Results and discussion 1. Continuous current delivery

The permeated amounts representative of each hour interval, total delivery (permeation +

40 deposition), and delivery efficiencies of PRA, RAS and HUP from donor compartment after transdermal iontophoresis at 0.15, 0.3, and 0.5 mA/cm² are presented in Figure 3a-c. With the increase in current density, significantly larger amounts of drugs were permeated at each time points. At 0.15 mA/cm²,timely increase in flux of all drugs was observed. As the current density increased to 0.3 and 0.5 mA/cm² the steady state flux was achieved in short time.

Statistically insignificant decrease in permeation was observed at 0.5 mA/cm² after 4 h which could be due to the depletion effect that is the decrease in drug concentration in the donor compartment over time. After termination of current, the delivery from the donor compartment was also stopped and therefore, the permeation after 6h was due to the passive diffusion of already deposited drug from the skin in to the receiver compartment.

Cumulative permeation after iontophoresis for 6 h at 0.15, 0.3 and 0.5 mA/cm² was 1227.5 ± 118.5, 2891.6 ± 156.6 and 4231.7 ± 228.6 nmol/cm², respectively for PRA; 924.4 ± 104.7, 1278.6 ± 189.2 and 1807.9 ± 98.6 nmol/cm², respectively for RAS; and 507.7 ± 63.2, 895.5 ± 66.9 and 1189.8 ± 87.7 nmol/cm², respectively for HUP. Drug extraction from the skin samples also showed significant amounts of drugs retained within the membrane at each current density. One obvious reason of the observed difference in the total delivery of each drug under similar current conditions could be due to the differences in the concentration of drug solutions in the donor compartment: PRA (20 mM), RAS (10 mM), and HUP (4 mM).

The total delivery of PRA, RAS and HUP with respect to the electric charge transported during the 6 h continuous constant current application at 0.15, 0.3 and 0.5 mA/cm² is represented in Figure 4; good linear correlation was observed in between charge transferred versus total delivery.

A statistically significant increase in the delivery efficiency was also observed for each drug as a function of current density (Figure 3d). The delivery efficiencies were found similar to the earlier reported results on the iontophoretic delivery PRA, RAS and HUP [6,7,17]. Our results further confirm that PRA, RAS and HUP are ideal candidates for iontophoretic delivery.

41 Figure 3. Change in flux of the (a) PRA (20 mM), (b) RAS (10 mM), and (c) HUP (4 mM) (in columns, primary axis) as a function of time at 0.15, 0.3 and 0.5 mA/cm² of current densities (in lines, secondary axis) after transdermal iontophoresis for initial 6 h + 2 h without current application; (d) represents the respective total delivery (cumulative permeation + skin deposition) and delivery efficiency for each drug. (Mean ± SD; n ≥ 5.)

42 Figure. 4. Total delivery of (a) PRA (20 mM) (b) RAS (10 mM), and (c) HUP (4 mM) as a function of amounts of charge transferred (3.24, 6.48, and 10.8 C/cm²) corresponding to transdermal iontophoresis at 0.15, 0.3 and 0.5 mA/cm² of current density for initial 6 h + 2 h without current application. (Mean ± SD; n ≥ 5.)

3.2. Multi-phasic current delivery

The permeation profiles of PRA, RAS and HUP under multi-phase current Profiles 1, 2 and 3 are presented in Figure 5. The permeated amounts of drugs were found to vary under the influence of variable current profiles. Permeation of all the drugs increased gradually upon increasing the current density. Similarly, the permeation reduced with decrease in current density, though the effect was not immediate due to the diffusion of drug from skin deposits.

Visibly, the rise or fall in the permeation could be seen after 30 min of modulating the current density. Current application was stopped at t = 360 min and the drugs permeation level fell progressively. This illustrates that iontophoresis provided good control over the drug delivery

43 kinetics. Transdermal iontophoresis is a multistep process beginning with the delivery into the epidermis from the donor compartment and then to the dermis before entering into capillary network. Drug may also diffuse passively into deeper tissues depending on the partitioning and affinity of the molecule to remain in the subcutaneous tissues.

Profile 1 and 2 transported same amounts of electric charge therefore, total delivery of drugs from profile 1 and 2 was found nearly similar albeit the current was delivered in a different pattern. The cumulative permeated amounts of PRA, RAS and HUP for Profile 1 (2757.7 ± 222.3, 1211.8 ± 122.66 and 804.6 ± 60.1 nmol/cm², respectively) and Profile 2 (2485.3 ± 186.6, 1309.5 ± 137.2, and 840.6 ± 102.0 nmol/cm², respectively) were found statistically similar (t-test, p < 0.05). Similarly, Profile 3 that delivered only half electric charge of Profile 1 and 2, delivered correspondingly less amounts of drugs in comparison to Profile 1 and 2.

The linear equation obtained in Figure 4 was used to predict the delivery obtained from Profile 1, 2 and 3 based on the amounts of charge transferred which were then compared with the observed experimental values. The obtained experimental values were very close to the predicted values with accuracy of 100 ± 15 % (Table 2). As determined earlier by Phipps and Gyory, the applied current density linearly influences the flux of an ion and this relation holds for almost all ions [18]. Having said that, the input kinetics of a drug can be controlled, either increased or decreased, to achieve better efficacy under electrically assisted delivery technique.

44 Figure 5. Flux of PRA (20 mM), RAS (10 mM), and HUP (4 mM) (continuous lines, primary axis) at different multi-phasic current profiles (Profile 1, 2, and 3) obtained by varying current density from 0.15 to 0.3 and 0.5 mA/cm² (grey bars, secondary axis) after transdermal iontophoresis for initial 6 h + 2 h without current application. Total delivery (cumulative permeation + skin deposition) of PRA, RAS, and HUP from Profile 1, 2, and 3 is presented at the bottom. (Mean ± SD; n ≥ 5.)

45 Table 2. Comparison of predicted vs. experimental total delivery of PRA, RAS and HUP after transdermal iontophoresis following multi-phasic Profile 1, 2 and 3.

C/cm² (Q) Total delivery (nmol/cm²)

PRA RAS HUP

6.84 (Profile 1)

Predicted 3556.6 ± 355.7 1824.0 ± 182.4 1223.9 ± 122.4 Experimental 3831.7 ± 207.8 1987.2 ± 353.5 1143.1 ± 43.6

Accuracy (%) 92.8 91.8 107.1

6.84 (Profile 2)

Predicted 3556.6 ± 355.7 1824.0 ± 182.4 1223.9 ± 122.4 Experimental 3574.0 ± 116.4 2143. 4 ± 134.0 1318.9 ± 29.1

Accuracy (%) 99.5 85.1 92.8

3.42 (Profile 3)

Predicted 2087.0 ± 208.7 1277.2 ± 127.7 862.9 ± 86.3 Experimental 1933.0 ± 160.9 1499.4 ± 32.7 831.9 ± 45.3

Accuracy (%) 108.0 85.2 103.7

In transdermal systems, it is difficult to achieve high delivery efficiency (maximum ~20%).

Therefore, an optimized iontophoretic transdermal system would provide maximal amounts of drug delivery to offer an efficient alternative to oral delivery route. Additionally, the ability of these drugs to respond to the different densities of electric current would provide highly individualized treatment by modulating the delivery rate as per the need.

3.3. Skin biodistribution

The biodistribution profile i.e., the amount of drug present as a function of depth within the skin was determined to study the effect of current density and duration of current application on delivery behavior (Figure 6). The amounts of PRA present in nine lamellae each with a thickness of 100 μm going from the skin surface to a depth of 900 μm was quantified. PRA present in the remaining dermis was also quantified to determine total deposition. Drug biodistribution profiles in the skin following 0.15 mA/cm² of current application showed increase in amount of PRA deposition due to the increase in duration of current application

46 (Figure 6a). Out of total delivery, 16.6, 24.7 and 33.0 % of PRA permeated as the duration of current application increased from 1 to 2 and 4 h, respectively. Current density represents the rate of charge transfer and therefore, under same intensity of driving rate, total delivery should be linearly proportional to the duration of current application assuming that there is no change in the skin properties and drug concentration in the donor compartment during iontophoresis.

The deposition in the skin was also found to increase with increase in current density from 0.15 mA/cm² to 0.3 mA/cm² and 0.5 mA/cm² (Figure 6b). The application of higher current density migrates PRA molecules faster into the deeper skin layers and that is why more amount of PRA can be seen in the skin biodistribution profile (Figure 6b). From the figure, it is clear that the slope of PRA biodistribution increased until 300 µm with the increase in current density. At 0.15 mA/cm² of current density the migration of PRA is slower than 0.3 and 0.5 mA/cm², and therefore the biodistribution profile is much flatter than the other two. It may be considered that at 0.15 mA/cm² the diffusion rate of PRA in the deeper layers of the skin is close to the rate of electrotransport of the drug itself. Electromigration and electroosmosis are the two different mechanisms that contribute to total electrotransport.

However, based on the earlier study, it is clear that electromigration is the principal mechanism involved in the electrotransport of PRA [17]. Tendency of the drug to deposit in the skin in high amounts can possibly be exploited to provide a sustained post-iontophoretic delivery into the bloodstream. Opportunity of creating skin depots provides the possibility of reducing the current density during treatment while still providing uniform plasma concentration. Accordingly, shorter duration current application may also be used and hence reduce the risk of skin irritation due to patch components or exposure to current.

Figure 6c presents the comparative biodistribution profile of PRA delivered under different current settings where nearly similar amounts of electric charges were transferred. The biodistribution profile from current application at 0.15 mA/cm² for 4 h and 0.3 mA/cm² for 2 h were found statistically similar with total deposition of 1128.7 ± 118.6 nmol/cm² and 978.74

± 182.3 nmol/cm², respectively (t-test, p < 0.05). At 0.5 mA/cm² for 1 h, less amounts of charge were transferred and therefore we see little less delivery with total deposition of 788.2

± 120.3 nmol/cm². Since the current was applied for only 1 h, non-uniform distribution of PRA in the skin was attained where comparatively higher amounts of PRA were quantified in

47 the top layers than the deeper layers of skin. However, much uniform distribution in the skin was observed at 0.15 mA/cm² for 4 h and 0.3 mA/cm² for 2h due to the availability of more time for the drug to diffuse in the deeper layers.

Figure 6. Comparative skin biodistribution of PRA in the skin to a total depth of 900 μm at a resolution of 100 μm after transdermal iontophoresis (a) for different durations (1, 2, and 4 h) at current density of 0.15 mA/cm²; (b) at different current densities (0.15, 0.3, and 0.5 mA/cm²) for 1 h; and finally (c) using conditions that provided similar amounts of charge transferred. (Mean ± SD; n ≥ 5.)

48 Table 3. Comparing the effect of duration (t) and the intensity of current application (Id) to deliver different amounts of charge (Q) in coulombs on the transdermal iontophoretic delivery of PRA.

Id

(mA/cm²)

t (h) PRA Total delivery (nmol/cm²) Q (C/cm²) Experimental

Condition 1 0.15 1 0.54 418.0 ± 68.7

Condition 2 0.15 2 1.08 715.9 ± 56.6

Condition 3 0.15 4 2.16 1548.2 ± 87.3

Condition 4 0.3 1 1.08 609.0 ± 49.3

Condition 5 0.3 2 2.16 1406.1 ± 153.8

Condition 6 0.5 1 1.8 943.0 ± 94.4

Different current application conditions used to study skin biodistribution were calculated in terms of the charge transferred to establish relation with the PRA total delivery (Table 3). The range of PRA linear equation (Figure 4) was extended by deriving a new linear equation from the total delivery values of biodistribution studies added to previous continuous current studies for 6 h. Increasing the amounts of charge transferred (c) resulted in statistically significant linear enhancements of PRA total delivery (tdel) evaluated from new regression line using least square method – tdel = 471.92c + 262.2 (r² = 0.989), clearly an advantage for controlled drug delivery. Figure 7a shows the linearity analysis of the prediction versus experimental values after applying the model equation where the yielded correlation coefficient (r²) was 0.989. From the regression line the deviation of the obtained delivery values at their respective amounts of the charge transferred were then analyzed to determine precision and accuracy (Figure 7b). Only the data points of 1h studies i.e. 0.54 C (0.15 mA/cm²), 1.08 C (0.3 mA/cm²) and 1.8 C (0.5 mA/cm²), presented in grey, were out of limit while, in the other studies, where current was applied for more than one hour all the data points were found within the limit (presented in black). A plot of charge transferred versus total delivery after iontophoresis for one hour revealed that there was linear enhancement of PRA delivery evaluated using least square method – tdel = 419.22c + 178.76 (r² = 0.995), (Figure 8).

49 Figure 7. (a) Comparison of predicted vs. experimental delivery of PRA. (b) Data points represent deviation of the experimental delivery from the predicted delivery where grey data points designate 1 h studies; black data points designate studies of more than 1 h duration; and orange data points designate experimental values of 6 h iontophoresis studies of Profile 1, 2 and 3. Green line represents the lower and the upper limit and black circles are the mean of the data points (Mean ± SD; n ≥ 4.)

This suggests that to predict the delivery under short duration iontophoresis more data sets are required to establish a good fit. The experimental values of multi-phase current conditions were not a part of the linear regression analysis and therefore, these values were compared with the predicted values to validate the model. Predictions for all three multi-phase current profiles were found to be very accurate where all experimental values were within ± 20 % of the predicted values (presented in orange) (Figure 7b).

R² = 0.9889

50 Figure 8. Linear relationship in between total delivery of PRA and the amounts of charge transferred during transdermal iontophoresis for 1 h at 0.15, 0.3 and 0.5 mA/cm² of current density. (Mean ± SD; n ≥ 5.)

Under in vitro conditions, skin is exposed to the aqueous solutions on its both sides; dermis and epidermis. It is evident that longer the skin remains in contact with water, more it becomes hydrated. A fully hydrated skin presents lesser diffusional resistance to the drugs.

This phenomenon changes the characteristics of the skin as a barrier and thus, the iontophoretic delivery from low duration experiment is expected to be lower than the longer duration experiment. In condition 2, iontophoresis was carried out at 0.15 mA/cm² for 2 h and in condition 4 at 0.3 mA/cm² for 1 h; both delivering 1.08 coulombs of charge. However, total delivery of condition 2 (715.9 ± 56.6 nmol/cm²) was found to be little higher than condition 4 (609.0 ± 49.3 nmol/cm²). Similar observation was made with condition 3 and 5, where condition 3 at 0.15 mA/cm² for 4 h and condition 5 at 0.3 mA/cm² for 2 h delivered same 2.16 coulombs of charge but delivery from condition 3 (1548.2 ± 87.3 nmol/cm²) was little higher than condition 5 (1406.1 ± 153.8 nmol/cm²) (t-test, p < 0.05). These observations states that the linear enhancement of the delivery as a function of amounts of charge transferred is true for iontophoretic delivery and it would be more applicable to consider time while making the predictions.

4. Conclusion

We have demonstrated here the delivery of PRA, RAS and HUP under the influence of different current profiles – continuous and multi-phasic. High delivery efficiencies were obtained from continuous current anodal iontophoresis with linear correlation of the total

51 delivery and the amounts of charge transferred at each current density for PRA, RAS and HUP. The permeation also found to vary with the alteration in current density as in multi-phasic Profile 1, 2 and 3 where the total delivery was equivalent to the amounts of the charge transferred. This modulation of current density for determined time period can provide simple means to control electrotransport of drug to personalize dosing. Skin biodistribution study revealed the distribution behavior in response to the duration and the density of the current application. For longer period of current application PRA distributed much uniformly then the short duration experiments. Further, the intensity of current affected the rate of PRA delivery in the skin where at lower current density the skin distribution was slower and faster at the higher current densities. The prediction of drugs delivery based on the amounts of charge transferred was found to be very accurate for all three drugs tested using multi-phasic current profiles. However, the prediction was not found to be true for the short duration iontophoresis experiments. Nevertheless, the observed linear enhancement for one-hour iontophoresis at 0.15, 0.3 and 0.5 mA/cm² indicates that for short duration iontophoresis the development of a new model would be more appropriate to obtain more accurate predictions.

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.

52 References

[1] F. Stocchi, The concept of continuous dopaminergic stimulation: what we should consider when starting Parkinson's disease treatment, Neurodegener. Dis., 7 (2010) 213-215.

[2] G. Small, B. Dubois, A review of compliance to treatment in Alzheimer's disease:

potential benefits of a transdermal patch, Curr. Med. Res. Opin., 23 (2007) 2705-2713.

[3] B. Winblad, A.K. Kawata, K.M. Beusterien, S.K. Thomas, A. Wimo, R. Lane, H. Fillit, R.

Blesa, Caregiver preference for rivastigmine patch relative to capsules for treatment of probable Alzheimer's disease, Int. J. Geriatr. Psychiatry, 22 (2007) 485-491.

[4] Y.N. Kalia, A. Naik, J. Garrison, R.H. Guy, Iontophoretic drug delivery, Adv. Drug Deliv.

Rev., 56 (2004) 619-658.

[5] D.R. Kalaria, P. Patel, V. Merino, V.B. Patravale, Y.N. Kalia, Controlled iontophoretic delivery of pramipexole: electrotransport kinetics in vitro and in vivo, Eur. J. Pharm.

Biopharm., 88 (2014) 56-63.

[6] D.R. Kalaria, P. Patel, V. Patravale, Y.N. Kalia, Comparison of the cutaneous iontophoretic delivery of rasagiline and selegiline across porcine and human skin in vitro, Int.

J. Pharm., 438 (2012) 202-208.

[7] D.R. Kalaria, P. Patel, V. Merino, V.B. Patravale, Y.N. Kalia, Controlled iontophoretic transport of huperzine A across skin in vitro and in vivo: effect of delivery conditions and comparison of pharmacokinetic models, Mol. Pharm., 10 (2013) 4322-4329.

[8] M.F. Piercey, Pharmacology of pramipexole, a dopamine D3-preferring agonist useful in treating Parkinson's disease, Clin. Neuropharmacol., 21 (1998) 141-151.

[9] M. Horstink, E. Tolosa, U. Bonuccelli, G. Deuschl, A. Friedman, P. Kanovsky, J.P.

Larsen, A. Lees, W. Oertel, W. Poewe, O. Rascol, C. Sampaio, Review of the therapeutic management of Parkinson's disease. Report of a joint task force of the European Federation of Neurological Societies and the Movement Disorder Society–European Section. Part I: early (uncomplicated) Parkinson's disease, Eur. J. Neurol., 13 (2006) 1170-1185.

[10] P.A. LeWitt, MAO-B inhibitor know-how: back to the pharm, Neurology, 73 (2009) 2048.

[11] L.W. Elmer, J.M. Bertoni, The increasing role of monoamine oxidase type B inhibitors in Parkinson's disease therapy, Expert Opin. Pharmacother., 9 (2008) 2759-2772.

[12] P. Jenner, Preclinical evidence for neuroprotection with monoamine oxidase-B inhibitors in Parkinson’s disease, Neurology, 63 (2004) S13-S22.

53 [13] R. Wang, H. Yan, X.C. Tang, Progress in studies of huperzine A, a natural cholinesterase inhibitor from Chinese herbal medicine, Acta Pharmacol. Sin., 27 (2006) 1-26.

[14] X.C. Tang, P. De Sarno, K. Sugaya, E. Giacobini, Effect of huperzine A, a new cholinesterase inhibitor, on the central cholinergic system of the rat, J. Neurosci. Res., 24 (1989) 276-285.

[15] B.S. Wang, H. Wang, Z.H. Wei, Y.Y. Song, L. Zhang, H.Z. Chen, Efficacy and safety of natural acetylcholinesterase inhibitor huperzine A in the treatment of Alzheimer's disease: an updated meta-analysis, J. Neural. Transm. (Vienna), 116 (2009) 457-465.

[16] A. Naik, Y.N. Kalia, R.H. Guy, Transdermal drug delivery: overcoming the skin’s barrier function, Pharm. Sci. Technol. Today, 3 (2000) 318-326.

[17] D.R. Kalaria, P. Patel, V. Merino, V.B. Patravale, Y.N. Kalia, Controlled iontophoretic delivery of pramipexole: Electrotransport kinetics in vitro and in vivo, Eur. J. Pharm.

Biopharm., (2014).

[18] J. Bradley Phipps, J.R. Gyory, Transdermal ion migration, Adv. Drug Deliv. Rev., 9 (1992) 137-176.

PART 1: TRANSDERMAL IONTOPHORETIC DELIVERY OF SMALL MOLECULES

CHAPTER 2

57

Controlled delivery of pramipexole from an iontophoretic transdermal