Results and discussion

The objective of the initial in vitro experiments was to optimize the formulation and iontophoretic conditions for the subsequent in vivo experiments. The initial experiments were conducted using the iontophoretic patch Model 1. Ag/AgCl electrodes are more commonly used in iontophoretic systems because of several advantages over inert electrodes such as required low operating potential and avoidance of changes in solution pH due to water hydrolysis [14]. Upon activation of the device, electron flow is converted to an ion flow at the respective electrode/formulation interface. At the anode, Ag⁺ ions react with Cl⁻ ions arriving from skin, if not present already in the formulation, to form insoluble AgCl at the electrode surface. Presence of insufficient amount of Cl⁻ leads to precipitation and then deposition of Ag⁺ on the skin surface; therefore, Cl⁻ is added externally to the drug formulation to avoid silver skin deposition. Hydrochloride salt forms of drugs are ideal candidates since this avoids the need to add external Cl⁻ that brings with it another cation, normally Na⁺, which can compete with the drug to carry current and so reduce the delivery efficiency (DE) of the system [15,16].

65 According to Faraday’s law of electrolysis, the total electric charge transferred per unit area expressed in coulombs (Q) through the electrodes is the product of current density (Id) in amperes and the duration of current application (t) in seconds. Total moles of charge transfer is calculated by dividing total charge transferred (Q) with the Faraday’s constant (F). This relation accounts for 224 µmol of chloride ions required for the iontophoresis using silver anode where the area of the patch is 2 cm² and the current of 0.5 mA/cm² is applied for 6 h. In Model 1, the PRA patch is in direct contact with the anode. Each mole of PRA carries two moles of chloride ions in a dihydrochloride salt form (that is, 20 µmol of PRA patch contains 40 µmol of chloride ions). Since the PRA patch is able to provide only 40 µmol of chloride ions, unavailability of remaining 184 µmol of chloride ions leads to the liberation of Ag⁺ from the anode and formation of black silver deposits on the skin surface (Figure 3).

Figure 3. Iontophoretic delivery of PRA across porcine skin from one-compartment iontophoretic patch after transdermal iontophoresis for 6 h at 0.5 mAcm². (a) Total delivery (permeation + deposition) and (b) cumulative permeation time profile of PRA from different patch compositions. (c) Images of porcine epidermal surface after completion of the iontophoretic treatment through respective patches (mean ± SD; n ≥ 5).

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66 To avoid this phenomenon, PRA patches with 224 µmol of Cl^- were developed. This was achieved by incorporating either (i) 184 µmol of sodium chloride and 20 µmol of PRA in the patch or (ii) excess amounts of PRA (112 µmol) in the patch. As shown in Figure 3c, both approaches resolved the issue of silver deposition on the skin by providing enough Cl^- at the electrode-patch interface. The addition of external source of Cl^- in the form of sodium chloride resulted in decrease in delivery due to the competition in between PRA and Na⁺ with the total DE (permeation + deposition) determined to be 6.8%. A 5.6-fold increase in patch loading from 20 to 112 µmol did not produce a significant increase in the cumulative amount of PRA delivered across the skin during a 6 h current application (2500.1 ± 257.3 and 2898.3

± 225.8 nmol/cm² for 20 and 112 µmol patch loads, respectively). For the PRA patch of 20 µmol total DE of 35% was obtained in comparison to 7% from the patch of 112 µmol.

Using the steady-state flux (Jss) obtained from the linear portion of the graph; it is possible to calculate the PRA transport number (tss) at steady-state from

𝑡𝑠𝑠 =^{𝐽𝑠𝑠 𝐹}_𝐼 (1)

where I and F represent the current density and Faraday’s constant, respectively. The PRA steady-state flux (Jss) for the 20 µmol patch load calculated from the linear portion of Figure 3 is 579.7 ± 64.2 nmol/cm²/h; thus, for a 2 cm² patch, this corresponds to a drug input rate of 1159.4 ± 99 nmol/h. Insertion of the appropriate values reveals a tss of ~0.03. Thus, approximately 3% of the charge passed during current application was transported by the drug. tss forhigh PRA 112 µmol patch was similar to 20 µmol i.e., ~0.03. The calculated tss

for patch containing NaCl decreased to ~0.004 because of the presence of other ions that competed with PRA to carry the current. Given that about 93% of PRA left undelivered at the end of iontophoresis from the PRA patch of 112 µmol, it was decided to develop a PRA patch with NaCl to avoid wastage of the drug. Since using NaCl reduced the DE, the patch design was optimized so that competition with the Na⁺ could be minimized while improving DE of the final patch.

Model 2 was developed as a two-compartment system where the drug compartment was separated from the anodal compartment using a NaCl patch (16 µmol/2 cm²). This lower concentration NaCl patch was there to make an electrical connection between PRA patch (20 µmol) and anodal compartment. Anodal compartment was provided with excess amounts of

67 NaCl for silver anode in the form of a separate NaCl patch. This way the drug compartment (2 cm²) had in total 25% of the required moles of Cl^- i.e., 56 µmol (40 µmol Cl^- from 20 µmol of drug and additional 16 µmol from NaCl patch) required to transfer charge at 0.5 mA/cm² of current density for 6 h through a patch of 2 cm². The delivery from the Model 2 was twice to the amounts obtained from Model 1 in the presence of NaCl. The total delivery from the Model 2 was found to be 1403.1 nmol/cm² (DE = 14%). Further, separating the PRA compartment with the anodal compartment protected the skin from silver deposition (Figure 4(b)). These results suggest that new optimized design of the patch managed to reduce the competition between PRA and Na⁺. The steady-state flux from the Model 2 (272.0 ± 54.8 nmol/cm²/h) calculated from the linear portion of Figure 4(b) was nearly half of PRA 20 µmol patch (Model 1); thus, corresponded to a drug input rate of 544.0 ± 109.6 nmol/h for a 2 cm² patch. Insertion of the appropriate values reveals a tss of ~0.014.

Figure 4. (a) Total delivery (permeation + deposition) and (b) cumulative permeation time profile of PRA after iontophoretic delivery across porcine skin from a two-compartment iontophoretic patch after transdermal iontophoresis for 6 h at 0.5 mAcm² (mean ± SD; n ≥ 5).

Skin image shows no deposition of silver on the surface.

Finally, the Model 2 was selected and the effect of current density on the PRA delivery was studied. The cumulative anodal iontophoretic delivery and steady state flux for PRA as a function of current density using the same iontophoretic patch system were significantly higher (Figure 5). Increasing the current density from 0.15 to 0.3 and 0.5 mA/cm², resulted in 2.7- and 6-fold increase in total delivery, from 233.8 ± 17.1 to 622.73 ± 73.0 and 1403.1 ± 174.2 nmol/cm²; respectively. The steady state flux showed corresponding 2.9- and 6.7-fold increase. The ability of modulating the current density in order to control drug transport

68 kinetics is highly valuable to treat PD using dopamine agonists. As the disease progresses, dose escalation is required; in the case of PRA, the dose is gradually increased from 0.25 to 1.25 mg. This escalation of dose can be achieved by simply increasing the current density without any change in drug loading or patch size as required for passive transdermal systems.

Skin deposition also showed appreciable increase from 31.1 ± 4.4, to 58.0 ± 9.1 and 133.4 ± 24.2 µg/cm² as the current density was ramped from 0.15 to 0.3 and 0.5 mA/cm², respectively. These values suggest that PRA depot forms in the different skin layers and even after termination of current application drug would be released from the skin deposits into the systemic circulation.

Figure 5. Total delivery (permeation + deposition) and steady state flux of PRA from a two-compartment iontophoretic patch as a function of current density (at 0.15, 0.3, and 0.5 mA/cm²) across porcine skin after transdermal iontophoresis for 6 h (mean ± SD; n ≥ 5).

Based on the results from the in vitro experiments, it was decided to conduct the in vivo studies using the iontophoretic patch – Model 2. The objective of the initial in vivo studies were to investigate the PRA levels in the bloodstream under continuous current iontophoresis at two different current densities: 0.15 and 0.5 mA/cm². In the second part of the in vivo experiments, three complex multistep current profiles were used to study the PRA levels in the bloodstream as shown in Figure 2. The objective of this in vivo study was to achieve a

‘‘step-function” modulation in PRA concentrations in the bloodstream; hence, the use of different current densities after each hour of current application.

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69 Figure 6. The observed PRA plasma concentration vs. time profile in rats after iontophoretic administration for 6 h + 2 h without current application (a) 0.15 mA/cm² and (b) 0.5 mA/cm² (Mean ± SD; n ≥ 4).

No skin erythema was observed at the site of patch application for all continuous and multi-phasic current applications. Figure 6 shows the mean plasma concentration–time curve of PRA during and following iontophoretic current application for six hours. Typically, upon initiating the iontophoretic treatment, the amount of drug increases in the skin from where the drug is gradually distributed into the systemic circulation giving an unsteady state plasma profile at the beginning before constant flux is achieved [17]. PRA was detected in the bloodstream within 30 min after application of 0.5 mA/cm² current treatment and the drug level was 0.50 ± 0.2 nmol/mL. At lower current density, 0.15 mA/cm², PRA was detected at 60 min where the drug level was 0.30 ± 0.1 nmol/mL; indicating relatively fast drug uptake at higher current density (Figure 6). The plasma levels of PRA rose gradually during iontophoresis and reached a maximum value of 1.01 ± 0.2 nmol/mL at 0.15 mA/cm² and 3.87

± 1.7 nmol/mL at 0.5 mA/cm² in 6 h when current application was terminated. Although drug levels decreased after current stopping, they remained quantifiable up to 8 h time-point.

The plasma profiles of PRA under multi-phasic current Profiles 1, 2 and 3 are presented in Figure 7. PRA level varied gradually with the change in current density. Clearly, the rise or fall in the plasma concentration was noticeable within 30 min of varying the current density.

At t = 360 min, current application was stopped and the PRA plasma level fell progressively.

This demonstrates that iontophoresis offered good control over the drug delivery kinetics.

Being a multistep process, transdermal iontophoresis begins with the delivery of drug into the

0

70 epidermal membrane from the donor compartment from where the molecule further migrates into the dermis before finally entering into the capillary network.

Figure 7. Plasma concentration profiles of PRA as a function of time from two-compartment iontophoretic patch at different multi-phasic current profiles (grey bars, secondary y-axis) (a) Profile 1, (b) Profile 2, and (b) Profile 3.

From Profile 1, a progressive increase in current density increased drug level where a maximum value of 5.13 ± 2.7 nmol/mL was achieved using current density of 0.5 mA/cm² at 240 min. As soon as the current density decreased, plasma concentration also reduced to 3.40

± 1.2 nmol/mL at 0.15 mA/cm² at 360 min. In the Profile 2, the objective was to demonstrate the effect of an initial bolus, followed by a progressive fall in input rates and then a subsequent second bolus on PRA levels in the blood. Due to commencing with the 0.5 mA/cm², maximum humanly tolerable current density, high PRA plasma concentration, 2.10

± 0.9 nmol/mL, was achieved in the initial 90 min, which later reduced due to the step-wise hourly reduction in applied current density. Although PRA levels subsequently fell in

0

71 response to a decrease in the applied current, they remained significant (1.68 ± 0.6 nmol/ml to 1.16 ± 0.6 nmol/ml) throughout the t = 120–240 min period indicating that after the initial

‘‘bolus”, levels of PRA can be maintained using a lower current input. At 240 min, current density was progressively increased from 0.15 to 0.5 mA/cm². In response, plasma levels rose from 1.16 ± 0.6 nmol/ml at t = 240 min to 1.58 ± 0.6 nmol/ml at t = 300 min and 2.17 ± 0.6 nmol/ml until the current was terminated at t = 360 min. Profile 3 was consisted of on-off current application periods where higher current density was provided at each subsequent ‘on’

periods. Similar to Profile 1, Profile 3 was also started with the low current density (0.15 mA/cm²) that resulted in the detection of plasma level only at t = 60 min. At the end of each

‘on’ period, increased PRA plasma levels was detected: 0.27 ± 0.03, 0.64 ± 0.5 and 0.92 ± 0.4 nmol/ml at t = 60, 180 and 300 min. The plasma concentration profiles obtained using continuous and multi-phasic current profiles in vivo highlights the control offered by iontophoresis and its exceptional ability to rapidly modify drug input rates. Apart from iontophoresis, plasma profiles as shown in Figure 7 could only be achieved by using far more invasive techniques like an infusion pump.

The summary of the distribution of PRA in several compartments of the rat body after iontophoretic delivery is presented in Table 1. Extraction of PRA from the skin after 2 h of no current application still resulted in PRA recovery. This tendency of forming skin depot could be exploited to provide post-iontophoretic delivery into the bloodstream and during the treatment provide the possibility of reducing the current density while still providing plasma concentration within therapeutic window. Hence, risk of skin irritation associated with patch components or exposure to current may be reduced by using consequently shorter duration current application. In comparison to skin, less amounts of drug was recovered from the underlying abdominal membrane that is highly conditioned with body fluids; easy elimination of a highly water soluble drug such as PRA. Iontophoresis using continuous current and Profiles 1 and 2 also enabled PRA delivery to the brain and CSF. Total current delivered from Profile 3 was comparatively half of Profile 1 and 2 and therefore very small amounts of PRA could be detected in the brain and none in the CSF. The amounts of PRA in the brain and CSF were found to have a positive correlation with the density and duration of current application.

Continuous 6 h current application at 0.5 mA/cm² resulted in the delivery of highest amounts of total current, therefore, PRA delivery to the brain (9.8 ± 2.2 nmol) and CSF (5.9 ± 2.3 nmol) was highest relative to rest of the current profiles. Application of low current density at

72 0.15 mA/cm² also managed to deliver PRA to the brain and CSF. Profile 1 and 2 delivered same amounts of total current that led to the statistically similar PRA delivery to the brain and CSF (Student’s t test; α = 0.05).

Table 1. The distributed amounts of PRA in the various body compartments of rat from iontophoretic delivery using two-compartment iontophoretic patch (Model 2) at continuous (0.15 and 0.5 mA/cm²) and multi-phasic current Profiles 1, 2 and 3.

Charge

In summary, we have developed a new strategy to develop an iontophoretic patch system by separating drug and the electrode compartment for the transdermal delivery of PRA to optimize the delivery and reduce the competition ion effect. This new model also enabled complete avoidance of silver precipitation in the patch and thus prevented from skin staining.

The in vivo data confirm that transdermal delivery of PRA is feasible using the in-house developed iontophoretic patch system. No signs of inflammation were observed in the animal at the patch application site. Temporal modulation of PRA plasma concentration observed using the animal model suggested that transdermal delivery in humans, using an iontophoretic patch system, could result in personalize the treatment and individualize the dose as per the need. Choice of an appropriate current profile would enable faster input of drug into the bloodstream (reducing lag time), sustained delivery of a low-level maintenance dose to avoid peaks and troughs that are common with oral administration and thus preventing from pulsatile stimulation of dopamine receptors – mimicking normal physiological conditions in

73 healthy individuals.

Acknowledgements

We would like to thank the Swiss Federal Commission for Scholarships for Foreign Students and University of Geneva for financial support and for providing teaching assistantships for MS.

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PART 1: TRANSDERMAL IONTOPHORETIC DELIVERY OF SMALL MOLECULES

CHAPTER 3

79

Controlled iontophoretic delivery in vitro and in vivo of ARN14140, a