compositions were created with the concentration of each chemical enhancer ranging from 0–2 % w/v, yielding a library of 25,000 candidate SCOPE formulations. About 20 % of this library (5,040 formulations) was screened using INSIGHT, the largest ever-cohesive screening study reported in the transdermal literature. Each formulation was tested at least four times in over 20,000 experiments (Karande et al. 2004). Using the traditional tools for formulation screening, it would have taken over 7 years to do these many experiments. With INSIGHT screening, the same task was accomplished in about 2 months with screening rate of 500–1,000 experiments per day.

Binary formulations exhibited a wide range of enhancements. The percent of randomly generated enhancer combinations that exhibit ER above a certain threshold decreases rapidly with increasing threshold (Fig. 8.6a). The inset shows a section of the main figure corresponding to high ER values. Less than 0.1 % of formulations exhibited more than 60-fold enhancement of skin conductivity. Discovery of such rare formulations by brute force experimentation is contingent on the throughput of the experimental tool. INSIGHT screening opens up the possibility of discovering such rare formulations.