Effect of lontophoresis on In Vitro Skin Permeation of an Analogue of Growth Hormone Releasing Factor in the Hairless Guinea Pig Model

SARAN KUMAR*', HING CHAR', SUNIL PATEL*, DAVID PIEMONTESE*, KHURSHID IQBAL*, A. WASEEM MALICK', ERIC NEUGROSCHEL*, AND CHARAN R. 6EHL*

Received April 16, 1991, from 'Pharmaceutical Research and Development and *Research Technical Support, Hoffmann-La Roche, Inc.. Nufky, NJ 071 10. Accepted for publication August 28, 1991.

Abstract 0 The shortened analogue of growth hormone releasing factor (GRF) Ro 23-7861 (1) has a molecular weight of 3929 daltons (equivalent to GRF (1-29)J and is more potent than the endogenous GRF (14). The in vitro hairless guinea pig model and vertical and horizontal diffusion cell assemblies were used to study the effect of iontophoresis on the permeability to skin of 1. The transport of 1 across the skin was studied by monitoring the rate of its appearance in the receiver compartment with a radioimmunoassay. No permeability of 1 was observed without iontophoresis, whereas with iontophoresis, the permeability of 1 was significant. For example, at a current density of 0.23 mA/cm2 and buffer concentration of 0.05 M, the flux of 1 was 56.8 2 8.21 ng/cm2 - h. The flux of 1 was independent of the design of the permeation apparatus, the electrodes, the donor and receiver volumes, the type of current (constant or pulsed), and the frequency of the pulsed current. The flux of 1 increased curvilinearly with the increase in salt concentration of the buffer and linearly with the increase in current.

Iontophoresis is a technique that uses low levels of current to enhance transdermal delivery of drugs. Such enhancement has been observed for both un-ionized and ionized mole- cules.'-' This technique is of particular benefit for the deliv- ery of peptide or protein drugs.- These drugs generally are inactive orally either because they are poorly absorbed be- cause of their polar nature or because of their enzymatic breakdown in the gastrointestinal tract. Various peptides and proteins have been listed as potential candidates for this type of delivery.6 Recently, iontophoresis was used successfblly in the transdermal administration of leuprolide, a leutenizing hormone releasing hormone analogue, in humans.6 Growth hormone releasing factor [GRF (1-4411 stimulates growth in humans and animals.9 The shortened analogue GRF (1-29) retains full activity with reduced potency.10 However, deriv- atives of GFtF (1-29) have yielded more-potent peptides.11-1s One such derivative is Ro 23-7861 (l), which has a molecular weight of 3929 daltons (see structure).

The objective of this study was to investigate various factors affecting the transdermal delivery of 1 with the in vitro hairless guinea pig model.14 Several investigators have used various designs of horizontal side-by-side diffusion cell as- semblies with either two1.6 or four16J6 electrodes. Some investigators have also used the vertical cell assernbly.17JS The vertical cell assembly mimics the clinical situation more

1 5 10 d e M H f l p A l a - A s p A l a - I l e ~ - A s r t - S e r ~

-Arq-Lys-Val-Lau-Ala4.n-lm-Ser-Ala-Arg 15 20

25 29 -Lys-m-L8u-Gln-AspIle+let-Ser-Arg~

RO 23-7861 (1)

closely and allows easier handling and testing of delivery systems. A direct comparison of these two diffusion cell setups was made in this study. Two modes of current outputs, constant and pulsed, are reported. Pulsed-current iontophore- sis provided transdermal absorption of metoprolol in humans and reduced skin irritation compared with constant-current iontophoresis.19 In rabbits, a greater efficiency of transdermal iontophoretic delivery of insulin by pulsed current, compared with constant current, has been observed in vivo.6 A direct comparison of the two modes of current output was made under in vitro conditions for the delivery of 1 in the present study. With the pulsed mode, the effects of salt concentration, current, voltage, and frequency on the permeation of 1 were also studied.

Experimental Section A summary of experimental conditions is given in Table I. Chemicals and Drug Solu t ioneAl l chemicals were used as

received. Ro 23-7861 (1) was obtained from Hoffmann-La Roche, Inc. The solubility of 1 in pH 4 acetate buffer is -20 mg/mL, as determined by visual observation of the cloud point. Acetate buffers of pH 4 with ionic strengths of 0.01,0.025, and 0.05 M were prepared by mixing0.2 M acetic acid and 0.2 M sodium acetate in a suitable proportion and diluting to the desired concentration. Drug solutions with a concen- tration of 3 mg/mL were prepared in buffers of each of the three ionic strengths.

Permeation Apparatus-For horizontal cell experiments, the side-by-side Valia-Chien cells obtained from Crown G l m Company were used. For the vertical cell experiments, the modified Franz cell was used. This apparatus consisted of receiver and donor compart- ments. The receiver side was placed in a chamber that allowed constant stirring of the receptor contents and also maintained a constant temperature. This chamber was especially designed and fabricated for use in a vertical setup. The receptor compartment of the vertical cell is equipped with a side arm that allows placement of the reference electrode and withdrawal of samples. The donor compart- ment is an open-faced cylindrical chamber that is placed on the flange of the receiver chamber and is held in place with a clamp. A polytetrafluoroethylene (Teflon) holder designed for holding the donor electrode was also placed on the donor side.

Electrode-Platinum electrodes were used in this study (Scheme I). In the horizontal cell setup, a wire electrode embedded in a g l m stopper was used in both donor and receiver compartments. In the vertical cell setup, the wire electrode embedded in a g l m stopper was used in the receiver side, but a plate electrode embedded in Teflon was used on the donor side (see Scheme I for dimensions of electrodes).

Current Output Generator-Constant- and pulsed-current out- puts were used in this study. The current output generators were built in-house (Scheme II). The constant-current unit was built to provide constant direct current, which varied from 0 to 1000 4. The unit had 12 output channels, with a current eetup dial for each channel and a liquid-crystal display for voltage readout. The pulsed- current unit was designed to provide adjustable peak voltages from 0 to 20 V and variable frequencies from 2 to 50 WIZ. The wave form used in the pulsed-current unit is shown in Scheme III. The odoff

OO22-3549/9H07OO-O635$OZ. 5010 8 1992, American Pharmaceutical Association

Journal of PharmaceotiCal Sciences 1 635 Vol. 81, No. 7, July 1992

Table CSummary of Experlmental Condltlonr Donsr Electrode (Teflon)

Receptor Electrode (G1.SS)

Variable Description or Value

Permeation cell

Animal skin

Skin area

Temperature Receiver and donor medium Receiver volume

Receiver sample size

Volume applied on donor

Number of replicates Current conditions:

Constant current

Pulsed unit

Assay

Electrodes

Anode (+) Cathode (-)

Modified vertical Franz cell; Horizontal side-by-side cell

Hairless guinea pig; 11 weeks old; whole skin; abdominal

1 cm2 (vertical cell); 0.64 cm2 (horizontal cell)

37 "C (receiver); 32 "C (donor) pH 4 Acetate buffer 7 mL (vertical cell); 3.5 mL

(horizontal cell) 1 mL (vertical cell); 0.5 mL

(horizontal cell) 0.5 mL (vertical cell); 3.5 mL

(horizontal cell) Radioimmunoassay 3 skins from 3 animals

Constant direct-current output and voltage readout

Pulsed voltage output with adjustable peak voltages, percent duly, and frequencies; average current readout.

Horizontal cell: Platinum wire (receptor and donor)

Vertical cell: Platinum wire (receptor) and platinum plate (donor)

Donor Receptor

ratio or the percent duty could be varied from 5 to 50%. This unit also had 12 channels and a liquidcrystal display that provided the average current readout for each channel. This average current readout is the current averaged over the cycle. At a setting of SO-kHz duty, for example, the duration of each pulse would be 20 p, but at a setting of 50% duty, the pulse would be on for 10 p and off for 10 pa. In some experiments, a pulsed-current unit with a single output channel and a fixed frequency and percent duty was used.

Permeation Proceduree-The h h l y excised skin membrane was sandwiched between the two compartments of the permeation cell. The two cell halves were assembled together with a clamp and placed on the drive unit. The receiver compartmenta were filled with pH 4 acetate buffer solutions of desired ionic strengths and maintained at 37 "C with constant stirring at 600 rpm. The donor side of the vertical and horizontal cells received 0.5 and 3.5 mL, respectively, of the drug solution in the buffers. Compound 1 is most stable at pH 4 and has an isoelectric point of 8.011 At pH 4, the pH selected for the donor compartment, the peptide was fully ionized. A platinum wire electrude was placed in the receptor side and served as the cathode for the vertical and horizontal cells. A platinum wire electrode was used in the donor side of the horizontal cell, which served as the anode. For the vertical cell, a platinum plate electrode embedded in Teflon and inserted through the Teflon holder on the donor side served as the anode (Scheme I). Alligator clamps were used to connect the anode and the cathode electrodes to the current output unit. The drug concentration in the receiver chamber was monitored at predetermined time intervals up to 5 h. A constant volume was always maintained in the receptor side.

Assay-Samples were assayed by radioimmunoassay with an antibody specific for the peptide. The assay sensitivity (limit of quantification) was 0.1 pg/mL. Briefly, 100 pL of the 1261-labeled peptide was mixed with 100 pL of sample or 500 p L of buffer and incubated with 100 pL of the peptide-specific antibody (raised in rabbits) for 18-24 h at room temperature. Then, 100 pi, of 4% normal rabbit serum and 100 p L of a 1 : 5 dilution of goat antirabbit gamma globulin were added, and the mixture was spun in a vortex mixer. ARer 15 min, 1 mL of 6% polyethylene glycol 8000 was added with vortex spinning, and the mixture was centrifuged at 3000 rpm (2000 x g) for 15-20 min. The supernatant was decanted, and the residue was counted in a Micromedic gamma counter.

P l a t i n & Plate f & mm dla: I IP t h i c k )

- 1 cm ( 0 . 5 rn t h i c k ) I P1.tl""rn Mil*

vertical (Franr) Cell

h Skin h

Scheme I-Diffusion cells and electrodes used for the permeation studies.

WPLITUDE CONTROL

SWITCHED DIGITAL 400 KHz DIGITAL DCIY CYCLE

OSCILLATOR DIVIDER OUTPUI AWLITUDE

DRIVER

SWIICHED

I I FREQUENCY DUTY CYCLE SWITCH SWITCH

MONITOR I (DPt4) I

Scheme ICBlock diagram of the pulsed current output generator.

0 Volt ITn Scheme IlCSchematic of the rectangular waveform used in the pulsed- output current generator. The percent duty (a) is determined by [(TIT) x 1001, where T = 1/F, Fis frequency (Hz), and Tis time (ps). D is variable in 5% increments from 5 to 50%, and Fcan be set to either 2, 4, 10, 20, or 40 kHz or 2.5, 5, 12.5, 25, or 50 kHz. Peak amplitude (A) is variable from 0 to 20 V.

Results and Discussion The amount of 1 appearing in the receiver compartment of

the diffusion cells was plotted as a function of time. The slopes of the resulting linear plots were computed. From these slopes, the flux (ng/cm2 * h) was calculated. In all experi- ments, the current or voltage was measured prior to with- drawing samples from the receiver compartment. These measurements and the flux values are presented in Tables 11-VII. The flux values were abstracted from these tables for different parameters and are summarized in Table VIII.

The transport of ions under the influence of a uniform electric field was first studied by Planck.20 The fundamental

636 I Journal of Pharmaceutical Sciences Vol. 81, No. 7, July 1992

Table ICEtfect of Apparatus (Horlzontal Cell versus Vertical Cell) on In Vltro Skin Permeation of 1 a

Total Amount of 1 Permeated f SD, ng/cm2 Measured Voltage f SO, V

Hour . Horizontal Cellb Vertical Cell" Horizontal Celld Vertical Celle

0 4.7 f 0.78 5.7 f 0.72 0 0 1 4.4 f 0.38 4.7 f 0.18 107f 14.6 17.7 2 30.7 2 4.6 f 0.18 4.6 f 0.20 122 f 32.0 56.9 f 57.5 3 4.6f 0.18 4.7f 0.20 179 2 11.9 106 f 50.1 4 4.6 2 0.27 4.8 f 0.29 187 2 11.5 139 2 27.0 5 4.6 2 0.34 4.7 f 0.27 208 f 8.02 156 f 25.0

~

Permeation measured under constant-current iontophoretic condi- tions with the hairless guinea pig model; current, 0.1 mA; salt concen- tration, 0.025 M; pH 4.0 buffer. bAvera e 4 6 + 0.10 V. "Average, 4.7 f 0.082 V. d F I ~ ~ , 38.2 f 0.818 ngkm h, lag time, -1.00 2 0.271 h; concentration of 1,3.0 mg/mL; donor volume, 3.5 mL; dose, 10.5 mg; skin area, 0.64 an2; number of replicates, 3. Flux, 39.8 f 3.02 ng/cm2 * h; lag time, 0.630 f 1.1 2 h; concentration of 1, 3.0 mg/mL; donor volume, 0.5 mL; dose, 1.5 mg; skin area, 1 .O cm2; number of replicates, 3.

!3.* : -

Table IlCEtteCt of Type of Current (Constant versus Pulsatile) on In Vltro Skin Permeation of 1 under lontophoretlc Condltlons wlth the Hairless Guinea Pig Model

Total Amount Permeated f SD, ng/cm2 Current f SD, mA

Hour Constant Pulsatile Constant Pulsatile Current

(set). (rneasuredIb Current" (constant voltage)d 0 0.1 0.115 2 0.035 0 0 1 0.1 0.140 f 0.018 17.7 2 30.7 45.8 f 43.0 2 0.1 0.148 2 0.007 56.9 f 57.5 74.8 2 62.8 3 0.1 0.142 f 0.006 106 2 50.1 147.0 f 77.0 4 0.1 0.137 f 0.009 139 2 27.0 165.0 f 69.0 5 0.1 0.133 f 0.012 156 f 25.0 214.0 f 54.2

__ _ _ ~ ~

Average, 0.1 mA. Average, 0.1 40 f 0.0056 mA. " Flux, 39.8 f 3.02 ng/cm2 * h; lag time, 0.630 f 1.1 2 h; concentration of 1,3.0 mg/mL; donor volume 0.5 mL; dose, 1.5 mg; skin area, 1 .O cm2; salt concentration, 0.025 M; duty, 100%; voltage, 6 V. Flux, 44.7 f 12.9 ng/cm2 h; lag time, 0.1 56 2 0.61 1 h; concentration of 1,3.0 mg/mL; donor volume, 0.5 mL; dose, 1.5 mg; skin area, 1 .O cm2; salt concentration, 0.025 M; duty, 50%; frequency, 50 kHz; voltage, 12 V.

Nernst-Planck equation is widely used to describe the mem- brane transport of ions:21.22

In eqs 1 and 2, J is the flux of ions across the membrane, D is the diffusion coefficient, dcldx is the change in concentration as a function of distance in the membrane, C is the concen- tration of ions with valence z and electron charge e, E is the electric field, k is the Boltzmann constant, and T is the absolute temperature. This equation describes the flux of an ion under the influence of both a concentration gradient and an electric field. The experiments without any electric field in the present study showed no detectable transport of 1 across the membrane (Table VIII). Therefore, any flux values re- ported from experiments with an electrical field are due to the influence of iontophoresis.

A comparison of the flux values of the horizontal (side-by- side) and the vertical F r a u cell did not show any difference, even though the cells and the electrodes used were quite different (Tables I1 and 111). The measured voltages from these

two experiments were also very similar (Table II). This result indicates that the iontophoretic enhancement is minimally influenced by cell and electrode design and donor and receiver volumes.

A comparison of the iontophoretic flux was made with two modes of current output, constant and pulsed. Both units were set to provide similar levels of current output. The constant- current unit had a setting of 0.1 mA, and the pulsed-current unit was set a t 12 V with 50% duty at 50 kHz frequency (this setting provided an average current output of 0.14 mA; Table 111). Under these conditions, no significant difference was observed between the flux values from the two units.

By using the pulsed current at 12 V, 50% duty cycle, and 50 kHz frequency, the effect of salt (buffer) concentration on the iontophoretic flux of 1 was determined (Table IV); these results show a curvilinear increase in flux with increasing molar concentration of the salt (Figure 1). Wearley et al.16 also found a similar increase in the flux of verapamil hydro- chloride, and the increase was more pronounced at the higher concentrations of the drug. The authors have provided a theoretical explanation based on the original Nernst-Planck equation (eq 1). The fraction of current carried by a particular ion is its transference number (t) and may be calculated by:

(3)

In eq 3, Z, is the total current; Zi is the current carried by ion i; and D, z, and C are diffusivity, valence, and concentration, respectively, of ions i, j, . . . n. On the basis of eq 3, the flux of ion i will decrease as competing ions are added to the medium. However, the presence of more-mobile ions in the medium can influence the flux of ion i in a positive way, by convective flow. Gangarosa et al.2 showed that skin permeation of nonionic drugs could be enhanced by convective flow, and Burnette and Ongpipattanakuls demonstrated that the flux of mannitol across human cadaver skin is enhanced by convective flow. Wearley et a1.16 have stated that such convective flow and its coupling to ionic flux have been neglected in the Nernst- Planck equation.

Recently, Vanorman et al. 123 using capillary electrophore- sis as a physicochemical model, found electroosmotic flow to be the mechanism of transport of neutral molecules during iontophoresis. Iontophoretic transport across skin is the cumulative result of three factors: convective solvent flow induced by the current (electroosmosis), as discussed earlier; an increase in skin permeability induced by the field; and a direct effect of the field on charged permeants (Nernst-Planck effectl.8 The effect of the second factor, increase in skin permeability induced by the field, was not determined. The influence of this factor is quite different for hairless m o w skins and human skin24 under constant, direct-current-field conditions. In this study however, the increased flux may be mostly related to the observed increase in the current (Table V; Figure 21, consistent with the Nernst-Planck equation (eq 1). The current applied across the skin serves as a driving force for drug permeation. Similar observations have been made by other researchers for alkanols and alkanoic acids,1 verapami1,le benzoic acid,% and thyrotropin releasing hor- mone.25 The observed curvilinear increase in flux with in- creasing salt concentration would therefore imply that an optimum salt concentration for iontophoretic permeability exists.

The observed average current values increased linearly with the molar concentration of the buffer (Figure 3). At zero molar concentration (water in donor and receiver), the cur- rent was -0.03 mA for an applied voltage of 12 V.

The iontophoretic flux of 1 was also determined at 9 and 20 V, which provided effective voltage outputs of 2.7 and 6.0 V,

Journal of Pharmaceutical Sciences I 637 Vol. 81, No. 7, July 1992

Tabk IV-Effect of Salt Concentratlon on In Vltro Skln Pmneatlon of 1 under lontophomtlc Condltlonr wlth the Halrku Qulneo Plg Model and Pulratllo Current

Hour Measured Current (mA)

at the Indicated Salt Concentration Total Amount Permeated ( n g / d ) at the Indicated Salt Concentration.

0.010 Mb 0.025 MC 0.050 M" 0.010 Me 0.025 M' 0.050 Mg

0 0.058 f 0.20 0.1 15 5 0.035 0.223 f 0.014 0 0 0 1 0.085 f 0.003 0.140 5 0.018 0.237 f 0.006 0 45.8 f 43.0 36.5 f 36.5 2 0.099 f 0.006 0.148 5 0.007 0.239 f 0.005 31.0 f 53.7 74.8 f 62.8 96.1 f 68.5 3 0.098 f 0.006 0.142 * 0.006 0.231 f 0.013 39.0 f 67.5 147 f 77.0 190 f 65.0 4 0.103 f 0.012 0.137 5 0.009 0.224 f 0.007 58.0 f 100.4 165 f 69.0 199 f 70.3 5 0.104 f 0.014 0.133 5 0.012 0.216 f 0.010 95.6 f 74.5 214 f 54.2 270 f 56.1

~ ~ ~~ ~~ ~ ~~~

Concentration of 1,3.0 mg/mL; donor volume, 0.5 mL; dose, 1.5 mg; skin area, 1 .O cm2; frequency, 50 kHz; voltage, 12 V; duty, 50%; number of replicates, 3. bAverage, 0.098 f 0.0076 mA. 'Average, 0.140 * 0.0056 mA. "Average, 0.230 f 0.0095 mA. a Flux, 21.8 f 19.6 ng/cm2 - h; lag time, 2.82 f 2.05 h. 'Flux, 44.7 f 12.9 ng/cm2.h; lag time, 0.156 f 0.6 h. OFlux, 56.8 f 8.21 ng/cm2. h; lag time, 0.514 T 0.15 h.

Tabk V-Effecl of Current on In Vltro Skln Permeation of 1 under lontophontlc Condltlona wlth the Halrleu Oulrna Plg Model and Pul$atlk, Current.

Total Amount Permeated (ng/cm2) at the Indicated Current

0.098 mAb 0.14 mA' 0.23 mA" Hour

0 0 0 0 1 0 45.8 f 43.0 36.5 2 36.5 2 31.0 2 53.7 74.8 f 62.8 96.1 f 68.5 3 39.0 f 67.5 147 f 77.0 190 f 65.0 4 58.0 f 100.4 165 f 69.0 199 f 70.3 5 95.6 f 74.5 214 f 54.2 270 f 56.1

~~ ~ ~

Concentration of 1,3.0 mg/mL; donor volume, 0.5 mL; dose, 1.5 mg; skin area, 1 .O cm2; voltage, 12 V; frequen 50 kHz; duty, 50%; number of replicates, 3. Flux, 21.8 2 19.6 ng/cmT h; lag time, 2.82 f 2.5; salt concentratlon, 0.010 M. ' Flux, 44.7 12.9 n@m2 - h; lag time, 0.156 f 0.61 1 h; salt concentration, 0.025 M. Flux, 56.8 f 8.21 ng/cm2 - h; lag time, 0.514 f 0.151 h; salt concentration, 0.050 M.

Tabk Vl-Effect of Applld Voltage on Current Denrlty and In VHto Skln Pewmeatlon of 1 under lontophomtlc Condklonr wlth the Halrleu Gulnm Pig Skln Mock1 and Pul$atlb Cumnt

Measured Current (mA) at Total Amount Permeated (ng/cm2) at Indicated

Voltage. Hour Indicated Voltage

9 Vb 20 vc 9 V" 20 ve ~ ~ ~ ~~

0 0.020 f 0.010 0.090 f 0.020 0 0 1 0.013 f 0.006 0.096 f 0.005 0.009 f 0.008 40.8 2 43.2 2 0.013 f 0.006 0.11 f 0.010 12.6 f 21.8 75.4 5 72.4 3 0.013 f 0.006 0.097 5 0.010 18.0 5 31.2 87.6 2 58.9 4 0.010 f 0.WO 0.097 f 0.006 11.7 f 20.2 117 5 51.8 5 0.010 f 0.WO 0.096 f 0.006 24.6 f 42.7 1675 37.9

a Concentration of 1,3.0 mg/mL; donor volume, 0.5 mL; dose, 1.5 mg; skin area, 1 .O an2; salt concentration, 0.025 M; frequency, 40 kHz; duty, 30%; number of replicates, 3. Average, 0.012 f 0.002 mA. ' Average, 0.099 f 0.006 mA. "Flux, 4.84 f 8.37 ng/cm2. h; lag time, -1.37 f 1.39 h. eFlux, 36.8 f 10.3 ng/cm2- h; lag time, 0.315 f 1.55 h.

respectively, at 30% duty setting and a frequency of 40 kHz. Under these conditions, a twofold increase in voltage output resulted in a 7.6-fold increase in the flux (Table VI). In a two-electrode cell system, the voltage applied across the electrodes to drive a given current density is a complex fundion of the resistance of the skin, the electrolyte, and the two electrod+eledrolyte interfaces. The last factor is domi- nant in determining the overall voltage drop, and although the voltage across the skin is the relevant fador in determin- ing transport rates, the two are not linearly related.26 How- ever, the measured increase in the current was eightfold. Therefore, the flux increase is because of an increase in the

Table VICEffect of Frequency on In Vltro Skln Permeation of 1 under lontophomtlc Condltlona wlth the Halrku Qulnea Plg Skln Model and Pulmtlb Current

Total Amount Permeated (ng/cm2) at Indicated

Frequency' Measured Current (mA) at

Hour Indicated Frequency

40 kHzb

0 0.090 f 0.020 1 0.096+0.005 2 0.110 5 0.010 3 0.097 f 0.010 4 0.097fO.006 5 0.096 f 0.006

50 kHz'

0.115 f 0.035 0.140 f 0.018 0.148 f 0.007 0.142 f 0.006 0.137 f 0.009 0.133 f 0.012

40 kHzd

0 40.0 f 43.2 75.4 f 72.4 87.6 f 58.9 117 f 51.8 167 f 37.9

50 kHz*

0 45.8 f 43.0 74.8 f 62.8 147 f 77.0 165 f 69.0 214 f 54.2

Concentratlon of 1,3.0 mg/mL; donor volume, 0.5 mL; dose, 1.5 mg; skin area, 1 .O cm2; salt concentration, 0.025 M; duty, 30% at 40 kHz and 50% at 50 kHz; number of replicates, 3. Average, 0.099 f 0.006 kHz. 'Average, 0.14 f 0.0056 kHz. Flux, 36.8 f 10.3 ng/m2 - h; lag time, 0.31 5 f 1.55 h; voltage, 20 V. a Flux, 44.7 f 12.9 ng/cm2 * h; lag time, 0.156 -e 0.61 1; voltage, 12 V.

Tabk VllCSummary of the In Vltro Skln Pemaatlon of 1 under lontophomtlc Condltha wlth the Halrkrr Q u l m PIg Model

Variable Flux, ng/cm2 * h

Control (no iontophoresis) Permeation apparatus

Ho,rizontal cell Vertical cell

Type of current constant Pulsatile

Salt concentration 0.010 M 0.025 M 0.050 M

0.098 mA 0.14 mA 0.23 mA

Voltage

20 V (= 6.0 v) Frequency

40 kHz 50 kHz

Current

9 v (= 2.7 v)

0.00

38.2 * 0.818 39.8 f 3.02

39.8 f 3.02 44.7 f 12.9

21.8 2 19.6 44.7 +- 12.9 56.8 f 8.21

21.8 2 19.6 44.7 f 12.9 56.8 f 8.21

4.84 f 8.37 36.8 f 10.3

36.8 f 10.3 44.7 f 12.9

current density, an observation consistent with the results in Figure 3.

The flux values of the peptide were independent of fre- quency. No difference in the flux of 1 was observed when the frequency was changed from 40 to 50 kHz (Table VID.

838 I Journal of Pharmaceuticel Sciences Vol. 81, No. 7, July 1992

~-

0.000 o.mo 0.040 0- Buffu ~~ (U)

mum 1-In vltro flux of 1 acm hairless guinea plg skin versus molar concentration of the buffer.

loo n i

cwrnt (4 Flgun 2-In vltro flux of 1 a m hairless gulnea pig skin versus current.

0.2% r

f 0.1% Y o'200 1

t o.Oo0 ' J

0 . W 0.020 0.040 0.060 8uff.r ConcrnhUon (U)

Flgum +Current versus molar concentration of buffer during ionto- phoresis of 1.

However, a more pronounced decrease in blood glucose levels was found in vivo in diabetic rate when the fresuency was i n d twofold during the iontophoretic delivery of insu- lin.6

The pH of the donor and receiver chambers was determined at the end of each experiment. The donor pH was 2.3-3.8, and the receiver pH was 4.14.7. Although the pH of the donor solution was significantly lowered because of pmduction of hydronium iona at the electrode, it is believed that the degradation of the drug would have been minimal during the

short course (5 h) of the experiment, on the basis of the available stability information for these analogues.10-13 The pH for optimum stability is 3-5.

Conclusions Results of experiments with an in vitro hairless guinea pig

model indicate that 1 does not permeate through the skin by passive diffusion. However, permeation of the peptide was significantly enhanced by iontophoreais. The flux of 1 was independent of the design of the permeation apparatus, the electrodes, the donor and receiver volumes, the type of current (constant or pulsed), and the frequency of the pulsed current. The flux of 1 showed a curvilinear increase with an increase in the salt concentration of the buffer and a linear response with an increase in current.

References and Notes 1. DelTem, 9.; Behl, C. R.; Nash, R. A. Phonn. Res. 1989,6,86-90. 2. Gangaroea, L.; Park, N.; Wiggine, C.; Hill, J. J. Pharmucol. Exp.

3. Bumette, R.; Ongpipattanakul, B. J. Phwm. Sci. 1987,76,76&773. 4. Banga, A.; Chien, Y. W. J. Controlled Release 1988, 7, 1-24. 6. Chien, Y. W.; Siddi ui, 0.; Sun, Y.; Shi, W. M.; Liu, J. C. Ann.

Ther. 1980,212,377481.

N.Y. A d . Sci. 198?,507.32-51. 6.

7.

8.

9.

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Journal of Phannaceufical sciences I 838 Vol. 81, No. 7, July 1992