Transdermal iontophoretic permeation of luteinizing hormone releasing hormone: Characterization of electric parameters

journal o f

ELSEVIER

controlled release Journal of Controlled Release 40 (1996) 187 - 198

Transdermal iontophoretic permeation of luteinizing hormone releasing hormone: Characterization of electric parameters Li-Lan H. Chen, Yie W. Chien * Controlled Drug-Delive~ Research Center, Rutgers Universi~, College of Pharmacy, 41-D Gordon Road, Piscataway, NJ 08854, USA Received 21 April 1995; accepted 25 October 1995

Abstract A single-compartment iontophoretic permeation cell incorporated with two polyacrylamide hydrogel reservoir devices was used to characterize the effect of several electrical parameters on transdermal iontophoretic permeation of LHRH through hairless rat skin. Receptor solutions and skin regions were selected before evaluating the effect of electrical parameters, which included different application patterns, various waveforms and o n / o f f ratios. Iontophoretic permeation of LHRH was higher while current was applied continuously rather than in an intermediate pattern. The difference among various waveforms was not significant. However, triangular wave seems to have the least iontophoresis enhancing effect and different voltage profiles. The o n / o f f ratios of pulsed direct current showed a significant effect on transdermal iontophoretic permeation of LHRH, and demonstrated that the higher the o n / o f f ratio (duty cycle), the greater the skin permeation of LHRH. Duration of current application was more important than amplitude and intensity of current in terms of transdermal iontophoresis. The voltage profiles (including onset, apparent and offset voltage) for different o n / o f f ratio with either same or different current amplitude demonstrated that the voltage reduction was very slow for the o n / o f f ratio with a lower value. This implies that the skin resistance remained high during current application resulting in lower skin permeation of LHRH for lower o n / o f f ratio. Keywords: Iontophoresis; LHRH; Polyacrylamide hydrogel; Waveform; O n / o f f ratio

1. Introduction The skin has been used for several decades as the site for topical administration of dermatological drugs to achieve localized pharmacological action in skin tissues [1]. In recent years, the potential of using skin as a site for systemic delivery of therapeutic agents has been recognized [1,2]. Several biomedical benefits have been derived from transdermal delivery of drugs, among them are: (i) elimination of variables

* Corresponding author. Fax: + 1 908 4456175. 0168-3659/96/$15.00 Published by Elsevier Science B.V. SSD1 0 1 6 8 - 3 6 5 9 ( 9 5 ) 0 0 1 8 1 - 6

accompanying oral administration, such as pH, transit times, and presence of food and enzymes; (ii) improvement of systemic bioavailability resulting from bypassing of hepatic first-pass metabolism; (iii) better patient compliance due to ease of application and removal, and elimination of injection requirement; and (iv) essentially constant rate of delivery. Since the inception of genetic engineering, therapeutic application of p e p t i d e s / p r o t e i n s has received increasing attention [1,3-7]. The lack of systemic bioavailability from oral administration, and the frequent injection requirements of parenteral adminis-

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L.-L.H. Chen, E W. Chien / Journal of Controlled Release 40 (1996) 187-198

tration have prompted scientists to search for viable routes of nonoral and nonparenteral delivery of biotechnologically produced peptides/proteins [1,8]. The need for novel techniques to efficiently deliver these drugs has been increasingly recognized, particularly as more and more therapeutically useful peptides/proteins have been produced by bioengineering. The potential of transdermal systemic delivery of proteins/peptides has been recognized, since skin has the lowest enzymatic activity of the various delivery routes [1]. However, the barrier properties of the stratum corneum generally limit permeation by passive diffusion to lipophilic drugs of small molecular size [8]. The major obstacle to transdermal systemic delivery of proteins/peptides is the impermeability of hydrophilic and ionic macromolecules across the lipophilic stratum corneum by passive diffusion. Various skin permeation-enhancing techniques have been employed to improve skin permeation of proteins/peptides. Among these permeation-enhancing techniques, iontophoresis has been recognized as a tool to increase skin permeation of charged and hydrophilic macromolecules. The term 'iontophoresis' is used in medical literature to indicate the process of increasing penetration of charged drugs into surface tissues by the application of an electric current for therapeutic purposes. Iontophoresis has the potential to overcome several limitations of conventional transdermal systems, and could be used to deliver ionic, hydrophilic and high-molecular-weight drugs [9]. Iontophoresis-facilitated transdermal delivery has several advantages over other skin permeation-enhancing techniques and holds promise for controlled delivery of a variety of compounds. Iontophoretic delivery of drugs in a pulsatile pattern will be most useful in treating conditions which required higher serum drug concentrations only at particular times, as for the physiological secretion of endogenous hormones such as LHRH. Luteinizing hormone-releasing hormone, [LHRH, also called gonadotropin releasing hormone (GnRH)], is a decapeptide (1182 Da) with three ionizable amino acid residues (His 3, Tyr 5 and Arg 8) [10]. It is known to be secreted from the hypothalamus in a pulsatile pattern (one pulse every 60-120 min) to activate pituitary release of luteinizing hormone (LH) and follicle stimulating hormone (FSH), which in turn act on sex organs to stimulate production of

gonadal steroids such as testosterone and progesterone/estradiol [11]. Natural LHRH and its analogs were successfully delivered into pigs [12] and human volunteers [13-16] by transdermal iontophoresis. However, the evaluation of electric parameters on transdermal iontophoresis of LHRH was not conducted. The objectives of this article were to investigate and characterize the effects of electrical parameters on transdermal iontophoresis of LHRH using polyacrylamide hydrogel as a reservoir matrix. This work was presented at 7th AAPS Annual Meeting, San Antonio, TX, in 1992.

2. Experimental 2.1. Materials Unless otherwise stated, all chemicals were ACS reagent grade or better, and were used as received. All solutions were prepared using distilled water (18 MQ) which had been filtered through a Barnstead purification system (Dubuque, IA). Luteinizing hormone-releasing hormone (LHRH-AcOH 2.5 H20) was purchased from Bachem Bioscience, Inc. (Philadelphia, PA). Platinum foils (99.95% purity), which were used to fabricate electrodes, were obtained from Johnson Mattey (Seabrook, NH). Acrylamide, N'N'-methylene bis-acrylamide, ascorbic acid, ferrous sulfate and 30% hydrogen peroxide, which were used to synthesize polyacrylamide hydrogel, were ordered from Sigma Chemical Co. (St. Louis, MO). High performance liquid chromatography (HPLC) grade acetonitrile (Fisher), potassium phosphate and sodium hydroxide (Sigma) were used to prepare HPLC mobile phase. 2.2. Animal model Female hairless rats (6-8 weeks old, Harlan Sprague Dawley Inc., Indianapolis, IN) were used as the animal model. The rat was first sacrificed by asphyxiation just before the initiation of each experiment. Full-thickness skin specimens were then freshly excised from the abdominal and dorsal regions, then cut into pieces of 4 cm 2 each for mounting onto the openings (0.64 cm z available surface area) of a

L.-L.H. Chen, Y. W. Chien / Journal of Controlled Release 40 (1996) 187-198

m

189

hydrogel disc

~

Teflon holder

Reservoir device

Electrode housing (platinum/tin-pvc/ plastic holder,

--

ii~,~iiii!

Fig. 1. A schematic illustration of the assembly of an iontophoretic reservoir device consisting of a transparent, crosslinked polyacrylamide hydrogel disc, one Teflon holder and one electrode housing unit.

specially designed single-compartment skin permeation cell for transdermal iontophoretic studies [17].

2.4. Preparation of LHRH-loaded polyacrylamide

2.3. Power supply

Polyacrylamide hydrogel was directly synthesized from a monomer (acrylamide) and a crosslinker (N'N' methylene bis-acrylamide) at room temperature under acidic conditions [18]. 15% ( w / v ) of acrylamide, 1.05% ( w / v ) of methylene acrylamide (7% ( w / w ) of monomer), 0.1% ( w / v ) of ascorbic acid, and 0.0025% ( w / v ) of ferrous sulfate were dissolved in citrate phosphate buffer of pH 3.6 to become the monomer solution. LHRH-loaded

The programmable Keithley 500 A control and measurement system (Keithley, Cleveland, OH) was used as a power supply in this investigation. The Keithley 500 A was controlled by a computer, and was able to generate four channels of simultaneous output with different current intensities, waveforms, frequencies and o n / o f f ratios.

a

hydrogel

Jaode m p l i n g port

1. a b d o m i n a l skin 2. L H R H r e s e r v o i r c

water jacket

• dorsal skin • blank reservoir device

central compartment

synchronous motor Fig. 2. A schematic illustration of in vitro iontophoretic skin permeation setup, which contained one single-compartment iontophoretic permeation cell with two openings for mounting skin specimens and reservoir devices.

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monomer solution (5 m g / m l ) was prepared and 175 ixl of the solution was pipetted into a specifically designed Teflon holder. Polymerization was then initiated by adding 0.03% of hydrogen peroxide to the holder. Polymerization was completed within one min at room temperature. LHRH-loaded and transparent crosslinked polyacrylamide hydrogel disc with a diameter of l0 mm was formed inside the Teflon holder.

2.5. Fabrication of LHRH reservoir det~ice As shown in Fig. 1, the LHRH-loaded or blank crosslinked polyacrylamide hydrogel disc was polymerized inside the Teflon holder and then put into an electrode housing device which consisted of a platinum electrode foil, a tin-pvc patch and a plastic holder.

2.6. Analytical method All samples were analyzed by HPLC method. A HP 1090 system with a multi-wavelength UV detector and a MOS C 8 reversed phase column (100 × 4.6 mm i.d.) (Hewlett-Packard) was used. The wavelength at 220 nm was used to detect LHRH since Trp 3 and Tyr 5 have maximum UV absorption at 219 and 222 nm, respectively [19]. The mobile phase used was potassium phosphate buffer (0.5 M) with pH 6.5 and acetonitrile at a ratio of 70/30. The flow rate of mobile phase was 1 m l / m i n and the injection volume was 25 Ixl.

2.7. Experimental setup for iontophoretic permeation studies of LHRH Fig. 2 illustrates the experimental setup to conduct in vitro iontophoretic permeation studies [17]. Abdominal skin was mounted onto one opening of the iontophoretic permeation cell, and dorsal skin was mounted onto the other opening. The volume capacity of the central compartment was 4-4.5 ml. The maximum volume of 4.5 ml buffer (0.01 M of dimethylglutaric acid and 0.2% albumin in 0.86% of NaC1 with a pH 3.6) was pipetted into the central compartment as the receptor solution. A LHRHloaded reservoir device was mounted onto the abdominal skin, and a LHRH-free reservoir device was

attached to the dorsal skin to complete the experimental setup. An anodic electrode was applied to the LHRH-loaded reservoir device as a working electrode to deliver LHRH into the abdominal skin (LHRH was positively charged at pH 3.6). A cathodic electrode was applied to the LHRH-free reservoir device to complete the electric circuit. Samples (50 Ixl each time) were taken from the sampling port at a predetermined intervals, and replaced with the same volume of LHRH-free buffer to keep constant volume (4.5 ml) in the central compartment of the iontophoretic permeation cell.

2.8. Effect of loading doses Iontophoretic permeation of LHRH through the hairless rats' abdominal skin with two loading doses (175 and 875 txg) was compared by applying 3 h of pulsed direct current ( o n / o f f 1:1) with a current intensity 0.6 mA. Normal saline at pH 3.0 (adjusted by 1 M HC1) was used as the receptor solution. The experimental setup is shown in Fig. 2.

2.9. Effect of receptor solutions Three different buffer solutions (normal saline adjusted to pH 3.0, isotonic dimethylglutaric a c i d / s o d i u m chloride solution pH 3.6 and citrate/phosphate buffer pH 5.0) were used as receptor solutions in the central compartment of the iontophoretic permeation cell to compare their effect on transdermal iontophoretic delivery of LHRH through abdominal skin of hairless rats. Direct current with a current intensity of 0.6 mA was continuously applied for three h.

2.10. Effect of skin sites The thickness of skin specimens was measured before mounting onto the iontophoretic permeation cell openings. A standard procedure, in which the whole skin specimen was sandwiched between two microscope glass slides, was used to determine skin thickness. The skin thickness was calculated by subtracting the thickness of two glass slides from the total thickness of the skin-slides sandwich, as measured by micrometer. The isotonic dimethylglutaric

L.-L.H. Chen, E W. Chien / Journal of Controlled Release 40 (1996) 187-198

acid buffer was used as the receptor solution, and the loading dose of LHRH in each anodic reservoir assembly was 875 ~g for all studies. Direct current with a current intensity of 0.6 mA was applied continuously for 3 h. The study was continued for another 3 h after current was withdrawn. Samples (50 OA each) were taken from the sampling port and analyzed by HPLC. The iontophoretic permeations of LHRH through the abdominal skin and the dorsal skin of hairless rats were compared.

2.11. Effect of current application patterns Three different current application patterns with a direct current (0.6 mA) were compared for their effect on iontophoretic permeation of LHRH through abdominal skin of hairless rats.

li

(A)

central compartment

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R(s)

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electrode 1

(c) @

2.13. Effect of on / off ratios Various o n / o f f ratios with the same current amplitude or same current intensity output at frequency 1 kHz were continuously applied for 3 h, and their effects on the iontophoretic permeation of LHRH through the abdominal skin were studied.

2.14. Measurement of voltage difference

3.1. Effect of loading doses

(B) R'(s)

Four different waveforms, at the same frequency (1 kHz) and current intensity (0.6 mA), were continuously applied for 3 h, and their effects on the iontophoretic permeation of LHRH through abdominal skin of hairless rats were compared.

3. Resultsand discussions

G R'(g)

2.12. Effect of waveforms

Voltage difference between two reservoir devices was measured by Simpson digital multi-meter for apparent voltage and by LBO-522 Oscilloscope for onset and offset voltages during the course of iontophoretic permeation. Fig. 3 is the schematic illustration of electrostatic potential drop (C) and equivalent electric circuit (B) between two electrodes according to the in vitro experimental setup (A).

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/

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Fig. 3. A schematic illustration for measurement of voltage (A), the equivalent electric circuit for the voltage measurement (B) and the electrostatic potential drop between two electrodes (C).

The effect of loading doses on iontophoretic permeation of LHRH was very significant, and is shown in Fig. 4. As expected, iontophoretic permeation of LHRH was greater with higher loading dose (875 p~g/175 ~1) due to the higher driving force (electrochemical potential). The concentration of LHRH in the central compartment with 175 p~g/175 Ixl as the loading dose was too low to be detected by our HPLC method.

3.2. Effect of receptor solutions Iontophoretic fluxes of LHRH were significantly influenced by buffers used as receiving media, as shown in Table 1. Among the three different receptor solutions, normal saline solution (0.9% NaC1) seems

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L.-L.H. Chen, Y. W. Chien / Journal of Controlled Release 40 (1996) 187-198 20

due to the lack of buffer capacity. There was a very slight pH increase for isotonic D M G A solution which contained 0.01 M dimethyl glutaric acid and 0.86% NaC1. C i t r a t e / p h o s p h a t e buffer was able to maintain constant pH owing to its high buffer capacity but L H R H was unstable at pH higher than 5 [20] and skin integrity could not be maintained for buffer pH lower than 5. D M G A solution was chosen as the receptor solution because of its ability to maintain the stability of L H R H in aqueous solution, the consistency of solution pH, and the physical integrity of skin.

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Fig. 4. The effect of loading doses in polyacrylamide hydrogel discs on transdermal iontophoretic permeation of LHRH through abdominal skin of hairless rats. to give the highest iontophoretic flux with no lag time and there was no significant difference between D M G A and phosphate buffers. However, the postactive flux (after current application), which demonstrated the significant difference which implies two possibilities. The first possibility is that the desorption phenomena of L H R H from the skin depends on the buffer used. Another reason might be due to the different degree of degradation in the receptor medium after permeation [20]. A significant pH shift (increased by 2.56 units) was observed when normal saline (pH adjusted to 3 by adding HC1) was used

3.3. E f f e c t o f skin sites

Iontophoretic permeation of L H R H through abdominal skin was much higher than that through dorsal skin (the iontophoretic permeation profiles are shown in Fig. 5). Table 2 summaries the fluxes during and after current application, lag time, thickness of skin specimens and diffusivities for abdominal and dorsal skins. The iontophoretic permeation rate of L H R H through the hairless rats' abdominal skin was two times higher than that through the dorsal skin; however, permeation of L H R H after iontophoresis was not significantly different between skin sites, which indicates that the desorption of L H R H from dorsal skin was the same as from abdominal skin. The lag time for L H R H to reach the

Table 1 The effect of receptor solutions on the iontophoretic permeation of LHRH through the abdominal skin of female hairless rats Flux a (/xg/cm 2 h) Flux b (ixg/cm2 h) Lag time (h) pH shift (pH unit) DMGA(pH 3.6) Normal saline(pH 3.6) Phosphate buffer(pH 5.0)

2.26( + 0.09) 2.62( _+0.29) 2.19( _+0.16)

0.99( + 0.10) 0.73( _+0.06) 0.41 ( _+0.08)

0.31 ( ± 0.06) 0.25( + 0.08)

0.42( + 0.07) 2.56( _+0.76) 0.02( _+0.01)

a Flux during application. b Flux after current application. Table 2 The comparison of iontophoretic permeation of LHRH through the abdominal and the dorsal skins of female hairless rats Flux a(Ixg/cm2 h) Flux b(Ixg/cm2 h) Lag time(h) Thickness(mm) Diffusivity(mm~-/h) Abdominal skin Dorsal skin

2.26( _+0.09) 1.22(+0.39)

Flux during current application. b Flux after current application. a

0.99( + O.10) 1.15(_+0.26)

0.31( + 0.06) 1.53(_+0.22)

0.33( +_0.01) 0.55(_+0.01)

0.059 0.033

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L.-L.H. Chen, Y.W. Chien/ Journal of Controlled Release 40 (1996) 187-198

skin. Diffusivity of L H R H through both skin specimens was calculated according to the lag time method, and proved that the diffusivity of hairless rat dorsal skin was much lower than that of abdominal skin. Abdominal skin was used for the rest of the investigation due to its higher permeability.

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3.4. Effect o f current application patterns

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As shown in Fig. 6, the total cumulative amount of L H R H was highest for current application pattern A, in which 0.6 m A direct current was continuously applied for 3 h and then withdrawn. Application pattern C gave the least iontophoretic permeation of L H R H among the three application patterns even though the total duration of current application was the same. According to the results and tracks, one should be able to predict and expect a lower iontophoretic permeation of L H R H with a more frequent application pattern. The possible cause of lower

r

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2

3

4

Time

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(hour)

Fig. 5. Transdermal iontophoretic skin permeation profiles of LHRH with a loading dose of 875 Ixg through abdominal and dorsal skin. steady state through dorsal skin was five times longer than through abdominal skin, even though the dorsal skin was only two times thicker than the abdominal

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Fig. 6. Comparison of transdermal iontophoretic skin permeation profiles of LHRH by three different application pattems with a current intensity of 0.6 mA.

194

L.-L.H. Chen, Y.W. Chien/ Journal of Controlled Release 40 (1996) 187-198 3.5. Effect o f waveforms equilibrium concentration gradient C t (max)

The permeation fluxes of L H R H during and after current application and A U C for different waveforms are summarized in Fig. 8. The postactive fluxes (after current application) were much lower than the active fluxes (during iontophoresis) which indicates the electrical current enhancement of transdermal transport of LHRH. The iontophoretic fluxes among direct current, sinewave and squarewave showed no difference. However, desorption of L H R H from the skin for direct current was significantly higher than that for squarewave. Triangularwave seems to have the lowest iontophoretic permeation and postactive permeation compared to others from A U C data. The results indicate that a higher iontophoretic flux followed a greater desorption rate. Fig. 9 depicts voltage profiles among the three different waveforms. The apparent voltage showed no difference among three waveforms during the period of current application. However, the triangularwave seems to have lower onset and higher offset voltage profiles compared to squarewave and sinewave. Especially, the offset voltage of triangular wave was extremely different from, and significantly higher than the other waveforms. This may be the cause of lower iontophoretic permeation of L H R H when triangularwave was used at frequency 1 kHz with a higher offset voltage profile. More studies are needed to

tF,,l

C t (0)

\

Distance (thickness of skin)

Fig. 7. Schematic illustration of different concentration gradients across skin during various durations of current application. The transdermal iontophoretic skin permeation of LHRH is proportional to the concentration gradient, whose equilibrium concentration gradient is time-dependent.

iontophoretic permeation for frequent application patterns might be due to time-dependency of the change in chemical potential (no current application, E = 0) and electrochemical potential (during current application), as illustrated in Fig. 7.

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sinewave

squarewave triangularwave (on/off 1:1)

Fig. 8. Effect of various waveforms on permeation fluxes of LHRH during/after current application and AUC of permeation profiles.

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L.-L.H. Chen, Y.W. Chien / Journal of Controlled Release 40 (1996) 187-198

Square wave

Sine wave

Triangular wave

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Fig. 10. Differences in transdermal iontophoretic permeation profiles of LHRH for different on/off ratios with the same current amplitude.

Fig. 9. Comparison of voltage profiles for different waveforms during current application.

characterize the effect of different electric waveforms on transdermal iontophoretic permeation of macromolecules. 3.6. Effect o f o n / o f f ratios

Fig. 10 shows the effect of o n / o f f ratios (DC, 1 / 1 and 1 / 3 ) with the same current amplitude which gave different current intensity outputs (0.6, 0.3 and 0.15 mA) on iontophoretic permeation of LHRH. The lower iontophoretic permeation with a lower o n / o f f ratio in Fig. 10 might be simply due to the lower average current intensity output. The voltage potential across the whole in vitro skin permeation setup for various o n / o f f ratios with the same current amplitude is shown in Fig. 1 l. The results indicate that the slowly-reducing voltage profiles (including onset, apparent and offset voltages) during current application for lower o n / o f f ratio, which in turn implies the reduction of skin resistance maintained almost constant during current application. This might also contribute to the cause of lower iontophoretic permeation of L H R H for lower o n / o f f ratio. Fig. 12 shows iontophoretic permeation profiles of L H R H for different o n / o f f ratios and current amplitudes with the same current intensity output (0.6 mA) and frequency (1 kHz). Iontophoretic per-

meation of L H R H was higher for the greater o n / o f f ratio (higher duty cycle). Fig. 13 summaries the permeation fluxes of L H R H during and after current application for the various o n / o f f ratios. Except for the 1 / 3 o n / o f f ratio, which gave the lowest iontophoretic flux, there was no significant iontophoretic flux difference among them. However, the 8 / 1 o n / o f f ratio seems to have the highest postactive permeation flux. The AUC results demonstrate

Direct current Pulsed current 20

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L.-L.H. Chen, Y. W. Chien / Journal of Controlled Release 40 (1996) 187-198

20 • © •

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Fig. 12. Comparison of transdermal iontophoretic permeation profiles of LHRH for various on/off ratio with the same current intensity. the faster the voltage decrease, which indicates faster skin resistance reduction, which yielded the greater iontophoretic permeation. Fig. 15 compares the iontophoretic permeation profiles of continuous direct current and pulsed direct current ( l : l ) when two different receptor solutions were used. Both indicate that continuous direct current enhanced iontophoretic transport of L H R H

that the higher the o n / o f f ratio, the greater the iontophoretic permeation of LHRH. Fig. 14 shows the voltage drop as a function of time for various o n / o f f ratios with the same apparent current intensity, and illustrates different voltage profiles including onset, apparent and offset voltages during current application even though the average current intensity output was the same. The higher the o n / o f f ratio,

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Fig. 13. Summary of skin permeation fluxes during and after current application for various on/off ratios.

L.-L.H. Chen, KW. Chien / Journal of Controlled Release 40 (1996) 187-198 on/off = 8 : 1

on/off = 4 : 1

on/off = 1 : 1

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better than pulsed direct current even though the current intensity was the same. It is possible that the major transport pathways through skin for ions or charged molecules are transappendageal and intercellular, which can be considered as two parallel resistors (R' and R"), as shown in Fig. 16A. The transcellular pathway, which is not favored for the transport of hydrophilic charged molecules can then be assumed to be a parallel capacitor (C) due to its lipophilicity and ability to store electrons. The capacDMGA/saline

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itor for the transcellular pathway is an open-circuit when continuous direct current is applied. Therefore, current only flows through two parallel resistors and allows the transport of charged molecules through

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2

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Fig. 15. C o m p a r i s o n o f effects o f continuous direct current a n d pulsed direct current (1:1) with the s a m e current intensity (0.6 m A ) on transdermal iontophoretic skin p e r m e a t i o n of L H R H using two different buffers.

Fig. 16. S c h e m a t i c illustration o f a skin equivalent electric circuit a c c o r d i n g to: (A) three transport p a t h w a y s and their p h y s i c o c h e m ical characteristics; (B) current flow through three transport pathw a y s while continuous direct current is applied; a n d (C) current flow three transport p a t h w a y s while pulsed direct current is used.

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L.-L.H. Chen, E W. Chien / Journal of Controlled Release 40 (1996) 187 198

transappendageal and intercellular p a t h w a y s as s h o w n in Fig. 16B. W h e n pulsed direct current is used, s o m e current f l o w s through the lipophilic transcellular p a t h w a y without carrying any charged m o l e c u l e s , thereby r e d u c i n g transport efficiency o f L H R H through intercellular and shunt pathways, as depicted in Fig. 16C.

4. Conclusions Selection o f receptor solutions (pH, concentration and buffer species) and skin sites is v e r y important and needs to be standardized or indicated before starting any e x p e r i m e n t s and drawing any conclusions f r o m in vitro iontophoretic studies. Transdermal iontophoretic skin p e r m e a t i o n of L H R H was higher w h e n current was applied continuously in instead o f in an intermediate pattern due to the t i m e - d e p e n d e n t e l e c t r o c h e m i c a l potential during current application. The difference a m o n g various w a v e f o r m s was not significant. H o w e v e r , triangular w a v e seems to have the least p e r m e a t i o n - e n h a n c i n g effect. M o r e studies are needed to characterize the effects o f different w a v e f o r m s . The significant effect of o n / o f f ratio d e m o n s t r a t e d that the h i g h e r the o n / o f f ratio, the greater the iontophoretic p e r m e ation, and also indicated the i m p o r t a n c e o f duration o f current application at each cycle e v e n though current amplitude or current intensity output was the same. The difference in transdermal iontophoretic p e r m e a t i o n o f L H R H created by various o n / o f f ratios m i g h t also be due to their different electroc h e m i c a l potentials, which were t i m e - d e p e n d e n t at a m i c r o s c o p i c time scale. Duration of current application seems to be m o r e important than the amplitude and intensity o f current applied. The apparent voltage across the w h o l e e x p e r i m e n t a l setup could be a helpful tool to explain and support the effects o f different electric parameters on the transdermal iontophoresis studies.

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