Investigation of properties of human epidermal membrane under constant conductance alternating current iontophoresis

Journal of Controlled Release 89 (2003) 31–46 www.elsevier.com / locate / jconrel

Investigation of properties of human epidermal membrane under constant conductance alternating current iontophoresis Honggang Zhu a , Kendall D. Peck b , David J. Miller c , Mark R. Liddell a , Guang Yan a , William I. Higuchi a , S. Kevin Li a , * a

30 S 2000 E RM 201, Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT 84112, USA b Department of Chemistry, Brigham Young University-Idaho, Rexburg, ID 83460, USA c Aciont, Inc., 350 W. 800 N. Suite 250, Salt Lake City, UT 84103, USA Received 27 November 2002; accepted 8 January 2003

Abstract Previous studies in our laboratory have shown that enhanced, constant permeant fluxes across human skin can be achieved by applying an alternating current (AC) to maintain skin electrical conductance at a constant level. Relative to conventional direct current (DC) iontophoresis, for which current is maintained at a constant level, this newly developed constant conductance alternating current (CCAC) method achieves constant fluxes with less inter- and intra-sample variability. The present study focused upon further investigating the permeability properties of human skin during CCAC iontophoresis at a variety of target resistance / conductance values. A three-stage experimental protocol was used with flux measurements determined on 3 consecutive days. Stage I was an AC only protocol (symmetrical AC square-wave signal), stage II was an AC plus DC protocol (AC square-wave with DC offset voltage), and stage III was a repeat of stage I. During this three-stage protocol, the skin electrical resistance was maintained at a constant target value by manually adjusting the applied AC voltage. Radiolabeled mannitol and urea were model permeants in all experiments. Their fluxes were determined and used to characterize the permeability properties of human skin. The results from the present study established that: (i) the CCAC protocol made it possible to reduce HEM electrical resistance to different target levels as low as 0.8 kV cm 2 and maintain the specific resistance level throughout the flux experiment, (ii) permeant fluxes are proportional to skin electrical conductance, (iii) under the studied CCAC passive conditions, membrane pore size tends to increase as skin resistance decreases, and (iv) as the membrane breaks down, its pore sizes become larger.  2003 Elsevier Science B.V. All rights reserved. Keywords: Transdermal; Iontophoresis; Constant conductance; Human epidermal membrane; Flux variability

1. Introduction

*Corresponding author. Tel.: 11-801-581-4110; fax: 11-801585-1270. E-mail address: [email protected] (S.K. Li).

Transdermal drug delivery is a promising alternative to traditional oral or intravenous routes of drug delivery due to elimination of the first-pass effect, better patient compliance, and other advan-

0168-3659 / 03 / $ – see front matter  2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0168-3659(03)00032-4

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H. Zhu et al. / Journal of Controlled Release 89 (2003) 31–46

tages [1–4]. However, most drugs do not have high enough passive transdermal fluxes to achieve therapeutic effects due to the barrier properties of human skin, particularly the stratum corneum. Therefore, physical and chemical enhancers have been widely used to increase skin permeability. Among the physical enhancers, iontophoresis has been shown to significantly increase fluxes of both ionic and polar neutral compounds through one or a combination of the following mechanisms: electrophoresis, electroosmosis, and electroporation [5–8]. Although iontophoresis enhancement methods have been extensively studied, flux variability [9–11] has remained an obstacle that hinders iontophoresis from being widely used. Our recent research [12,13] has focused upon the development of a new constant conductance alternating current (CCAC) iontophoresis protocol. The unique aspect of this method is that the skin electrical conductance / resistance is controlled and maintained at a constant target value by manually adjusting an applied AC voltage. The cited results [12] showed that under the constant conductance condition, constant permeant flux was achieved with significantly less inter- and intra-sample variability relative to conventional constant current iontophoresis. This novel approach may be the basis for the development of iontophoretic devices with more controllable and predictable rates of drug delivery or endogenous material extraction. In order to better understand the human skin barrier / permeation properties under constant conductance conditions, flux studies were conducted at four target constant resistance values: 0.8, 1.6, 3.2 and 4.8 kV cm 2 . For each target resistance condition, a three-stage protocol was performed, with each stage conducted on consecutive days. Stage I was AC alone (symmetric AC square-wave signal), stage II was AC with a 400-mV DC offset voltage, and stage III was a repeat of stage I. Mannitol and urea fluxes at each experimental condition were simultaneously determined and this information was used to characterize human epidermal membrane (HEM) transport properties at each condition. The information gained from this study will add to our mechanistic understanding of human skin barrier / permeation properties during iontophoresis and will potentially have a major impact on future practical applications of iontophoresis.

2. Materials and methods

2.1. Materials Radiolabeled [ 3 H]mannitol and [ 14 C]urea (radiochemical purity determined to be greater than 99% by HPLC) were purchased from New England Nuclear (Boston, MA) and American Radiolabeled Chemicals (St. Louis, MO). HEM was prepared by heat separation [14] of split-thickness human skin obtained from skin banks and Watson Pharmaceuticals (Salt Lake City, UT) and frozen for later use. Millipore GVWP filters were obtained from Millipore (Bedford, MA). Phosphate-buffered saline (PBS) (pH 7.4) was prepared from reagent grade chemicals at an ionic strength of 0.1 M [5].

2.2. Experimental procedure All the experiments were carried out in PBS at 37 8C using two-chamber, side-by-side diffusion cells with diffusion surface area of |0.8 cm 2 . HEM was mounted between the two half-cells of the diffusion cells next to a Millipore filter to support the HEM sample [15]. Following diffusion cell assembly, HEM samples were allowed |24 h to equilibrate with the PBS at 37 8C before starting the transport experiments. The HEM initial resistance was measured by applying 100-mV electrical potential across the membrane using a four electrode potentiostat system (JAS Instrumental System, Salt Lake City, UT) as previously described [5,15]. HEM samples with an initial resistance in the range from 25 to 100 kV cm 2 were used in the present experiments. A three-stage experimental protocol utilizing the same HEM sample in three successive experiments was employed in the present study. This protocol minimizes the influence of HEM sample-to-sample variability in data interpretation and allows the assessment of reproducibility of HEM using the data obtained from the same HEM sample as its own control. With this protocol, reproducibility and parameter change following extended exposure to iontophoresis can be determined and meaningful results can be obtained in a minimum number of experiments.

H. Zhu et al. / Journal of Controlled Release 89 (2003) 31–46

2.3. Three-stage experimental protocol 2.3.1. Stage I: constant conductance ‘ AC only’ experiments A 30-V peak-to-peak 2000-Hz square-wave AC voltage generated from a custom-made function generator (EmTech, Salt Lake City, UT) was applied for 1–5 min to reduce the HEM resistance to a target value of 0.8, 1.6, 3.2, or 4.8 kV cm 2 . Once the target resistance was achieved, this resistance was maintained throughout the flux experiment by manually adjusting the output AC voltage. The details of the instrumentation and the resistance measurement and resistance control procedures have been described previously [12,13]. Briefly, the driving Ag /AgCl electrodes connecting the HEM were in series with a known resistance fixed resistor in the circuit. HEM electrical resistance was determined by the slope of DC offset voltage across HEM versus DC offset voltage across the fixed resistor, the resistance value of the fixed resistor, and Ohm’s law. In this ‘AC only’ stage, the applied AC square-wave was symmetric and no DC offset voltage was applied (i.e. zero DC offset voltage across HEM). When the target resistance was obtained, tracerlevel [ 3 H]mannitol and [ 14 C]urea were added to the donor chamber and flux experiments were conducted for |8 h. Samples (1 ml) were taken from the receiver chamber approximately every 30 min and replaced with fresh buffer solution. Samples (10 ml) were taken from the donor chamber every hour. Samples were mixed with 10 ml scintillation cocktail (Ultima Gold姠, Packard Instrument, Meriden, CT) and assayed by liquid scintillation counting (Parkard TriCarb姠 Model 1900 TR Liquid Scintillation Analyzer). 2.3.2. Stage II: constant conductance ‘ AC plus DC’ experiments After stage I, the donor and receiver chambers were rinsed with fresh PBS solution several times and the HEM sample was allowed to recover overnight. Prior to the stage II flux experiment, HEM electrical resistance was measured using the fourelectrode potentiostat system described earlier. The same target resistance level studied in stage I for a given HEM sample was obtained during stage II. The method of obtaining the target resistance and maintaining that resistance was as described for stage

33

I. The permeants and sampling procedure during stage II were also identical to stage I. Stage II differed from stage I in that a 400-mV DC offset voltage was applied across HEM in addition to the AC voltage during the flux experiment.

2.3.3. Stage III: constant conductance ‘ AC only’ experiments Following stage II, both chambers were again thoroughly rinsed and the HEM samples were allowed to recover overnight. The stage III protocol was identical to stage I both in terms of the electrical and flux protocols. Stage III experiments were performed to assess reproducibility and parameter changes following extended exposure to iontophoresis. Five to seven different HEM samples were analyzed by the three-stage protocol for each target resistance level (0.8, 1.6, 3.2, and 4.8 kV cm 2 ). Each HEM sample for a specific target resistance level was taken from a different skin donor. 2.4. Theoretical considerations In the present study, the data were analyzed by the theory of transport across a porous membrane. The porous or polar pathway, in parallel with the lipoidal pathway, in HEM has been studied and discussed previously [16]. Although the literature has shown the existence of the polar pathway in passive and iontophoretic transport across HEM [17–19], the locations or sites of the porous structure in human skin are still controversial. The following are the theories and models used to analyze the data in the present study. Stages I and III incorporated AC square-wave applied voltages only. Under this condition, fluxes are essentially passive with electroporation being the only iontophoretic contribution to flux enhancement. Therefore, permeant fluxes during stage I and stage III, ‘AC only’, can be expressed by the following equation HAC DCD ´AC JAC 5 ]]]] Dx

(1)

where JAC , ´AC , and HAC are the permeant flux, combined membrane porosity and tortuosity factor, and hindrance factor for the permeants, respectively. Dx is the membrane thickness, D is the diffusion

H. Zhu et al. / Journal of Controlled Release 89 (2003) 31–46

34

Table 1 Diffusivities and radii of permeant and background electrolyte ions at 37 8C Permeant

Diffusion coefficient (310 25 cm 2 / s)

Radius c ˚ r (A)

Na 1 Cl 2 Urea Mannitol

1.78 a 2.72 a 1.75 b 0.903 b

2.5 1.9 2.7 4.1

a

Data from Lide [34]. Data from Peck et al. [18]. c Rw r Hydrated radius calculated using r 5s1.5 3 ] 1 ]] 3 r r 1 2 3 Rwd R SE [29], in which R w is the radius of water molecule and R SE is kT the Stoke-Einstein radius calculated using R SE 5 ] , in which k 6Dph is the Boltzmann constant, T is the temperature, D is the diffusion coefficient, and h is the viscosity. b

coefficient of the permeant provided in Table 1, and CD is the permeant concentration in the donor chamber. The hindrance factor, H, is a function of membrane pore size and permeant size [20–22] 6pF H 5 ]] Kt

(2)

where

F 5s1 2 ld 2

(3)

and l is the ratio of the permeant radius to effective pore radius. Kt is expressed as

F O 2

9 ] Kt 5 ] p 2Œ2s1 2 ld 25 / 2 1 1 a ns1 2 ld n 4 n 51

Oa

G

n 13

ln

(4)

n50

The coefficients in Kt can be found in the literature [21,22]. Eq. (2) assumes cylindrical pore geometry in the membrane and is derived from the asymptotic centerline results. When the ratio of solute radius to pore radius ( l) is small (,0.4), it is equivalent to the Renkin equation commonly used in the literature. The membrane pore radius can be estimated from the ratio of permeability coefficients of the two permeants, urea and mannitol, used in the present study PAC(mannitol ) HAC(mannitol ) Dmannitol ]]]] 5 ]]]]]] PAC(urea) HAC(urea) Durea

J P5] CD

(6)

Permeant flux, J, was determined from permeant transport data. During stage II, a 0.4-V DC offset voltage was applied leading to iontophoretic permeation enhancement due to electroporation and electroosmosis. Under these conditions, the permeability coefficient can be expressed as Wn´AC1DC PAC1DC 5 ]]]] s1 2 exp(2Pe))

(7)

where W is the hindrance factor for convection, n is the solvent flow velocity, ´AC1DC is the combined porosity and tortuosity factor during AC plus DC (stage II) iontophoresis, and Pe is the Peclet number Pe 5 Wn (Dx) /(HD). The electroosmotic solvent flow velocity, n, is a function of the effective membrane pore size, pore charge density, solution ionic strength and applied voltage [23,24]. Pe is directly proportional to solvent flow velocity and therefore is indicative of the contribution from electroosmosis to flux. Pe relates to the permeability enhancement factor due to electroosmosis, E, through the following relationship: PAC1DC Pe E 5 ]]] 5 ]]]] PAC 1 2 exp(2Pe)

4

1

where P is the permeant permeability coefficient, and can be calculated from the following equation

(5)

(8)

The hindrance factor, W, is also a function of the membrane pore size and permeant radius [21,22]. With the same assumptions stated in Eq. (2)

Fs2 2 FdKs W 5 ]]]] 2Kt

(9)

where

F O

2 9 ] Ks 5 ] p 2Œ2s1 2 ld 25 / 2 1 1 b ns1 2 ld n 4 n51

Ob

G

4

1

n13

ln

(10)

n50

The coefficients in Eq. (10) can be found in the literature [21,22]. Pore size during AC plus DC iontophoresis (stage II) can be estimated using the following ratio

H. Zhu et al. / Journal of Controlled Release 89 (2003) 31–46

Wmannitol PAC1DC(mannitol ) ]]]]]] 1 2 exps 2 Pe mannitold ]]]]] 5 ]]]]]]. PAC1DC(urea) Wurea ]]]]] 1 2 exps 2 Pe uread

(11)

(12)

where R is the membrane electrical resistance, H9 and D9 are the average hindrance factor and average diffusion coefficient for the conducting ions (Na 1 and Cl 2), respectively, and k is a conversion constant. Assuming that ´ and Dx are the same for permeants and conducting ions, Eqs. (1), (6), and (12) can be used to derive an expression relating the ‘AC only’ permeant permeability coefficient, PAC , and the electrical resistance HAC D´AC HAC D PAC 5 ]]] 5 ]] ? ]] Dx RH9 kD9

(13)

Taking the logarithm of both sides of Eq. (12) yields

S D

S D

HAC D logsPACd 5 log ]] 1 log ]] RH9 kD9

Wn´AC1DC Wn 1 PAC1DC 5 ]]] 5 ]] ? ]] Dx RH9 kD9

(15)

Taking the logarithm of both sides of Eq. (15) yields

Previous studies have shown that there is a strong correlation between membrane electrical resistance and permeant permeability with small neutral permeants [12,13,19,25,26]. The most recent of these studies have also shown that the membrane parameters that affect permeant transport (i.e. porosity, effective pore size, and effective pore charge density) are maintained essentially constant when the electrical resistance is maintained constant for these permeants. The average permeability coefficient of current conducting ions is inversely proportional to membrane resistance as expressed by 1 H9´D9 ] 5 kP 5 k ? ]] R Dx

35

(14)

If the molecular size of the permeant equals that of the background electrolyte, HAC will equal H9 and the uslopeu expressed in Eq. (14) will equal 1. If membrane pore size remains constant at all resistance levels, the ratio of HAC to H9 will be constant and the linear relationship between log(PAC ) and log R will also exist with a uslopeu of 1. During stage II AC plus DC experiments, if electroosmosis dominates permeant flux, Eq. (7) can be rewritten as

S D

S D

Wn 1 log(PAC1DC ) 5 log ]] 1 log ]] RH9 kD9

(16)

If membrane pore size and pore charge remain constant at all target resistance levels, W, v, and H9 will remain constant, and the absolute value of the slope of log(PAC1DC ) vs. log R plot will be 1. Experimental slope values from plots associated with the discussion of Eqs. (14) and (16) have the potential to yield insight into the fundamental membrane properties associated with permeant transport.

3. Results and discussion Already mentioned in the Introduction, we have recently shown that it is possible to achieve a constant permeant flux with much less inter- and intra-sample variability relative to conventional constant current iontophoresis by maintaining HEM electrical resistance at a constant value using AC [12]. The practical objective and potential application of this technology is to design transdermal iontophoretic devices with improved controlled drug delivery and endogenous substance extraction rates. Optimization of this new technique will rely upon a more fundamental understanding of the barrier properties of HEM during CCAC iontophoresis and how these properties are influenced by changing the CCAC conditions. The present study was designed around obtaining a better understanding of the HEM parameters that affect transport, such as pore size and pore charge, under constant conductance conditions. Due to the insight that can be gained by measuring permeability values at varied HEM resistance levels [Eqs. (14) and (16)], the present permeation studies were conducted at four target resistance levels: 0.8, 1.6, 3.2, and 4.8 kV cm 2 . For each resistance level, a three-stage consecutive permeation protocol was carried out where stage I was an AC only stage (essentially passive permeation), stage II was AC

36

H. Zhu et al. / Journal of Controlled Release 89 (2003) 31–46

plus a 0.4-V DC offset voltage (passive plus electroosmotic transport), and stage III was a repeat of stage I. Over a 3-day period, the three permeation stages were conducted at a given resistance level using a single HEM sample. Fig. 1a–c shows HEM electrical resistance and applied AC voltage data for a representative HEM sample (HEM sample I) during the course of the three-stage protocol. Again, the resistance of the HEM was maintained at the target level in these studies by manually adjusting the applied AC potential. The data presented in these figures indicate that the HEM electrical resistance was tightly controlled within 10–15% of the target resistance level with minimal adjustment to the applied AC voltage. The urea and mannitol transport data in Fig. 2a–c were also taken from HEM sample I and show that, after |180 min, the permeant fluxes for both permeants reached steady-state and remained relatively constant throughout the rest of the permeation experiment. These data are consistent with previous similar transport studies [12,13]. The lag times for stages II and III were shorter than stage I, most likely due to residual permeant from the prior stage remaining in the membrane. Experimental results for all four target resistance levels are summarized in Tables 2–5. These tables show the initial HEM resistances, R 0 , steady-state urea and mannitol permeability coefficients, and effective pore size estimates for each HEM sample from each experimental stage. The urea and mannitol Peclet numbers are also shown for stage II. Only HEM samples with R 0 values prior to stage I of at least 25 kV cm 2 were used for this study. Although the initial resistance values measured prior to the start of the transport experiments tended to decrease for each consecutive stage, significant resistance recovery was observed after each stage relative to the resistance level maintained during the transport experiment. The average P-values, pore size estimates and Pe-values along with the associated standard deviations and coefficients of variance are also shown in these tables for each stage. HEM sample L in Table 4 showed unusually high mannitol permeability for stages II and III and the estimated pore sizes for this sample and these stages were the highest observed under any conditions of the present study. These observations indicated a possible breakdown of this membrane during or following stage I.

Two additional HEM trials (HEM samples P and Q) were performed for the 3.2 kV cm 2 condition and an ESD Single-Outlier procedure was used to treat this group of data. The statistical analysis showed that the stage II mannitol P-value for HEM sample L is an outlier ( p,0.01). For this reason, the average values shown in Table 4 for permeability, pore size, and Peclet number were calculated excluding HEM sample L. One objective of this study was to analyze HEM parameters during CCAC iontophoresis and to assess how these parameters may change when the HEM resistance level is experimentally varied. Analyzing the data within a given table (Tables 2–5) yields transport properties at an experimentally fixed resistance level. Analyzing the data between tables gives an indication how these properties vary when HEM resistance is experimentally varied. As described in the Theoretical considerations section, it is expected that during the AC only stages, due to the similarity in urea size and the sizes of the conducting ions, Na 1 and Cl 2 , a linear relationship will exist between log PAC(urea) and log R with a uslopeu of 1 [see Eq. (14) and the accompanying discussion]. Table 1 shows the sizes of the permeants and ions. Despite the membrane alterations that may occur, this relationship is predicted due to the expectation that changes in the membrane (such as changes in pore size) caused by varying the resistance will affect the transport of urea and the conducting ions to the same extent. Another way to state this expectation is that the ratio of the hindrance factors for urea and the conducting ions will essentially remain constant. Mannitol is significantly larger than the conducting ions (Table 1). A linear relationship with a uslopeu of 1 for a plot of log PAC(mannitol ) versus log R will result only if changes in resistance are not accompanied by changes in effective pore size. A uslopeu for the described plot that varies from 1 will give an indication of how pore size varies as resistance is altered. Fig. 3 shows the results of a computer simulation (Scientist, Micromath, Salt Lake City, UT) illustrating how H varies in a pore size range ˚ for each permeant and conducting from 6 to 10 A ˚ leads to ion. Increasing the pore size from 6 to 10 A a two-fold increase in H for urea, Na 1 and Cl 2 . The corresponding increase in H for mannitol is greater than three-fold. While the ratios Hurea /HNa 1 and

H. Zhu et al. / Journal of Controlled Release 89 (2003) 31–46 37

Fig. 1. Representative HEM electrical resistance (kV cm 2 ) and applied AC voltage (V) versus time (min) profile. Data shown in the figures are taken from HEM sample I. (a) Stage I; (b) stage II; (c) stage III.

38 H. Zhu et al. / Journal of Controlled Release 89 (2003) 31–46 Fig. 2. Representative urea and mannitol cumulative amount in receiver chamber normalized by donor concentration (cm 3 ) versus time (min) profile. Data shown in the figures are taken from HEM sample I. (a) Stage I; (b) stage II; (c) stage III.

Table 2 Summary of the experimental data obtained at 0.8 kV cm 2 HEM sample

Stage I: AC only

Stage III: AC only

R0 (kV)

Purea (310 27 cm/s)

Pmannitol (310 27 cm/s)

Pore size ˚ (A)

R 90 (kV)

Purea (310 27 cm/s)

35 33 91 33 43

12 12 10 12 11

1.8 1.7 1.6 1.9 1.8

8.7 8.5 9.0 8.9 9.1

10 10 12 14 8.1

28 21 19 16 31

2.0 1.3 1.5 0.60 2.6

7.6 5.2 4.8 3.3 9.1

11

1.8

8.8

23

1.6

0.11 6.5%

0.2 2.7%

6.0 26%

0.8 26%

Average S.D. Coefficient of variance (CV)

0.89 7.8%

Pe (urea)

Pore size ˚ (A)

R 990 (kV)

Purea (310 27 cm/s)

6.0 2.9 2.8 1.2 5.0

6.0 6.1 6.0 6.1 6.0

7.7 6.4 6.8 12 6.3

14 15 12 11 12

2.4 2.5 1.9 2.0 2.1

9.3 9.2 8.9 9.6 9.4

6.0

3.2

6.0

13

2.2

9.3

2.3 37%

1.4 45%

0.1 0.9%

1.6 13%

Pmannitol (310 27 cm/s)

Pe (mannitol)

Pmannitol (310 27 cm/s)

Pore size ˚ (A)

0.26 12%

0.3 2.8%

Pmannitol 27 (310 cm/s)

Pore size ˚ (A)

Table 3 Summary of the experimental data obtained at 1.6 kV cm 2 HEM sample

Stage I: AC only R0 (kV)

Purea 27 (310 cm/s)

40 50 33 83 36

6.1 6.2 5.5 6.8 6.7

Average

6.3

S.D. Coefficient of variance (CV)

0.5 8.3%

F G H I J

Stage II: AC with 400-mV DC offset

Stage III: AC only

Pore size ˚ (A)

R 09 (kV)

Purea 27 (310 cm/s)

0.73 0.82 0.64 0.85 0.79

7.9 8.2 7.8 8.0 7.9

21 26 13 11 12

16 13 18 12 13

2.4 1.7 3.1 1.3 1.5

4.1 3.5 4.6 2.1 2.4

0.77

8.0

14

2.0

0.2 2.5%

2.4 17%

0.7 35%

Pmannitol 27 (310 cm/s)

0.083 11%

Pe (urea)

Pore size ˚ (A)

R 099 (kV)

5.6 4.2 7.2 2.2 2.9

5.8 6.1 5.7 5.4 5.5

10 14 16 6.7 8.3

3.3

4.4

5.7

1.1 32%

2.0 46%

Pmannitol 27 (310 cm/s)

Pe (mannitol)

0.3 4.8%

Purea 27 (310 cm/s) 8.0 6.3 5.6 8.2 7.7

1.1 0.90 0.68 1.1 1.3

7.2

1.0

1.1 16%

0.24 23%

8.4 8.5 7.9 8.3 9.2

H. Zhu et al. / Journal of Controlled Release 89 (2003) 31–46

A B C D E

Stage II: AC with 400-mV DC offset

8.5 0.5 5.6%

39

40

Table 4 Summary of the experiment data obtained at 3.2 kV cm 2 HEM sample

Stage I: AC only R0 (kV)

Purea (310 27 cm/s)

34 33 111 90 33 33 83

a

Pore size ˚ (A)

R 90 (kV)

21 23 29 40 26 13 22

2.2 3.1 3.2 3.1 2.9 2.9 3.2

0.27 0.47 0.33 0.25 0.33 0.31 0.34

8.0 8.7 7.5 7.0 7.8 7.6 7.6

2.9

0.31

7.6

0.38 13%

0.037 12%

Average S.D. Coefficient of variance (CV)

Pmannitol (310 27 cm/s)

0.4 4.4%

Purea (310 27 cm/s)

Pe (urea)

Stage III: AC only Pmannitol (310 27 cm/s)

Pe (mannitol)

Pore size ˚ (A)

R 099 (kV)

Purea (310 27 cm/s)

21 16 31 42 25 9.3 12

2.5 3.4 2.9 2.9 3.0 2.9 3.2

0.36 0.73 0.30 0.30 0.53 0.60 0.47

8.5 11 7.5 7.5 9.4 10 8.6

2.9

0.43

8.6

0.13 29%

1.0 12%

Pmannitol (310 27 cm/s)

Pore size ˚ (A)

4.9 7.4 4.7 5.3 3.9 3.7 5.2

1.9 2.1 0.8 1.2 0.6 0.5 1.1

1.7 4.6 0.78 1.0 0.80 1.2 1.8

6.3 9.8 2.1 3.9 2.1 3.8 5.3

6.7 10 5.7 5.7 6.6 10 7.6

4.6

1.0

1.2

3.9

7.1

0.67 14%

0.5 50%

0.45 37%

1.7 43%

1.6 23%

0.22 7.9%

Pmannitol (310 27 cm/s)

Pore size ˚ (A)

Data not included in averages.

Table 5 Summary of the experiment data obtained at 4.8 kV cm 2 HEM sample

Stage I: AC only R0 (kV)

Purea (310 27 cm/s)

56 42 50 142 34

2.0 1.9 1.9 2.2 2.2

Average

1.9

S.D. Coefficient of variance (CV)

0.15 7.4%

R S T U V

Stage II: AC with 400-mV DC offset Pore size ˚ (A)

R 90 (kV)

0.22 0.20 0.20 0.19 0.27

7.7 7.6 7.6 7.1 8.0

50 42 42 42 16

0.21

7.6

Pmannitol (310 27 cm/s)

0.032 15%

0.3 4.3%

Purea (310 27 cm/s)

Pe (urea)

Stage III: AC only Pmannitol (310 27 cm/s)

Pe (mannitol)

Pore size ˚ (A)

R 990 (kV)

Purea (310 27 cm/s)

70 39 27 29 12

2.1 2.3 2.1 2.5 2.4

0.27 0.24 0.38 0.29 0.35

8.1 7.5 9.6 7.8 8.6

0.29

8.3

4.5 2.5 2.8 3.4 3.9

1.9 0.58 0.83 0.95 1.3

0.80 0.34 0.96 0.64 1.0

3.5 1.2 4.8 3.3 3.6

5.4 5.4 8.6 5.9 6.2

3.3

1.1

0.70

3.3

6.3

2.2

0.81 24%

0.5 46%

0.27 36%

1.3 40%

0.4 6.9%

0.18 7.8%

0.058 19%

0.8 9.9%

H. Zhu et al. / Journal of Controlled Release 89 (2003) 31–46

K La M N O P Q

Stage II: AC with 400-mV DC offset

H. Zhu et al. / Journal of Controlled Release 89 (2003) 31–46

Fig. 3. Simulation of hindrance factors for permeants, urea and mannitol, and conducting ions, Na 1 and Cl 2 , as a function of pore size during AC only (stages I and III) experiments.

Hurea /HCl 2 remain relatively constant, the ratios Hmannitol /HNa 1 and Hmannitol /HCl 2 essentially double ˚ Based upon when pore size increases from 6 to 10 A. Eq. (14) and analysis of Fig. 3, a plot of log P vs. log R will yield a uslopeu of 1 for urea regardless of pore size changes and a uslopeu greater than 1 for mannitol if pore size increases as resistance decreases. Figs. 4 and 5 show experimental log PAC vs. log R data for stage I and stage III, respectively. A linear relationship exists between log PAC and log R for urea and mannitol for each stage. These relationships are consistent with the results obtained during passive and iontophoretic transport in previous studies [17,19,27]. From stage I data (Fig. 4), the uslopesu shown for urea and mannitol are 0.98 and 1.20, respectively. From stage III data (Fig. 5), the uslopesu shown for urea and mannitol are 1.01 and 1.13, respectively. A multivariance regression analysis was performed to test the difference between the uslopesu for urea and mannitol. This analysis showed that the difference in uslopeu for urea and mannitol is statistically significant ( p,0.05) for the stage I data. The correlation between permeability and resistance and the difference in the urea and mannitol slope are both consistent with previous observations [19,27]. The urea slope is in excellent agreement with predictions based upon theoretical considerations and permeant / ion sizes. The mannitol slope indicates that as resistance is reduced under the present

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Fig. 4. Logarithm of urea and mannitol P-values (cm / s) versus logarithm of HEM electrical resistance (kV cm 2 ) for stage I experiments.

experimental conditions, the effective pore size increases. Although the calculated mannitol slope is larger than the urea slope for stage III, there is more variability in the stage III permeability measurements; therefore there is no statistical difference between the slopes for the two permeants. The differences in the degree of HEM breakdown by the 3rd day of the protocol and the effects of this breakdown on specific membrane properties most

Fig. 5. Logarithm of urea and mannitol P-values (cm / s) versus logarithm of HEM electrical resistance (kV cm 2 ) for stage III experiments.

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likely are responsible for the increase in inter-HEM variability observed in stage III relative to stage I. HEM pore sizes for stages I and III were calculated using Eqs. (2)–(5). The individual pore size determinations for each trial are shown in Tables 2–5 and the average values are presented in Fig. 6. Due to possible pore size distributions in HEM during CCAC iontophoresis, the applicability of these pore size calculations is limited to the size range of the permeants used in the present study. For stage I, there is a general trend of increasing membrane pore size as HEM resistance decreases. The pore size changes evident from Fig. 6 are consistent with the analysis of the slopes determined from Fig. 4. The pore size changes were relatively small when resistance was lowered from 4.8 to 1.6 kV cm 2 , but more pronounced when resistance was decreased from 1.6 to 0.8 kV cm 2 . Pore size and hindrance are important issues even when considering transport of molecules as small as glucose and become more significant as the permeant size increases. Although the trend indicates slight increases in pore size with decreases in resistance, even at 0.8 kV cm 2 , the measured pore size corresponds to a very restrictive pore and significant hindrance for even small molecule permeants. A significant advancement would be to determine a means of effectively increasing the membrane pore size during iontophoresis, thus decreasing the effects of hindrance in terms of low permeability and variability associated with permeant size.

Fig. 6. Estimated HEM pore sizes during stages I and III experiments at different target resistance levels.

The stage III pore size estimates tend to be larger than the stage I estimates for the same HEM sample and experimental condition. It is evident from the R 0 values shown in Tables 2–5 that there is some deterioration in the HEM membranes from the beginning of stage I to the beginning of stage III. Although the changes are relatively small when these estimates are made with HEM samples that have been returned to the same target resistance level, it is likely that, during prolonged exposure to an electrical current without control of HEM resistance, the changes in the membrane parameters such as pore size will be more significant. These considerations, although not investigated by others attempting to develop iontophoresis devices, most likely play a role in the significant variability observed in both iontophoretic drug delivery and endogenous substance sampling. During stage II, a 400-mV DC offset voltage was applied. Therefore, both electroporation and electroosmosis contribute to stage II transport enhancement. Tables 2–5 show Pe values calculated for each skin sample at all target resistance levels. These values are indicative of the contribution of electroosmosis to the total permeant flux. Consistent with previous findings, the measured Pe values are always greater for mannitol than for urea as the molecular size of mannitol is larger than that of urea. Although the current densities varied from 0.08 to 0.5 mA / cm 2 at different target resistance levels (under the 0.8, 1.6, 3.2, and 4.8 kV cm 2 protocols with 400-mV DC offset), the average Pe values for urea and mannitol at these different target resistance levels remained relatively constant, ranging from 1.0 to 2.0 for urea and 3.2 to 4.4 for mannitol. This is consistent with previous observations that enhancement due to electroosmosis relative to passive is proportional to the applied voltage [28]. If membrane porosity is the same during passive transport and iontophoresis, or the changes in the membrane porosity during iontophoresis are properly accounted for, electroosmotic flux enhancement is only a function of the applied voltage and is independent of current or resistance. The relatively constant Pe values also indicate that the average membrane charge density was relatively constant at different target resistance levels. However, individual Pe values for different HEM samples showed significant inter-sample variability,

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ranging from 0.5 to 3.1 for urea and 1.2 to 7.2 for mannitol. An overall analysis of the determined Pe values indicates that there is a large inter-sample variability in membrane pore charge and that this variability is quite independent of the HEM resistance level. Pore sizes for stage II were estimated using Eqs. (7)–(11) and are listed in Tables 2–5. The pore size estimates for stage II (AC plus DC) are generally smaller than those from stages I and III (AC only). One possible explanation for the smaller pore sizes determined for stage II relative to the passive stages is that different equations must be applied to estimate pore size when electroosmosis contributes to flux. The validity of these equations has been demonstrated using synthetic membranes, but the smallest pore size synthetic membrane systems studied were ˚ (pore radius), which is greater than the |45 A effective pore size of HEM [29–31]. To be certain that the validity these functions extends to the pore size range of HEM, further studies must be conducted. Another interesting and probable explanation for the smaller pore size estimates obtained for stage II relates to the possible existence of a pore charge distribution in HEM with neutral, negative, and positive charged pores [32]. Flux enhancement from electroosmosis results from solvent flow in negatively charged pores. Therefore, the pore sizes calculated in stage II will be weighted heavily toward the effective size of the negatively charged pores. The ‘passive’ fluxes measured during stages I and III are independent of pore charge for the neutral permeants, therefore the pore size estimates determined from the ‘passive’ stages will be an effective estimate of the size of all the pores independent of the pore charge. Hence, the smaller pore sizes estimated for stage II might be an indication that the negatively charged pores are smaller in size than the average size of the neutral and positively charged pores. For stage II pore size estimates, there is no apparent influence or trend corresponding to HEM resistance level. Characterizing the HEM parameters that influence transport for the stage II data is complicated by the fact that both pore size, through an influence upon H and W, and pore charge, through an influence upon solvent flow velocity, v [23], must be taken into consideration. Fig. 7 shows a plot of log P vs. log

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resistance for the stage II data. As illustrated by Eq. (16), linear relationships with uslopeu values of 1 would indicate constant membrane parameters as resistance is varied. Relatively linear relationships are shown in Fig. 7 and the uslopeu values are essentially 1, indicating relatively constant membrane properties despite a relatively wide range in membrane resistance levels. However, significant variability exists in the stage II permeability measurements making detailed analysis of the data shown in Fig. 7 difficult. The primary objective of developing and characterizing CCAC iontophoresis is the necessity to minimize permeant flux variability during iontophoresis. Tables 2–5, show that during stage I (AC only), the coefficients of variance (CV) for P-values ranged from 7 to 13% for urea, and 7 to 15% for mannitol. In stage II (AC plus DC), corresponding CVs increase to 14–26% for urea and 32–37% for mannitol. In stage III (AC only), these numbers were 8–16% for urea and 12–29% for mannitol. Considering the wide range in initial resistance for the different HEM samples and the degree of electroporation necessary to bring the HEM samples to the target resistance levels and that there are other sources of experimental variability (e.g. pipetting and assay), the inter-sample variability in permeability for urea and mannitol during stage I is remarkably

Fig. 7. Logarithm of urea and mannitol P-values (cm / s) versus logarithm of HEM electrical resistance (kV cm 2 ) for stage II experiments.

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low. The inter-sample variability in permeability is significantly higher during stage II when electroosmosis contributes to flux (F-test for the equality of variance, p,0.025). The stage III variability is lower than stage II, approaching stage I levels for urea and slightly higher levels than stage I for mannitol (F-test, p,0.05, except stages II and III for urea at 1.6 kV cm 2 ). As previously discussed, the similarity in size between urea and the background electrolyte ions leads to very tight control of urea permeability when the HEM electrical resistance is brought to a specific target level. The importance of matched permeant and electrolyte ion size when quantitatively correcting for the influence of electroporation upon flux has previously been reported [25]. Even for mannitol, where the permeant is approximately two-fold larger than the background electrolyte ions, the stage I variability in permeability is very low relative to other flux studies when HEM resistance is not controlled as it was in this study. The low variability in mannitol permeability indicates minimization of inter-sample pore size variability when the electrical resistance was brought to the target levels. When the flux was enhanced by the application of a 400-mV DC potential during stage II, the CV values approximately double for urea and mannitol despite the fact that the membrane electrical resistance was maintained constant during this stage, as it was during stage I. During stage II, electroosmosis contributes to the measured permeability; therefore, variability in permeability during this stage that is greater than observed during stage I is an indication of variability in electroosmotic solvent flow. Electroosmosis is a function of the applied voltage, membrane pore size, and effective pore charge density [7,14,28,33]. As the applied voltage was constant and the membrane pore size estimates showed little variability, the stage II variability is an indication of inter-sample variability in effective pore charge density. A key element of the experimental design for the present study was the measurement of flux at target resistance levels without and with the contribution of electroosmosis. This has allowed demonstration with relative certainty that variability in pore charge density contributes significantly to variability in flux when electroosmosis is an important factor influencing flux. Urea

being the smaller permeant, and therefore influenced less by solvent flow, shows less permeability variability during stage II than does mannitol. The practical outcome from this phase of the study is the insight that variability in electroosmosis is a significant source of flux variability for neutral permeants, even when HEM is brought to a constant level of electroporation. If sufficient flux levels can be obtained in applications such as glucose extraction by electroporation using an applied AC potential without a DC potential (i.e. no electroosmotic contribution to flux), flux variability could be minimized. Stage III experiments were performed to ascertain how closely permeability could be reproduced following extended exposure to iontophoresis and how the HEM parameters change during this exposure. Tables 2–5 also show that the stage III P-values and standard deviation for both urea and mannitol were generally slightly larger than stage I values. On a percentage basis, mannitol permeability increased more than urea indicative of an increase in pore size from stage I to stage III. Fig. 6 and Tables 2–5 confirm this general increase in pore size. Our laboratory recently reported results from a constant current DC iontophoresis study where mannitol flux varied significantly between HEM samples and during the course of flux experiments for a given HEM sample, despite the constant current condition [12]. In those experiments, the applied current density was maintained constant but skin electrical resistance varied from sample to sample, and also varied with time during the course of the experiment. The large inter- and intra-sample variability in constant current mannitol flux can be explained based upon the present study revelations that membrane pore size changes with changes in HEM resistance and large inter-sample variability in pore charge density exists.

4. Conclusions The present study shows that AC voltages can be used to reduce and maintain HEM electrical resistance at target levels as low as 0.8 kV cm 2 . Low inter-sample variability in permeability was observed when HEM samples were brought to the same

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resistance level. Electrical resistance is a good indicator of the extent of electroporation and can be used quantitatively to predict permeant permeability when the background electrolyte ion size matches the permeant ion size as in the case of Na 1 , Cl 2 and urea. Results from the AC only and AC plus DC iontophoresis experiments showed that permeability determined during AC only experiments showed less inter-sample variability than permeability determined during AC plus DC experiments. Although resistance was maintained constant for both conditions, variability in the AC plus DC experiments indicates variability in electroosmotic solvent flow as a result of significant variability in effective HEM pore charge density. If tight control of iontophoretic permeant delivery or extraction rate is required, constant conductance AC only iontophoresis may be a superior protocol as long as required transport rates can be achieved. The present study also suggests that there is a trend of increasing pore size during AC only experiments as HEM electrical resistance decreases and as the HEM begins to breakdown after extended periods of time. Acknowledgements The authors would like to thank Dr Aniko Szabo for her suggestions in the statistical treatment of the data and Watson Laboratories-Utah for kindly providing some of the human skin samples. This work was supported in part by NIH Grant GM 063559. References [1] B.K. Chen, B.B. Cunningham, Topical anesthetics in children: agents and techniques that equally comfort patients, parents, and clinicians, Curr. Opin. Pediatr. 13 (2001) 324– 330. [2] G.W. Creasy, L.S. Abrams, A.C. Fisher, Transdermal contraception, Semin. Reprod. Med. 19 (2001) 373–380. [3] G.K. Gourlay, Treatment of cancer pain with transdermal fentanyl, Lancet Oncol. 2 (2001) 165–172. [4] C.T. Sweeney, R.V. Fant, K.O. Fagerstrom, J.F. McGovern, J.E. Henningfield, Combination nicotine replacement therapy for smoking cessation: rationale, efficacy and tolerability, CNS Drugs 15 (2001) 453–467. [5] S.K. Li, A.H. Ghanem, C.L. Teng, G.E. Hardee, W.I. Higuchi, Iontophoretic transport of oligonucleotides across human epidermal membrane: a study of the Nernst-Planck model, J. Pharm. Sci. 90 (2001) 915–931.

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