Results and discussion 1. Stability studies

ARN14140 was stable in the presence of porcine and human skin with recovery of 99.9 ± 0.5%

and 100.4 ± 1.1%, respectively. After current application for 6 h at a current density of 0.5 mA/cm², the ARN14140 concentration in solution was 97.1 ± 3.1% of that measured initially, confirming the stability of the drug in the presence of electric current.

86 3.2. Effect of increasing ARN14140 concentration on transport

Control experiments confirmed that passive permeation of ARN14140 across porcine skin was below the LOD, 0.7 nmol/mL, corresponding to a cumulative permeation (QPERM) less than 4.2 nmol/cm². In contrast, anodal iontophoresis of ARN14140 at 0.5 mA/cm² using a 1, 2, and 4 mM buffered solution resulted in QPERM of 149.8 ± 24.0, 288.9 ± 70.0, and 383.8 ± 62.9 nmol/cm², respectively. ARN14140 total delivery (QT,DEL) and steady-state iontophoretic flux (Jss) as a function of donor concentration are presented in Figure 2a. Doubling ARN14140 concentration in the solution from 1 mM to 2 mM resulted in a proportional increase in QPERM

and Jss. However, increasing ARN14140 concentration from 2 to 4 mM resulted in a ~1.3 fold increase in QPERM. Increasing ARN14140 concentration in the formulation resulted in an increase in Jss even in the absence of competing ions due to saline bridges. This observation was consistent with results obtained with Huperzine A which also resulted in linear increase in cumulative delivery upon increase in concentration (anodal iontophoresis at 0.5 mA/cm² for 6 h resulted in QPERM of 82.5 ± 12.5, 202.9 ± 5.2, and 318.4 ± 90.1 μg/cm² using a 1, 2, and 4 mM buffered solution, respectively) in the absence of competing ions [16]. It has previously been seen that in the absence of other co-ions, the iontophoretic flux is independent of drug concentration and is largely dependent on the ratio of drug diffusivity in the skin and the diffusivity of predominant counter-ion in the receiver compartment i.e. on the opposite side of the membrane (Cl⁻) [17]. It was concluded that at low drug to Cl⁻ ions ratio there is a dependence on the concentration of the permeant despite the absence of competing ions. Our results with ARN14140 further confirm this phenomenon of concentration dependent permeation under low ARN14140/Cl⁻ ratio. It should be noted that MES is zwitterionic and this may provide some degree of competition to carry current. Increasing the ARN14140 concentration in the formulation further decreased the effect of drug depletion in the donor compartment where at 4 mM concentration continuous increase in ARN14140 flux can be seen until 5 h in comparison to 1 mM and 2 mM where no further increase in flux observed at 3 and 4 h, respectively (Figure 2b). The results also showed that there was a significant increase in skin deposition (QDEP) of ARN14140 upon increasing its concentration in the formulation (Figure 2a).

87 Figure 2. Effect of concentration on ARN14140 delivery after transdermal iontophoresis for 6 h at 0.5 mAcm² (2 mM in 10 mM MES; pH 6). (a) Total delivery (QT,DEL = QPERM + QDEP) and steady state flux (Jss), (b) change in flux (J) and (c) comparison of ARN14140 biodistribution (QDEP) in porcine skin to a total depth of 1000 μm at a resolution of 100 μm (Mean ± SD; n ≥ 5).

3.3. Effect of increasing current density on ARN14140 permeation

With the increase in current density from 0.15 to 0.3 and 0.5 mA/cm², QPERM of ARN14140 (2 mM in 10 mM MES; pH 6.0) linearly increased from 54.5 ± 11.9 to 127.1 ± 25.6 and 288.9 ± 70.0 nmol/cm², respectively (QPERM = 677.2id – 57.6 (r² = 0.98)) (Figure 3). The fraction of total delivery permeated across the skin i.e., QPERM/QT.DEL found to increase from 13 to 22 and 42 % at current density 0.15, 0.3 and 0.5 mA/cm², respectively. This signifies that increasing current density can preferentially increase drug transport into the systemic circulation. As determined earlier by Phipps and Gyory, the applied current density linearly influences the flux of an ion (𝐽_𝑖^𝐸𝑀) due to electromigration [18]. 𝐽_𝑖^𝐸𝑀 across a given interface for a particular ion (𝑖) can be determined using equation (1) [18,19].

0

88 𝐽_𝑖^𝐸𝑀 = 𝑡_𝑖 𝐼

𝑍_𝑖𝐹𝐴 (1) where, 𝑡_𝑖 denotes transport number (fraction of the current carried by the 𝑖^th ion), 𝐼/𝐴 is applied current density, 𝑍_𝑖 is the charge of an ion and 𝐹 represents the Faraday constant. Thus, the drug input kinetics can be easily controlled, by either increasing (or, if necessary, decreasing), the current density. The current density also found to have an impact on lag time i.e., time taken to reach pseudo-steady state as it was decreased upon increase in current density (Table 1).

Table 1. Iontophoretic transport kinetics of ARN14140 and the relative contributions of electromigration and electroosmosis^a. steady state flux (nmol/cm²/h); JEM: electromigration flux (nmol/cm²/h); JEO: electroosmotic flux (nmol/cm²/ h); %EM and %EO are the % contributions of EM and EO to total electrotransport; IF: inhibition factor; Tlag: lag time in h.

Figure 3. Effect of current density on ARN14140 delivery after transdermal iontophoresis for 6 h at 0.5 mAcm² (2 mM in 10 mM MES; pH 6). (a) Total delivery (QT,DEL = QPERM + QDEP) and steady state flux (Jss), and (b) comparison of ARN14140 biodistribution (QDEP) in porcine skin to a total depth of 1000 μm at a resolution of 100 μm (Mean ± SD; n ≥ 5).

89 3.4. ARN14140 skin biodistribution

Only 4.7 ± 1.5 % of the ARN14140 out of applied amounts in the donor compartment was deposited in the lateral regions of the skin using different current densities and concentrations, which states that the ARN14140 was in particular delivered through 2 cm² of the permeation area. The ARN14140 biodistribution profile, that is, the amount of ARN14140, present as a function of depth within the skin, was determined to study the effect of current density and drug concentration on drug accumulation in the skin. The amount of ARN14140 present in ten lamellae each with a thickness of 100 μm going from the skin surface to a nominal depth of 1000 μm was quantified. Comparison of the ARN14140 biodistribution profiles in skin following 0.5 mA/cm² of current application showed proportionally more ARN14140 deposition upon increasing ARN14140 concentration (Figure 2c). The fraction of total delivery deposited in the skin i.e., QDEP/QT.DEL was similar at each concentration (60 ± 2 %). This states that after transdermal iontophoresis at 0.5 mA/cm² for 6 h upto 40 % of the drug permeated across the skin and 60 % deposited within the skin.

Increase in current density significantly increased QT.DEL = 402.9 ± 42.2, 571.6 ± 37.9 and 684.0

± 31.3 nmol/cm² at 0.15, 0.3 and 0.5 mA/cm², respectively (P < 0.05). The QDEP in the skin increased upon increasing current density from 0.15 mA/cm² to 0.3 mA/cm² (P = 0.01).

However, no significant difference was observed in the biodistribution profile between 0.3 and 0.5 mA/cm² (P = 0.22) (Figure 3b). The QDEP/QT.DEL found to decrease from 86 to 78 and 58

% at current density 0.15, 0.3 and 0.5 mA/cm², respectively (QPERM/QT.DEL = 13, 22 and 42 % at 0.15, 0.3 and 0.5 mA/cm², respectively).This further confirmed that current density provides greater control over the drug input rates by preferentially improving permeation due to increased migration of drug ions across the skin in the same time. The tendency of ARN14140 to deposit in the skin can possibly be exploited to provide a sustained post-iontophoretic delivery into the bloodstream. Accordingly, shorter duration current application may be used and hence reduce the risk of skin irritation due to patch components or exposure to current.

3.5. Mechanism of ARN14140 transport

The contributions of EM and EO to ARN14140 electrotransport were estimated by co-iontophoresis of ACE at each concentration (1, 2, and 4 mM). Iontophoretic transport of uncharged drug candidate such as ACE is exclusively dependent on the electroosmotic solvent flow, and is used to report on EO [20,21]. Calculation of the ACE iontophoretic flux enabled

90 determination of the linear solvent velocity at 0.5 mA/cm . This enabled the estimation of the EM and EO contributions to ARN14140 electrotransport [16]. The results showed that EM contributed to >98% of ARN14140 delivery and EO played only a very minor role (Table 1).

ARN14140 has a secondary amine with a pKa of 11.18 and a tertiary amine with a pKa of 8.47, implying that it is almost completely ionized at pH 6 and hence is well-suited to delivery by EM. The inhibition factor (IF) described as the ratio of ACE electrotransport in the absence and presence of ARN14140 was 0.84, 1.07 and 1.72, for 1, 2 and 4 mM concentrations, respectively.

The convective solvent flow was slightly inhibited at 4 mM concentration by ARN14140 electrotransport in comparison to 1 mM and 2 mM concentrations (Table 1). Thus, ARN14140 at higher concentration was found to interact with the fixed negative charges in the skin and hence affected skin permselectivity. Furthermore, this factor could have contributed to the nonlinear increase in ARN14140 delivery from 2 mM to 4 mM in comparison to 1 mM and 2 mM. Given that ARN14140 has pKa of 8.47 and 11.18, it will remain completely ionized in the donor solution (pH 6) and receiver solution (pH 7.4) therefore, it is possible to have ion-pair formation in the transport channels between cationic ARN14140 and endogenous anionic species of skin. ARN14140 has logD of −0.5 at pH 7.4 but, it is possible that after interacting with the skin ARN14140 may become unionized (logP of 4.24) that further explains its retention in the skin due to its high affinity to partition into skin membranes; this gives more time to ARN14140 to interact with negatively charged surfaces of skin.

3.6. Delivery and transport efficiency

The delivery efficiency of ARN14140 i.e., the percentage of drug from the donor compartment delivered by iontophoresis, was extremely high. This confirmed that ARN14140 is an ideal candidate for iontophoretic delivery (Figure 4a and b). In transdermal systems, it is difficult to achieve high delivery efficiency. Therefore, an optimized iontophoretic transdermal system for ARN14140 should provide maximal amounts of delivery to offer an efficient and cost-effective alternative to oral administration. The transport efficiency in iontophoresis is described as the percentage of the total charge carried by an ion species in presence of other ions [14,18]. Low transport efficiency of ARN14140 was observed at the different current densities. This was attributed to the significantly higher Cl⁻ concentrations present in the skin and the receiver (133 mM; ~30- to ~130-fold higher). Given that Cl⁻ also has greater mobility, it follows that itis the principal charge carrier.

91 Figure 4. Delivery and transport efficiencies of ARN14140 as a function of (a) concentration (at 1, 2, and 4 mM in 10 mM MES; pH 6) and (b) 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). The delivery efficiency is described as the fraction of the ARN14140 delivered to the amount applied in the donor compartment at time zero. The transport efficiency is defined as the fraction of the total charge carried by the ARN14140 in reference to the other co-ions [19].

3.7. Validation with human skin

Electrotransport kinetics of ARN14140 across porcine skin were compared to the human skin [22]. ARN14140 QT.DEL (2 mM in 10 mM MES, pH 6.0) into and across porcine and human skin after iontophoresis at 0.5 mA/cm² for 6 h was found to be statistically equivalent (684.0 ± 31.3 and 633.7 ± 60.9 nmol/cm², respectively (P = 0.25). The component skin deposition (QDEP.HUMAN = 373.7 ± 56.1 nmol/cm², QDEP.PORCINE = 395.1 ± 71.8 nmol/cm²) and cumulative permeation (QPERM.HUMAN = 260.0 ± 38.1 nmol/cm², QPERM.PORCINE = 288.9 ± 70.0 nmol/cm²) were also equivalent.

3.8. Permeation studies with hydroxyethyl cellulose gel

After performing preliminary iontophoretic delivery studies of ARN14140 using aqueous solutions, hydroxyethyl cellulose gel (3 %) formulation was developed as a drug reservoir that could be used in an iontophoretic patch system[23]. The gel formulation was found to be stable;

the ARN14140 content was uniform with no signs of ARN14140 precipitation after 1 month.

Iontophoretic delivery from the gel formulation (QT.DEL = 495.4 ± 52.9 nmol/cm²) was 28 % lower in respect to the buffered solution (2 mM in 10 mM MES) (QT.DEL = 684.0 ± 495.4 nmol/cm²); respectively (Figure 5). A statistically significant difference between QPERM of ARN14140 from the gel and buffered solution was observed after iontophoresis for 6 h at 0.5

(a)

92 mA/cm (188.0 ± 40.3 and 288.9 ± 70.0 nmol/cm , respectively) (P = 0.01). It is assumed that the lower transport seen with the gel is due to the retarding effect of polymeric matrix that is possibly providing an additional barrier to diffusion. This may have contributed to the decreased partitioning of ARN14140 from the gel formulation to the stratum corneum[24].

Although ARN14140 delivery from the gel formulation was lower than from solution, the delivery efficiency from the gel remained very high, with nearly ~50% of the amount applied in the donor compartment being delivered.

Figure 5. Comparison of total iontophoretic delivery (QT,DEL = QPERM + QDEP) and delivery efficiency of ARN14140 from aqueous solution and hydroxyethyl cellulose gel (3% w/v) formulation each containing 2 mM of ARN14140 in 10 mM MES (pH 6) after iontophoresis at 0.5 mA/cm² for 6 h (Mean ± SD; n ≥ 5).

3.9. Iontophoretic delivery kinetics of ARN14140 in vivo

Earlier pharmacokinetic studies involving i.v. administration to rats, showed that ARN14140 had a large volume of distribution (30.1 ± 2.2 L/kg) and that the plasma clearance was 1.7 ± 0.2 L/h/kg. As a consequence of the observed high volume of distribution, the estimates of plasma clearance may not reflect the actual systemic clearance. Therefore, instead of elimination processes, distribution may be more important in determining the ARN14140 plasma concentration in rats.

93 Figure 6. The observed ARN14140 plasma concentration vs. time profile in rats after iontophoretic treatment for 6 h at (a) 0.15 mA/cm² and (b) 0.5 mA/cm² (Mean ± SD; n ≥ 4).

No erythema was observed at the site of iontophoresis for either current density after iontophoresis for 6 h. The steady state was reached after 4 h at 0.15 mA/cm² and was achieved more rapidly, after 2 h, at 0.5mA/cm² (Figure 6). Since, steady state plasma concentration for both the studied current densities reached without showing any rise in plasma concentration, it is clear that ARN14140 was quickly distributed in the body with only close to quantifiable amount of ARN14140 remaining in the plasma. From the back calculation of the amount of ARN14140 left in the donor compartment after iontophoresis for 6 h, the delivery efficiency was found to be 29.1 ± 3% (equivalent to QT.DEL = 426.7 ± 42 nmol/cm²) and 76.3 ± 5%

(equivalent to QT.DEL = 1118.3 ± 73 nmol/cm²) for 0.15 mA/cm² and 0.5mA/cm², respectively.

This accounts to delivery rate of ~71 and ~186 nmol/cm²/h at 0.15 mA/cm² and 0.5 mA/cm², respectively. This signifies that modulation of the current density can be used to provide control over the drug input rates into the body.

The biodistribution of ARN14140 in different compartments of the rat body after administration by i.v. and iontophoresis is presented in Table 2. Of the total amount delivered after iontophoresis at 0.15 mA/cm² and 0.5 mA/cm², approx 50% and 40%, respectively was found in the skin and the underlying abdominal muscular layer. Iontophoresis at 0.5 mA/cm² enabled ARN14140 to be detected in the brain. The ability to quantify the drug in the brain could be linked to the higher QT.DEL at 0.5 mA/cm² than at 0.15 mA/cm² of current density. Therefore, to achieve the delivery to the brain compartment at 0.15 mA/cm², larger active surface area can be used for iontophoretic treatment that would permit ARN14140 delivery in greater amounts.

The brain-to-plasma ratio (1.2), confirmed that more ARN14140 was present in the brain than

0.00

94 in the plasma pointing to its preferential distribution in the brain. The results with ARN14140 are consistent with an earlier study by Beconi et al., where dose-related distribution studies of memantine, reported brain-to-plasma ratios > 3 [25].

The reported inhibition constant (Ki) against the NMDA receptor of memantine (1.16 ± 0.07 µM) was half of ARN14140 (2.32 ± 0.43 µM) and the IC50 against AChEof galantamine (2.55

± 0.69 µM) was at least three times more than that of ARN14140 (0.69 ± 0.03 µM)[6]. Since there are no reports on the pharmacokinetic-pharmacodynamic (PK-PD) correlation for ARN14140 and higher Ki values than memantine for NMDA receptor and lower IC50 than galantamine for AChE enzyme, the delivery in therapeutic amounts is difficult to estimate using PK-PD parameters of memantine and galantamine. A complete PK-PD study of ARN14140 is required to determine the desired drug input rates using an iontophoretic system for the management of AD.

Table 2. Biodistribution of ARN14140 in different tissue after intravenous administration and iontophoresis (2 mM ARN14140 in 10 mM MES; pH 6) for 6 h at 0.15 and 0.5 mA/cm².

Administration route I Skin (nmol/cm²)

Abdominal wall (nmol/cm²)

Brain (nmol)

Intravenous NA NA NA 0.189 ± 0.04

Iontophoresis 0.15 116.0 ± 49 101.3 ± 26.5 <LOQ Iontophoresis 0.5 186.5 ± 22 241.4 ± 96.3 0.115 ± 0.03 I: current density (mA/cm²); NA: not applicable; LOQ: limit of detection. (Mean ± SD; n ≥ 5) 4. Conclusion

The results demonstrated the feasibility of using transdermal iontophoresis to deliver ARN14140 non-invasively across the skin and into the brain. The studies performed in vitro using porcine skin demonstrated the effect of iontophoretic parameters on electrotransport and established that constant-current anodal iontophoresis was able to deliver ARN14140 with high delivery efficiencies (~77%). Modulation of current density and concentration would provide simple means to control drug delivery kinetics and hence personalize dosing in vivo. The in

95 vivo studies confirmed the observations of drug pharmacokinetics made in earlier experiments (rapid clearance from blood and into the tissue) and demonstrated that iontophoresis enabled ARN14140 to be delivered and quantified in the brain. This is the first iontophoretic study to demonstrate the non-invasive delivery of a drug directly to the brain in vivo. The next steps will involve further preclinical studies in larger mammals to determine the PK-PD parameters and the development of an iontophoretic patch system. Based on the in vivo iontophoretic delivery, we conclude that transdermal iontophoresis could provide a useful means of delivering ARN14140 for the management of AD.