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Jun 1, 2018 - In this regard, the drug in adhesive patches of memantine (ME) co-loaded with donepezil (DO) was prepared using an ethylene vinyl acetat...
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Article Cite This: Mol. Pharmaceutics XXXX, XXX, XXX−XXX

In Depth Analysis of Pressure-Sensitive Adhesive Patch-Assisted Delivery of Memantine and Donepezil Using Physiologically Based Pharmacokinetic Modeling and in Vitro/in Vivo Correlations Naresh Mittapelly,†,‡ Gitu Pandey,† Sachin Laxman Tulsankar,† Sadaf Arfi,† Rabi Sankar Bhatta,†,‡ and Prabhat Ranjan Mishra*,†,‡ †

Pharmaceutics and Pharmacokinetics Division, CSIR-Central Drug Research Institute, Lucknow 226031, India Academy of Scientific and Innovative Research (AcSIR), New Delhi 201002, India

‡

S Supporting Information *

ABSTRACT: The objective of this work was to evaluate the feasibility of transdermal delivery of two widely prescribed dementia drugs for the Alzheimer’s disease. In this regard, the drug in adhesive patches of memantine (ME) co-loaded with donepezil (DO) was prepared using an ethylene vinyl acetate polymer and characterized for drug content, the crystallinity of drugs in the polymer matrix, and in vitro permeation. To understand the different physical and chemical processes underlying the percutaneous absorption, it is required to employ a comprehensive model that accounts for the anatomy and physiology of the skin. A transdermal physiologically based pharmacokinetic (TPBPK) model was developed and was integrated in a compartmental pharmacokinetic model to predict the plasma drug concentrations in rats. The model predictions showed a good fit with the experimental data, as evaluated by the prediction error calculated for both drugs. It was evident from the simulations that the drug diffusivity and partition coefficient in the polymer matrix are the critical parameters that affect the drug release from the vehicle and subsequently influence the in vivo pharmacokinetic profile. Moreover, a correlation function was built between the in vitro permeation data and in vivo absorption for both ME and DO. A good point-to-point in vitro/in vivo correlation (IVIVC, Level A correlation) was achieved by predicting the plasma concentrations with convolution for the entire study duration. The results of our study suggested that the implementation of mechanistic modeling along with IVIVC can be a valuable tool to evaluate the relative effects of formulation variables on the bioavailability from transdermal delivery systems. KEYWORDS: memantine, donepezil, transdermal patches, PBPK, IVIVC, skin absorption, pharmacokinetics

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INTRODUCTION Alzheimer’s disease (AD) is a severe chronic neurological disorder which predominantly affects the elderly above the age of 65 years. The disease manifests a wide variety of clinical symptoms, including memory defects and behavioral and psychological signs and symptoms. Loss of cholinergic neurons caused by the excessive neuronal firing of cortical is found be one of the reasons for the symptoms of AD. Presently, there are two different categories of drugs available for the symptomatic treatment of AD, i.e., n-methyl-D-aspartic acid (NMDA) receptor antagonists and choline esterase (an enzyme that degrades acetylcholine (ACh)) inhibitors. Memantine (ME) is the only approved NMDA receptor antagonist used for the treatment of mild to moderate AD. Donepezil (DO) is a second generation choline esterase inhibitor used for mild, moderate, and severe AD. These two drugs are often prescribed together in a clinic, and a fixed dose combination is also available on the market.1,2 However, in chronic disease conditions like AD, individuals often suffer comorbidities like © XXXX American Chemical Society

depression, anxiety, psychosis, agitation, and aggression, which further complicate the pharmacotherapy by the caregivers. Therefore, it is necessary to develop nongastrointestinal delivery systems, such as transdermal delivery systems (TDDS) that can be applied to the patients without any difficulty. TDDS are patient-friendly and noninvasive systems for direct delivery of therapeutic agents into the blood circulation. They have particular advantages like avoidance of the first-pass extraction of drugs, reduction of the incidence of side effects, increase in the therapeutic efficacy as a result of enhanced treatment adherence/patient compliance, etc. Although the transdermal route of administration is promising, the drug candidates need to have specific physicochemical characterReceived: February 16, 2018 Revised: April 30, 2018 Accepted: May 22, 2018

A

DOI: 10.1021/acs.molpharmaceut.8b00176 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics

purchased from a local market. Other chemicals used in the study were of reagent or higher grade. Methods. Preparation of ME and DO. ME hydrochloride (1 g) and DO hydrochloride (1 g) were separately dissolved in 25 mL of distilled water and the pH of the solution was adjusted to 11 using sodium hydroxide (NaOH, 1 N); the organic layers were extracted twice with 50 mL of chloroform and ethyl acetate, respectively, using a separating funnel. The combined organic layers were dried over sodium sulfate to remove the residual water followed by solvent evaporation to collect the product. TDDS Preparation. Drugs containing adhesive patches were prepared by a solvent evaporation technique.21 The 10% w/w drug(s), enhancers, and Emdilith DM-45 (PSA) were mixed with isopropyl alcohol and stirred until a clear solution was obtained. The solution was poured onto a release liner and allowed to dry for 3 h at 55 °C. The dried film was then laminated with a backing film. R-Limonene (5% w/w) and oleic acid (5% w/w) were incorporated as enhancers in the formulation. The following codes were used in the entire article for two different formulations: Patch (−) enhancer contains 5% w/w of DO, 5% w/w of ME, and 90%PSA. Patch (+) enhancer contains 5% w/w of DO, 5% w/w of ME, 5% w/w of oleic acid, 5% w/w of R-limonene, and 80% w/w of PSA. Drug Content Analysis. A patch with a surface area of 9 cm2 was dissolved in methanol under sonication and was filtered. After appropriate dilutions, 25 μL of the sample was injected into an LC−MS/MS system. LC−MS/MS Method. The chromatographic separation was carried out on an HPLC system consisting of a binary pump (WATERS, 515HPLC) and an autosampler (WATERS, 2707), and the system was connected to an API 3200 triple quadruple mass spectrometer (ABI SCIEX, ON, Canada). The mobile phase consisted of methanol and 10 mM ammonium acetate, pH 5 (adjusted using acetic acid), in 92/8 v/v ratio pumped at a flow rate of 0.7 mL/min. The chromatography column used was a Thermo Syncronis-C18 column (100 mm × 4.6 mm id, 5 μm) and was maintained at room temperature. Differential Scanning Calorimetry (DSC) Analysis. Differential scanning calorimetry (DSC) measurements were performed using a thermal analyzer SIIO 6300 (Japan). The sample was placed in a standard aluminum pan and was scanned at a 10 °C/min rate under a nitrogen flow rate of 20 mL/min, with a scanning temperature range of 0−250 °C. In Vitro Permeation. A Franz diffusion cell (volume of 20 mL, effective diffusion area of 5.722 cm2) maintained at 32 ± 0.5 °C and clamped with rat abdominal skin was used for the in vitro permeation study. The receptor chamber was filled with media pH 7.4 phosphate buffer saline and was continuously stirred at 500 rpm. At different time intervals, an aliquot of 0.5 mL of sample was withdrawn and replaced with the same volume of fresh buffer solution to maintain the constant fluid volume. The cumulative amount permeated (Q, μg/cm2) through the skin was calculated by the following equation

istics, like a small molecular weight (less than 500 Da), an octanol−water partition coefficient in the range of 1−4,3 an aqueous solubility >1 mg/L, and a daily dose of less than 20 mg.4 The stratum corneum (SC), composed of the outermost 10−15 layers of skin containing dead corneocytes, is the principal barrier to the skin absorption of drugs. To overcome the resistance offered by the SC, different approaches, such as passive and active strategies, can be implemented. Examples of the passive strategies include the use of a chemical permeation enhancer (CPE), which spatially disrupts the ordered arrangement of the intercellular molecules thereby decreasing the diffusion resistance. Examples for the CPE are glycols (e.g., polyethylene glycol), terpenes (e.g., limonene), fatty acids (e.g., oleic acid), etc. The TDDS are classified into three main types, a single layer or multilayer drug in the adhesive (DIA), liquid reservoir, and polymer matrix. The DIA patch is most commonly used among different systems,4,5 it is mainly composed of a drug adhesive as an integral element, which helps in patch adhesion to the skin surface and also controls the rate of the drug release from the adhesive matrix. In this study, we employed ethylene vinyl acetate (EVAc) polymer as a pressure-sensitive adhesive (PSA) for the preparation of TDDS, as it has excellent adhesive strength and also is a low-cost polymer with a good skin compatibility.6 In the recent past, the research has been proceeding toward a mechanistic understanding of transdermal pathways, including the attributes of the solutes and their interaction with skin with the emergence of mechanistic models. In this area, several researchers have worked and developed mathematical models to understand the drug transport across different layers of skin. At present, we have model equations for the dermal disposition in the SC,7−9 the viable epidermis (VE),10,11 and dermis (DE).12−14As these equations are established along the diffusion constant, permeation coefficient, and other similar parameters, which are derived from the quantitative structure− property relationship (QSPR) models, they can be conveniently applied to almost all chemicals/drugs for simulating the skin absorption to solve the what if questions linked to the skin absorption of chemicals.15,16 There are some reports of the TDDS of ME and DO individually, but these studies lack an in vitro/in vivo correlation (IVIVC) and have restricted their research to single drug administration only.17−20 Hence, the outcome when these two drugs will be administered in combination is not known. To address these issues, we developed a transdermal patch incorporating both ME and DO along with a permeation enhancer and studied in depth the permeation behavior of both drugs from the combination patch. In continuation with this, we have also studied the percutaneous absorption of aforesaid drugs using a physiologically based pharmacokinetic (PBPK) model.

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EXPERIMENTAL SECTION Materials. ME hydrochloride and DO hydrochloride were received from M/S Torrent Research Center (Gujrat, India) and M/S Lupin Research Park (Pune, India), respectively, as a generous gift. Amantadine hydrochloride and R-limonene were purchased from Sigma-Aldrich, and oleic acid was procured from Merck Millipore. Isopropanol and tert-butyl methyl ether were purchased from SRL, India. Emdilith DM-45 is a widely used polyethylene vinyl acetate-based polymer for making stickon cosmetic bindis for application on the forehead; it was

n−1

Q=

CnV + Sv ∑i = 1 Ci A

(1)

where V is the volume of the receptor compartment, A is the active surface area, Sv is the sampling volume, and Cn and Ci are the concentration of the drugs in the media at time point “n” and time point “i”, respectively. The Q was plotted against the B

DOI: 10.1021/acs.molpharmaceut.8b00176 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics time (h) to calculate the flux. Each point is the mean ± SD of three determinations. In Vivo Pharmacokinetic Study. Sprague−Dawley (SD) rats weighing 220−250 g were used for the pharmacokinetic study. The rat dorsal hair was removed using depilation cream (elois, manufactured by M/s Prem Henna Pvt, Ltd.) purchased from a local market. After 1 day, the patches were applied on the dorsal surface and removed after 24 h of application. For the intravenous (IV) pharmacokinetic study, test article (1 mg/kg) was dissolved in water for injection and injected through the tail vein. Blood samples (0.25 mL) were collected into a tube coated with ethylene diamine tetraacetic acid (EDTA, 20 μL of 10% w/v solution) and centrifuged at 4000g for separating the plasma. The plasma samples were stored at −80 °C until the analysis. Sample Preparation. A single step liquid−liquid extraction (LLE) method reported elsewhere was employed for the extraction of DO, ME, and IS (amantadine hydrochloride) from the plasma.22 Briefly, to 50 μL of plasma spiked with the drugs, 10 μL of IS (equivalent to 10 ng/mL) and 50 μL of 0.1% (v/v) ammonia base were sequentially added, and the solution was vortex mixed for 1 min. To this, 2.5 mL of tertiary butyl methyl ether (TBME) solvent was added, and the mixture was vortex mixed for 10 min. After centrifugation at 5000g for 15 min at 10 °C, the supernatant organic phase was collected and dried under a stream of nitrogen gas at 40 °C using an evaporator (Turbovap, MA, USA). Then, the dried residue was reconstituted with 50 μL of the mobile phase, and 10 μL was injected into the LC-MS/MS system. IVIVC Development. A point-to-point (Level A) correlation between the in vitro permeation and in vivo absorption of drugs was investigated. The in vitro data were obtained from the permeation experiments, whereas the in vivo plasma concentration data were deconvoluted using a Loo−Riegelman method to get the fraction of drug absorbed at different time points. Additionally, convolution data were generated from the unit impulse response (UIR) calculated from IV data and compared with experimental plasma concentrations obtained after TDDS application. Mathematical Modeling of Skin Drug Absorption. We developed a PBPK model of skin to make predictions of drug disposition in different skin layers of rat from physicochemical properties and physiological data available in the literature. The skin (Figure 1) was divided into three anatomically different layers, i.e., SC, VE, and DE. Each compartment in this model has a specific partition coefficient, thickness, and diffusivity and is connected with each other in parallel, as shown in Figure 2. The drug disposition in these compartments is governed by diffusion, except for DE, in which dermal clearance is responsible for the transport of the drug to blood circulation. Further, the developed skin model was integrated to a compartmental pharmacokinetic model to study the plasma concentration time profiles. The model construction and simulations were carried out using MATLAB Simulink23 (ver. 8.2, The Math Works, Inc., MA, USA) software. The transport of a chemical in skin layers can be described by Fick’s first law of diffusion; the mass transfer equation for drug transport from the x- to y-compartment can be described by the following equation ⎛ C Cy ⎞ ⎟ M = Peff A⎜⎜ x − K y /w ⎟⎠ ⎝ Kx /w

Figure 1. Representation of skin anatomy.

Figure 2. Structure of transdermal PBPK (TPBPK) attached to a twocompartment pharmacokinetic model. K, D, and h are the partition coefficient, diffusivity, and thickness, respectively.

where M (mg/h) is the mass transfer rate, Peff is the effective PxPy

( cmh ) = P + P , A (cm ) is the area of application,

permeability

x

2

y

and Kx/w and Ky/w are solute partition coefficients of x- and ycompartments with respect to water. The equations for the calculations of diffusion, partition, and permeability are presented in the Appendix in the Supporting Information. Two-Compartment Pharmacokinetic Model for ME and DO. A two-compartment model was used to describe the kinetics of ME and DO in the body. In this model, the central and peripheral compartments have specific volumes V1 and V2, transfer constants K12 and K21, and an elimination constant K10.

(2) C

DOI: 10.1021/acs.molpharmaceut.8b00176 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 3. DSC thermogram of (A) ME and (B) DO. (C) In vitro permeation as cumulative amount permeated (μg/cm2) vs time from drug solutions and (D) different patch formulations containing ME and DO. DSC thermogram of a (E) blank patch and (F) patch (−) enhancer.

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and DO were 4.30 ± 0.87, 19.94 ± 2.63, 7.62 ± 1.7, and 11.301 ± 1.2 μg/cm2/h, respectively. The steady state fluxes of the free bases were 4.6-fold and 1.5-fold higher than those of the respective hydrochloride salt form of ME and DO, as shown in Figure 3C. Effect of Permeation Enhancers on Free Base Permeation from TDDS. The permeation study for the TDDS revealed that the addition of chemical enhancers to the system has a significant effect on the cumulative permeation amount, as shown in Figure 3D. The transdermal fluxes for ME and DO from the patch (−) enhancer were 6.104 ± 2.08 and 3.05 ± 1.04 μg/cm2/h, whereas from the patch (+) enhancer,

RESULTS

Characterization of the Free Base. The DSC analysis showed that both ME and DO had a lower melting point, i.e., 172.5 (Figure 3A) and 93.21 °C (Figure 3B), compared to those of their hydrochloride forms, which were 271.5 (Figure S1) and 335 °C (Figure S2), respectively. Free Base and Hydrochloride Salt Permeation. The initial permeation experiments for salts and free bases were carried out to see if there is any difference in terms of the cumulative permeation amount with respective time. The fluxes calculated for the ME hydrochloride, ME, DO hydrochloride, D

DOI: 10.1021/acs.molpharmaceut.8b00176 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics they readily increased to 11.076 ± 1.24 and 9.671 ± 0.37 μg/ cm2/h, respectively. Drug Content and DSC Analysis. As the content uniformity and crystallinity of drugs in the patch formulation have a direct effect on the drug release and subsequently on the in vivo performance,24 it is required to perform detailed analysis of the patch. In this regard, the study for the content uniformity of the patches was carried out, and it was found that 80−110% of the theoretical amount of individual drugs was present in all batches of the patch formulation. The status of the crystallinity in the formulations was evaluated using DSC, and it was found that the PSA exhibited an endothermic peak at 100.4 °C (Figure 3E). In the case of the drug-loaded patch, only a single endothermic peak was observed at 123.1 °C (Figure 3F), and the characteristic melting peaks of individual drugs disappeared. This indicates that the drugs are dispersed in the PSA uniformly in the form of an amorphous solid dispersion. In Vivo Pharmacokinetics and IVIVC. The pharmacokinetic studies of prepared formulations were carried out at a dose of 40 mg/kg of both ME and DO. Additionally, a separate IV pharmacokinetic study was also carried out at a dose of 1 mg/kg to describe the naive distribution and elimination of individual drugs, which were subsequently used to calculate the unit impulse response (UIR) for the development of IVIVCs. The mean plasma concentration vs time profiles of ME and DO after IV administration and transdermal administration are presented in Figure 4, and the parameters for the transdermal administration are summarized in Table 1.

where Ct is the concentration of the drug in the central compartment, K10 is the elimination rate constant, AUC0−t is the area under the plasma concentration curve from 0 to t, AUC0−inf is the area under the plasma concentration curve from 0 to infinity, Vc is the volume of the central compartment, (Xp)t is amount of drug in the peripheral compartment at time t, and F is the fraction bioavailability calculated from the reference IV dose. The equations were fit into a linear or polynomial secondorder function for different data sets derived from in vitro and in vivo experiments. The plots between the percent of in vitro permeation vs in vivo absorption are shown in Figure 5. The calculated percent absorption for DE and MO was 13.6% and 10.35% from the patch (−) enhancer and 22% and 21.04% from the patch (+) enhancer, respectively. The IVIVCs generated here supported the relationship between the in vivo absorption and in vitro permeation. Internal validation was performed to evaluate the robustness of the developed IVIVC model. For this purpose, the in vitro data was convoluted (UIR is presented in the Supporting Information) using the IVIVC models to obtain the estimated in vivo plasma concentrations. The resulted Cmax and AUC were then compared with the experimental results, and the predictability of this procedure was evaluated using the percentage prediction error (%PE). The results are presented in Figure 6 and Table 2. As per the FDA guidelines, it is required that, for the establishment of the predictability of IVIVC, the average absolute %PE for the internal validation of Cmax and AUC should be less than 20%. Here, the average absolute %PE for Cmax ranged from 0 to 20.9%, and for AUC, it ranged from 5.9 to 28.6% for the data obtained from the in vitro experimental conditions. This error collectively can be attributed to the intersubject variability in the pharmacokinetic study data and in vitro permeation data. Mathematical Modeling of Skin Drug Absorption. After establishing the systemic compartmental pharmacokinetic model for DO and ME, the developed transdermal PBPK (TPBPK) model was attached individually to predict the plasma drug concentrations. The observed and predicted mean concentration profiles for the transdermal administration of ME and DO are illustrated in Figure 7A,B. The developed TPBPK model (calculated macroscopic parameters are presented in the Supporting Information) adequately predicted the plasma concentration−time profiles of both drugs from the TDDS. The goodness of fit for simulated profiles was evaluated by the correlation coefficient and percent deviation between the observed and simulated pharmacokinetic parameters. As observed in Figure 7A,B, the model-predicted Cmax and Tmax are in agreement with the observed data. Likewise, the prediction of the remaining pharmacokinetic parameters, such as AUCs, and the fraction bioavailability are in good agreement with the experimental data. From the simulation results, the relative percentage of ME and DO in the VE (patch) and SC with respect to time can be obtained. It appears from these profiles, as shown in Figure 7C,D, that both drugs get deposited in the SC to a substantial extent. The percentage dose deposited in the SC after 24 h was found to be in the range 4.77−6% for ME and 36−54% for DO, respectively, for the two different formulations. After the removal of the patch, the release of the accumulated drugs from the SC could be distinctly understood in the plasma profile. In Figure 8, the sensitivity analysis with respect to the model input parameters in the vehicle is presented. To begin with, we varied the partition coefficient between the vehicle and water

Figure 4. Plasma concentration vs time profiles of (A) ME and (B) DO after transdermal (40 mg/kg) and IV administration (1 mg/kg). Each point represents the mean ± SD of n = 4.

The deconvolution analysis for both drugs from the in vivo plasma profiles of patch formulations was performed using the Loo−Riegelman method25 Absorption% =

C t + K10 AUC0 − t + (X p)t /Vc FK10 AUC0 − inf

× 100% (3) E

DOI: 10.1021/acs.molpharmaceut.8b00176 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics Table 1. Pharmacokinetic Parameters of ME and DO after Transdermal Administration in Rats (n = 4)a ME

a

DO

parameter

patch (+) enhancer

patch (−) enhancer

patch (+) enhancer

patch (−) enhancer

Cmax (ng/mL) Tmax (h) AUCt (ng·h/mL) AUMCt (ng·h2/mL MRTt (h)

149.1 ± 57.13 10 ± 0 5175.90 ± 1270 128 350 ± 27 143.33 24.79 ± 21.37

100.7 ± 32 36 ± 0 3774.66 ± 991.21 101 177.65 ± 26 096.63 26.8 ± 26.32

190.5 ± 101.65 24 ± 10 5953.68 ± 1650.24 133 392 ± 25 880.6 22.40 ± 15.68

119.1 ± 28.14 24 ± 0 3513.84 ± 827.75 89 197.28 ± 18 354 25.38 ± 22.17

Contains 10% w/w of permeation enhancers (1:1, oleic acid and R-limonene), data are the mean ± SD (n = 4).

Figure 5. (A, B) Plots between the in vitro permeation and in vivo absorption of two different formulations for ME and (C, D) for DO.

Figure 6. Internal validation of the IVIVC model for (A) DO and (B) ME.

from 1 to 100 times of the nominal value (K = 1) and studied its effect on the plasma profile. Here, the low and high partition

coefficient values mimic the drug release from the hydrophilic and the hydrophobic polymer system, respectively. We F

DOI: 10.1021/acs.molpharmaceut.8b00176 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Molecular Pharmaceutics Table 2. Internal Validation Results of IVIVCsa Cmax (ng/mL) drug name ME DO a

formulation patch patch patch patch

(−) enhancer (+) enhancer (−) enhancer (+) enhancer

AUC(0−24) (ng·h/mL)

Obs

Pred

%PE

Obs

Pred

%PE

119.1 190.5 90.0 149.1

94.2 190.5 81.0 169.2

20.9 0.0 10.0 −13.5

1664.2 3415.5 1616.3 2520.9

1188.9 2513.5 1521.1 2674.6

28.6 26.4 5.9 −6.1

Obs = observed value, Pred = predicted value, and % prediction error (PE) = (Obs − Pred) × 100/Obs.

Figure 7. Simulated and observed plasma concentration−time profiles for (A) ME and (B) DO. Simulated time vs percent dose curves in patch and SC for (C) ME and (D) DO.

only after decreasing the diffusion coefficient by 100-fold or more. Further reduction of the coefficient values makes the permeation rate comparable to the SC, hence, slowing the penetration, sustaining the release, and leading to low plasma concentrations.

observed a steep rise accompanied by a fast descent in the plasma levels when the partition coefficient was decreased to imitate a hydrophilic polymer system. On the other hand, retention of the drugs in the delivery vehicle and the resultant slow release of the drugs into the skin and blood circulation were predicted with the use of a high partition coefficient mimicking a hydrophobic polymer. However, it must be taken into consideration that the overall effect could be more complicated owing to the different water vehicle partition coefficient of the different solutes and the differential diffusion properties of the skin. Next, the effect of drug mobility in the vehicle was examined. The diffusion coefficient in the vehicle was brought down to 1/ 10, 1/100, 1/500, and 1/1000 for both drugs to get an insight into the controlled release. As evident from Figure 8, the overlapping plasma profiles with negligible impact on the pharmacokinetics of ME and DO were observed when the diffusion coefficient of the vehicle was reduced by 2 orders of magnitude. A reduction in the plasma profiles was observed

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DISCUSSION This work aimed to prepare a combination patch delivery system of two widely prescribed dementia drugs, DO and ME, for transdermal administration. In the AD, as the disease progresses, the patients display a wide variety of psychological symptoms which may prevent the administration of these drugs by conventional means. Also, DO is well-known for its gastrointestinal side effects, owing to the fluctuations in the plasma concentrations. These problems can be addressed collectively by making efficient use of the alternate routes of administration. We have reported the long-acting injectable formulations for ME and DO individually.26,27 Previously, Choi et al.17 studied the in vitro and in vivo percutaneous absorption G

DOI: 10.1021/acs.molpharmaceut.8b00176 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

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Figure 8. Results of the sensitivity analysis for (A, B) ME and (C, D) DO.

of DO, and Del Rio-Sancho et al.18 reported the in vitro permeation of ME from a propylene glycol solution. They found that the pretreatment of the skin with oleic acid and limonene resulted in a maximum flux of DO and ME hydrochloride salt, respectively, across the skin. In the present research work, we attempted to formulate a combination patch of these two drugs for transdermal administration and explored its in vitro and in vivo permeation behavior using IVIVC and PBPK for better mechanistic understanding. In this regard, we first converted the marketed salt forms of DO and ME into the corresponding base forms and studied them employing DSC. The base form displayed a lower melting point in comparison to the salt form for both drugs. Because of this lowering of the melting point, we anticipated that the base form will be more oil soluble5 and consequently more skin permeable. This assumption was backed by the results where we observed the higher permeation of the base forms of the drugs in comparison to the salt forms. Further, we investigated the in vitro percutaneous absorption of DO and ME in adhesive patches without and with permeation enhancers. Although the adhesive layer is a vital formulation variable that influences the rate of the release of the drugs, in this study, we only evaluated a single adhesive patch system. In the in vitro permeation experiments, a steady and continuous permeation was observed for both drugs, indicating that the drugs were dispersed uniformly in the patch. Also, we separately evaluated the content uniformity of the patches and

found that the coefficient of variation was less than 10%, indicating that they were suitable for further experimentation. The supersaturation of active pharmaceutical ingredients (APIs) in an adhesive layer may result in their precipitation or crystallization. To evaluate this, we performed DSC analysis of the patch system and found that there was no characteristic peak of drugs confirming that the drugs were dispersed in a molecular form at a concentration below supersaturation. In the adhesive layer of the DIA patch, penetration enhancers may be incorporated to overcome the barrier properties of the SC. Such penetration enhancers may be used alone or in a mixture of two or more. In this study, to prepare the enhancerloaded formulation, we used R-limonene and oleic acid at a concentration of 5% w/w, the effect of these permeation enhancers was confirmed by the in vitro permeation study. An enhancement ratio of nearly 2−3 was observed when both drugs were delivered from a formulation containing enhancers compared with the formulation without enhancers. This can be explained by the combined effect of limonene on the SC fluidity and perturbation of the SC lamellar lipids by both oleic acid and limonene.28 As these permeation enhancers have already been used in some of the marketed formulations, we did not anticipate any stability related issues pertaining to the enhancers, and our study results were at par with our assumptions. The in vivo pharmacokinetic study manifested that, after IV administration, the plasma concentrations of DO and ME H

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adjusting the drug diffusivity and partition coefficient in-vehicle compartment. Further, we also modeled different scenarios in the sensitivity analysis, like the effect of vehicle properties ranging from hydrophilic (a low partition coefficient) to hydrophobic (a high partition coefficient) on the drug input rate and the plasma kinetics of drugs. It was estimated that a hydrophobic vehicle would partition a smaller amount of ME and DO in the SC, thereby reducing the effective permeability between the vehicle and SC. This further translates into a lower flux in other compartments and, subsequently, a small plasma drug concentration. Vice versa is valid for a hydrophilic system for ME and DO. The effects are more complicated for different solutes in a given delivery system, as the vehicle water partition coefficient and the skin-derived partition will assume different values with a change in solute property. Furthermore, our simulation results suggested that there is a slow release of both drugs from the skin, even after removal of the patch (reservoir effect). This effect is more pronounced in the case of DO and less for ME; the relative difference can be attributed to a high SC water-partition coefficient of DO (337) in comparison to that of ME (49.6). Additionally it is noteworthy that any interactions are not foreseen between ME and DO, as the absorption in different skin layers is assumed to happen through a first-order nonsaturable process.

rapidly declined, while in the case of transdermal administration, the plasma levels were maintained for 24 h (patch was removed after this time point). Upon removal of the transdermal patch, a reservoir effect was observed in the case of DO followed by an elimination similar to that of IV administration. The IVIVC for the oral formulation is well established with the available guidance from the US Food and Drug Administration. At present, no such guidelines are available for developing IVIVC for transdermal products. To date, the majority of the established IVIVC for TDDS include the comparisons of single parameters, such as Css (Level C). Presently, there are only two reports of a point-to-point (Level A) IVIVC between the in vitro permeation data and in vivo data.29,30 The data sets in the current study have successfully demonstrated IVIVC from a single parameter comparison in terms of either Css or AUC and point-to-point IVIVC. The in vitro permeation test results showed that the method employed here was sensitive enough to discriminate the differences between the formulations. The results obtained here indicated the possibility of the in vivo performance of TDDS based on the permeation data collected from the in vitro studies. The objective of developing a skin absorption model here was to provide a comprehensive physiologically based in silico model to simulate the drug disposition after transdermal administration and, in addition, to make a prediction of the overall pharmacokinetics of drugs. The model was constructed using known physiological factors describing the skin in rats. The thickness of different skin layers, pH, and blood flow rate were incorporated into this model. The mass transfer process occurs as a drug exchange between individual compartments and systemic circulation. The kinetics of the drug transfer in the present model could be described in three stages: (1) drug partitioning between the vehicle and top SC (partition coefficient calculated); (2) partitioning between the SC and VE, and VE and DE; and (3) drug uptake into the systemic circulation depending upon the capillary permeation and blood flow. The correlation between the physicochemical properties, like partition coefficient, molecular weight, or molecular volume, with the in vivo partition coefficient and diffusivities is well established, except for the dermal clearance, for which only a limited amount of data is available. However, the clearance parameter can be indirectly estimated using a capillary permeability model, as described by Ibrahim et al. The drug diffusion model employed here for SC takes into account the drug diffusion in both lipid bilayers and corneocytes. The parameters, namely, fraction unbound and fraction un-ionized, were incorporated in the model while calculating the partition and diffusion coefficient for VE and DE. The fraction unbound can be determined experimentally or derived theoretically from a correlation proposed by Yamazaki et al.,31 while the fraction un-ionized at a particular pH can be calculated from the Henderson−Hasselbalch equation if the pKa is known. After construction of the model, the macroscopic parameters derived from individual physicochemical properties of drugs were incorporated into it, except for the vehicle compartment. As of now, the correlations for the calculation of the partition coefficient and diffusivity in the EVAc matrix for individual drugs are lacking. Thus, we carried out a numerical optimization in this compartment until the observed matched with the predicted plasma concentration−time profile by

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CONCLUSION The work presented here reports the successful formulation and characterization of TDDS containing ME and DO. Further, in in vivo experimentation, therapeutically relevant doses of the individual drugs were successfully delivered to rats, highlighting the potential for exploitation of this delivery route for administration. This promising system could simplify the administration of two different drugs for elderly patients. Before proceeding further, understanding of the transdermal absorption of chemical agents and the effects of the formulation characteristic on in vivo absorption is required. Thus, we have developed a four compartment TPBPK model nested to a compartmental pharmacokinetic model. The model used literature derived anatomy and physiology for predicting the plasma concentration of ME and DO after TDDS application. Additionally, IVIVCs were developed, and it was found that there was a strong correlation between the in vivo percent absorption and in vitro percent permeation in rats. Moreover, the model can be applied for predicting the plasma concentrations for different delivery rates from a TDDS.

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.8b00176. Appendix A of equations, Appendix B of the list of methods, DSC thermograms, intravenous pharmacokinetic data and calculated partition, and diffusion and permeability coefficients (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected], [email protected]. ORCID

Prabhat Ranjan Mishra: 0000-0002-7418-8283 I

DOI: 10.1021/acs.molpharmaceut.8b00176 Mol. Pharmaceutics XXXX, XXX, XXX−XXX

Article

Molecular Pharmaceutics Notes

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank CSIR, India, for financial assistance provided in the form of a GATE fellowship to N.M., G.P., and S.L.T. We are grateful to the Director of CSIR-CDRI for providing the necessary research facilities. We also thank Nishka Laboratories, Hyderabad, for providing analytical services.This is CDRI communication number 9692.

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DOI: 10.1021/acs.molpharmaceut.8b00176 Mol. Pharmaceutics XXXX, XXX, XXX−XXX