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Investigation and mathematical description of the real driving force of passive transport of drug molecules from supersaturated solutions Eniko Borbas, Balint Sinko, Oksana Tsinman, Konstantin Tsinman, Eva Kiserdei, Balazs Demuth, Attila Balogh, Brigitta Bodak, Andras Domokos, Gergo Dargo, György T. Balogh, and Zsombor K. Nagy Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00613 • Publication Date (Web): 09 Sep 2016 Downloaded from http://pubs.acs.org on September 12, 2016

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Molecular Pharmaceutics is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

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Molecular Pharmaceutics

Investigation and mathematical description of the real driving force of passive transport of drug molecules from supersaturated solutions Enikő Borbás1, Bálint Sinkó2, OksanaTsinman2, Konstantin Tsinman2, Éva Kiserdei1, Balázs Démuth1, Attila Balogh1, Brigitta Bodák1, András Domokos1, Gergő Dargó 1,3, György T. Balogh3, Zsombor K. Nagy1* 1

Budapest University of Technology and Economics, Department of Organic Chemistry and

Technology, Hungary 2

Pion Inc., USA

3

Gedeon Richter Plc., Compound Profiling Laboratory, Hungary

Keywords: in vitro dissolution- permeation test, microflux, flux, electrospinning, nanofiber, Meloxicam, Soluplus, supersaturation, co-solvent, DMSO

ABSTRACT

The aim of this study was to investigate the impact of formulation excipients and solubilizing additives on dissolution, supersaturation and membrane transport of an active pharmaceutical ingredient (API). When a poorly water-soluble API is formulated to enhance its dissolution, additives, such as surfactants, polymers and cyclodextrins have an effect not only on dissolution

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profile, but also on the measured physicochemical properties (solubility, pKa, permeability) of the drug while the excipient is present, therefore also affect the driving force of membrane transport. Meloxicam, a nonsteroidal anti-inflammatory drug was chosen as a poorly water-soluble model drug and formulated in order to enhance its dissolution using solvent-based electrospinning. Three polyvinylpyrrolidone (PVP) derivatives (K30, K90 and VA 64), Soluplus and (2Hydroxypropyl)-β-cyclodextrin were used to create five different amorphous solid dispersions of meloxicam. Through experimental design, the various formulation additives that could influence the characteristics of dissolution and permeation through artificial membrane were observed by carrying out a simultaneous dissolution-permeation study with a side-by-side diffusion cell, µFLUXTM. Although the dissolution profiles of the formulations were found to be very similar, in case of Soluplus containing formulation the flux was superior, showing that the driving force of membrane transport cannot be simplified to the concentration gradient. Supersaturation gradient, the difference in degree of supersaturation (defined as the ratio of dissolved amount of the drug to its thermodynamic solubility) between the donor and acceptor side was found to be the driving force of membrane transport. It was mathematically derived from Fick’s first law, and experimentally proved to be universal on several meloxicam containing ASDs and DMSO stock solution.

INTRODUCTION The increasing number of poorly water-soluble drug candidates is one of the most pressing issues of pharmaceutical industry. Namely, low aqueous solubility prevents the active pharmaceutical ingredients (APIs) from being bioavailable and biologically active. For that

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Molecular Pharmaceutics

reason dissolution enhancement of BCS II classified (poorly water-soluble, but highly permeable APIs) has gained primary importance in original and generic formulation development as well 1, 2

. Although in vitro dissolution test is a relatively simple and commonly used tool for the

evaluation of dissolution enhancement, and it is even used for biowaiver studies3, the results of this test do not always correlate with in vivo bioavailability 4. The lack of correlation is usually due to the fact that by using various formulation additives not only the dissolution of the drug, but also passive diffusion and partitioning to the membranes is altered. For understanding the effect of commonly used formulation excipients and methods on membrane transport it is inevitable to distinguish them by the mechanism of action as it was recently pointed out by Raina 5

. Most approaches for dissolution enhancement can be categorized by how the dissolution

enhancement is achieved. Based on that two groups can be created: one where the dissolution enhancement is achieved by supersaturation (using non-solubilizing additives inhibiting crystallization)6 and the second where dissolution enhancement is achieved by solubility enhancement (with the help of solubilizing additives)7,8. One of the approaches for dissolution enhancement achieved by supersaturation is creating amorphous solid dispersions (ASD). The amorphous state of the API leads to increased apparent solubility, higher than the equilibrium crystalline solubility 9, 10, meanwhile the thermodynamic solubility is not altered, meaning supersaturated solutions are created when dissolving ASDs. Increased fluxes, compared to results from saturated or subsaturated solutions, were reported in many cases when supersaturated solutions were used 5,8,11–17. These results show that ASDs have a great potential to improve the bioavailability of poorly water-soluble drugs.

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Electrospinning (ES), previously called electrostatic spinning, is an emerging way of generating ASDs. This process is able to create nano- and micro-scaled polymer-fibers from a viscous polymer solution containing a poorly water-soluble API with the help of drawing force of an electrostatic field. The efficiency of this technique in dissolution enhancement of BCS II classified APIs were proven by many formulations developed on laboratory scale flexibly scalable and easily integrable in continuous manufacturing

26–28

18–25

. Being

electrospinning is a

promising technology producing ASDs, from which fast dissolution of the drug can be reached owing to the high surface area, the wettability of the polymer mat and the amorphisation of the API29. Polymer additives, commonly used for electrospinning or any other formulation method producing ASDs, generally do not alter equilibrium crystalline solubility of APIs

12

. Polymers

are rather responsible for making the API’s supersaturation last long in solution by inhibiting precipitation and crystal growth 30–38

The use of solubilizing additives, such as surfactants, cyclodextrins and co-solvents enhance dissolution by increasing the thermodynamic or equilibrium crystalline solubility, which results in creation of subsaturated or saturated solutions. The effect of Fassif media (containing surfactants) was studied using dissolution-permeation cell with Caco-2 membrane, showing that even though the amount of drug solubilized in fasted state stimulated intestinal fluid (Fassif) media was increased due to the surfactant content, the flux did not increase 8. During in vivo studies in rats with increasing the amount of surfactant, decreasing flux was observed 39. The flux results of ASDs and solubilizing additive containing formulations clearly show, that the driving force of membrane transport cannot be simplified to the concentration gradient, especially in cases, where solubility is altered. Namely by increasing the solubility of API’s

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apparent permeability decreases phenomenon by Alex Avdeef

40

. This solubility-permeability interplay was a well described

41

, and Dahan & Miller42,43,40 and esential to take into

consideration when investigating the real driving force of membrane transport. Although the number of papers decribing this phenomenon is increasing, still the superior bioavailability enhancement caused by ASDs has not been fully understood. For that reason the aim of this paper was to investigate the universal driving force of membrane transport and provide a mathematical description of it , which is independent from the type of formulation. During the formulation development of a poorly water-soluble APIs it is usual to employ strategies that combine solubilizing and non-solubilizing additives44. Using Soluplus as a formulation additive combines different type of formulation approaches itself. Namely Soluplus is not only a polyethylene glycol- polyvinyl caprolactam- polyvinyl acetate graft copolymer, but also a non-ionic surfactant. So by being a polymer, it is able to inhibit nucleation and thereby stabilize supersaturated solutions, and by being a non-ionic surfactant it is able to form thermoreversible hydrogel. As the temperature in Soluplus containing solution increases the ethyleneglycol groups get dehydrated, and a hydrogel is formed, which by cooling the solution under the cloud point becomes a clear solution again

45

. The presence of kosmotropic salts

dehydrate the polymer, effectively lowering the cloud point and facilitating formation of a thermoreversible hydrogel 45.

Microenvironmental pH-modification is a possible solution for improving dissolution behavior of APIs with pH dependent solubility

46–48

. However the total concentration of the API in

solution might be increased, it is due to ionization of the molecules, which can hinder absorption. Namely only the uncharged form of the ionizable molecule is able to permeate by passive

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49–52

. This formulation strategy is rather unique, cannot be categorized as

solubilizing or non-solubilizing, because it is not altering thermodynamic solubility of the uncharged API, and does not use solubilizing additive either. In this study meloxicam (MEL), a nonsteroidal anti-inflammatory drug was chosen as a poorly water-soluble model drug and formulated in order to enhance its dissolution using solvent-based electrospinning. Three polyvinylpyrrolidone (PVP) derivatives (K30, K90 and VA 64), Soluplus and (2-Hydroxypropyl)-β-cyclodextrin were used to create five different amorphous solid dispersions of MEL with the same 15 w/w % API content in every formulation. Through experimental design the effect caused by the load of formulation and the quality of formulation additives, that can influence the characteristics of dissolution and permeation through artificial membrane, were observed by carrying out a simultaneous dissolution-permeation study with a side-by-side diffusion cell, µFLUXTM.

2. Experimental Section 2.1. Materials

Meloxicam (351.403 g/mol, structure shown in Figure 1), buffer components (KH2PO4, NaOH, KCl) and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich Co. LLC. (St. Louis, MO, USA). Soluplus® (structure shown in Figure 1) and PVP derivatives were obtained from BASF (Ludwigshafen, Germany). (2-Hydroxypropyl)-β-cyclodextrin (HP-ß-CD) with the molar substitution degree: 0.64 was purchased from Roquette Fréres. Gastrointestinal tract (GIT) lipid and ASB buffer were obtained from Pion Inc (Billerica, MA, USA). Neutral Linear Buffer (NLB) was purchased from Sirius Analytical Ltd. (East Sussex, United Kingdom).

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Figure 1. Chemical structure of meloxicam (a) and Soluplus (b) 2.2. Electrospinning

The polymer and MEL calculated amounts were added into 8 mL of the solvent (tetrahydrofuran (THF) and dimethylformamide (DMF) 1:1 v/v) and stirred magnetically (VELP Scientifica, Usmate, Italy) at 600 rpm until the dissolution was complete. The electrical potential applied on the spinneret electrode (inner diameter 0.8 mm) was 40 kV (NT-65 high voltage DC supply, MA2000 device, Unitronik Ltd, Nagykanizsa, Hungary). A grounded aluminum plate covered with aluminum foil was used as collector (20-30 cm from the spinneret). Polymer solutions were dosed with 4-6 mL/h at room temperature (25 C) with an SEP-10S Plus type syringe pump (Aitecs, Vilnius, Lithuania). The composition of polymer solutions are shown in Table 1. Table 1. Composition of the polymer solutions

Formulation named containing polymer

after API (w/w Polymer %) (w/w %)

THF DMF HP-β-CD (ml/g dry (ml/g dry (w/w %) matter ) matter)

ES_PVP K90

15

85

0

4

4

ES_PVP K90 HP-ß-CD

15

15

70

4

4

ES_PVP K30

15

85

0

4

4

ES_PVP VA64

15

85

0

4

4

ES_Soluplus

15

85

0

4

4

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2.3. Scanning Electron Microscopy Morphology of the samples was investigated with a JEOL JSM-6380LA (JEOL Ltd., Tokyo, Japan) type scanning electron microscope. Each specimen was fixed by conductive double-sided carbon adhesive tape and coated with gold–palladium alloy before examination. The applied accelerating voltage and working distance were between 15 and 30 kV and 10 and 12 mm, respectively.

2.4. Differential Scanning Calorimetry Differential scanning calorimetry (DSC) measurements were carried out using a Setaram DSC 92 apparatus (Caluire, France). (Sample weight: 10–15 mg, open aluminum pan, nitrogen flush.) The temperature program consisted of an isothermal period (1 min) at room temperature, with subsequent linear heating from 25° C to 300°C at the rate of 10 °C/min.

2.5. X-ray powder diffraction X-ray diffraction patterns of the samples were recorded by means of a PANalytical (Amelo, the Netherlands) X’pert ProMDP X-ray diffractometer using Cu-K" α radiation (1.542 Å) and a Ni filter. The applied voltage was 40 kV, while the current was 30 mA. The samples were analyzed between 4° and 42° 2θ.

2.6. Measurement of the thermodynamic solubility For measuring the thermodynamic solubility crystalline MEL (200 mg) were added to 900 mL of pH 6.8 phosphate buffer already containing the calculated amount of formulation additive, such as polymer, cyclodextrin or co-solvent (n = 3). The resulting mixtures were stirred at 37 °C

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Molecular Pharmaceutics

for 24 h (to the solution equilibrium). After filtering the concentration of the API in the buffer was determined by a Hewlett-Packard HP 8453G UV-VIS spectrophotometer (Palo Alto, USA) at a wavelength of 363 nm based on a former calibration.

2.7. pKa measurement Spectrophotometric determination of pKa values were performed using the fully automated SiriusT3™ instrument. In the case of the pure MEL powder sample the instrument’s default Fast UV assay was used: 5 µl of 10 mM DMSO solution was titrated from pH 2.0 to pH 12.0 in 1.5 ml ionic strength adjusted water (ISA-water: 0.15 M KCl) under N2 atmosphere at 25±0.5 °C. In case of the different kinds of formulations a modified Fast UV assay was used. The ISA-water solutions were prepared in advance from the formulation samples with a calculated concentration of MEL equal to the initial concentration during the measurement of MEL powder sample. The absorbance changes during the titrations were monitored by the Dip Probe Absorption Spectroscopy (D-PAS) method, evaluation and calculation of pKa values were performed by the Refinement Pro™ software attached to Sirius T3™. The spectral region of 260 – 300 nm was used in the analysis and the results were calculated from 3-9 replicates in each case (Table 4).

2.8. Potentiometric determination of partition coefficients Potentiometric titration of the logP value of pure MEL was performed using the SiriusT3™ instrument. Approximately 1.3 mg of MEL samples were titrated under the same conditions as in pKa determinations (from pH 2.0 to pH 12.0 in 1.5 ml ionic strength adjusted water (ISA-water: 0.15 M KCl) under N2 atmosphere at 25±0.5 °C) but in the presence of various amounts of the partitioning solvent, water-saturated n-octanol. The phase ratio applied was varied from 1.0 ml water – 0.5 ml n-octanol to 1.0 ml water – 0.6 ml n-octanol. From the n-octanol containing

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titrations the poKa (the apparent ionization constant in the presence of n-octanol) and then logP values were estimated and refined by a weighted non-linear least-squares procedure using the Refinement Pro™ software, where the aqueous pKa values (taken from the UV-pH titration) were used as unrefined contributions. 6 titrations at different phase volume ratios were measured, and the respective average logP values were calculated. The ion-pair partitioning of charged species was characterized, also from the titrations of different phase ratios. The relevant relationships between logP, pKa, and poKa, for mono- and multiprotic substances, including cases of ion-pair formation, have been described in details earlier 53.

2.9. In vitro dissolution-permeation study In vitro dissolution-permeation studies were carried out using a µFLUX (Pion Inc., Billerica MA, USA) apparatus (Figure 2). It consists of a donor and an acceptor chamber (20 mL volumes) separated by an artificial membrane (PVDF, polyvinylidenfluoride, 0.45 µm, 0.78 cm2). In the case of oral drug delivery, the donor chamber represents the gastrointestinal tract, while the acceptor chamber represents the blood circulation. At first 25 µL of GIT lipid (Pion Inc., Billerica MA, USA) was dribbled on the membrane surface, then 20 mL of pH 6.8 phosphate buffer was added into the donor chamber, and 20 mL of sink buffer was added into the acceptor chamber. The solubility of MEL is 267 µg/mL in the acceptor buffer, which meets the requirement for sink condition, i.e. the volume of medium in the acceptor chamber is at least three times greater than maximum achieved concentration of the acceptor chamber 54. The pure drug powder, formulations or DMSO stock solution (5 mg/mL) equivalent to API load 1.24 mg, 2.08 mg and 2.68 mg (meaning theoretical maximum concentration of 62 µg/mL, 104 µg/mL and 134 µg/ml) were placed in the donor chamber. Both chambers were stirred at 150 rpm at 37

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°C. In both chambers the API concentration was followed by immersed UV-probes at 363 nm (Pion Inc., Billerica MA, USA). The flux across the membrane was calculated using the following equation: 

J(t)=∗ where the flux (J) of a drug through the membrane is defined as the amount (n) of drug crossing a unit area (A) perpendicular to its flow per unit time (t).

Figure 2. Schematic figure of the µFLUXTM (Pion inc.) apparatus.

2.10. Parallel Artificial Membrane Permeability Assay Parallel Artificial Membrane Permeability Assay (PAMPA) method was used as previously described by Avdeef 51.

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3. RESULTS At first, the morphology of MEL containing ASDs are described. Then the physicochemical properties of the pure API are studied with and without the presence of formulation additives. At last, the results of simultaneous dissolution-permeation test are shown for the five different ASDs and DMSO stock solution carried out with three different API loads. 3.1. Characterisation of MEL containing formulations 3.1.1. Morphology

Table 2. Morphology of samples Formulation Morphology Diameter named after (nm) containing polymer ES_PVP K90 ES_PVP HP-ß-CD

fibers

2000-3000

K90 beaded fibers

250-450

ES_PVP K30

beads

3000-7000

ES_PVP VA64

beads

2000-5000

ES_Soluplus

beads

1000-4000

In case of PVP K90 containing ASDs nano- and microscaled electrospun fibers are created (Table 2). In contrast with these, the ES_Soluplus, ES_PVP K30 and ES_PVP VA 64 samples are not made of fibers, but spherical beads. This means that only in case of PVP K90 were sufficient entanglements of the linear polymer achieved in solution, this way enabling fiber formation 55, in the other cases electrospayed samples were produced. 3.1.2. X-ray powder diffraction

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Figure 3. X-ray diffractograms of pure MEL and its formulations (a) MEL, (b) physical mixture of ES_PVPK30, (c) ES_PVP K90, (d) ES_PVP K90 HPBCD, (e) ES_ PVPK30, (f) ES_PVP VA 64, (g) ES_Soluplus X-ray powder diffraction was used to characterize the morphological changes of MEL induced by electrospinning. Figure 3 shows the diffractograms of the ES samples compared to the crystalline API and the physical mixture. In the case of the physical mixture the characteristic peaks of the crystalline drug were clearly observed, while in the case of the formulation (b-f), no diffraction peaks were seen, but only a diffuse background scattering as a proof of the amorphous nature of all the samples.

3.1.3. Differential Scanning Calorimetry

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Figure 4. DSC thermograms of pure MEL and ES samples: (a) MEL, (b) physical mixture of ES_PVPK30, (c) ES_PVP K90, (d) ES_PVP K90 HPBCD, (e) ES_ PVPK30, (f) ES_PVP VA 64, (g) ES_Soluplus Differential scanning calorimetry analyses were also performed in order to study the physical state of the components in the samples. DSC thermograms (Figure 4) show that MEL has a melting endotherm with a maximum at 254°C, this melting peak can also be observed in case of the physical mixture. The absence of a melting peak of MEL in the containing formulations leads to the conclusion that the drug is in amorphous form according to DSC results. Based on the SEM, XRD and DSC results dissolution enhancement was expected because of the high surface area and the amorphous state of the API. 3.2. Physicochemical characterization of the pure API and effect of additives on physicochemical properties: 3.2.1. Thermodynamic solubility Table 3. Thermodynamic solubility of pure MEL in 0.05 mol/dm3 KH2PO4 buffer and in the presence of additives at 37 °C

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Additives

Thermodynamic Additive of concentration solubility MEL (µg/mL) (µg/mL)

pure MEL

0

PVP K90

351.3-759.3* 68.7 ± 4.1

66.8 ± 5.7

PVP K90 HPBCD 351.3-759.3* 68.5 ± 6.6 PVP K30

351.3-759.3* 66.3 ± 2.4

PVP VA64

351.3-759.3* 59.5 ± 3.0

Soluplus

351.3-759.3* 25.4 ± 2.9

DMSO stock

12.5**

202.5 ± 3.9

20.8**

237.0 ± 9.0

26.8**

257.2 ± 8.8

* in the concentration range of 351.3-759.3 µg/mL measurments were carried out using 351.3 µg/mL, 589.3 µg/mL, 759,3 µg/mL additive

** µL DMSO/ mL buffer From the thermodynamic solubility results shown in Table 3 it can be concluded that the addition of PVP derivatives (in the concentration range of 351.3-759.3 µg/mL) to the dissolution media does not change the thermodynamic solubility of the API. The addition of HP-ß-CD has no significant solubility enhancer effect in this concentration range either. In contrast with these Soluplus decreased the thermodynamic solubility of MEL significantly. This phenomenon could be explained by the polyethyleneglycol content of Soluplus. Namely, the polyethyleneglycol content enables the formation of a thermoreversible hydrogel by the dehydration of glycol groups. The cloud point of this hydrogel is effectively lowered by the presence of kosmotropic salts, like KH2PO4, a component of pH 6.8 phosphate buffer 45. This leads to the formation of a thermoreversible hydrogel with the cloud point below 37 °C in pH 6.8 phosphate buffer, having the unique behavior of becoming a clear solution by cooling below the cloud point. In this turbid

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hydrogel at 37 °C the solubility of the API is only 25.4 µg/mL, and it is not dependent from the concentration of Soluplus in the range of 351.3-759.3 µg/mL, meaning that the solubility of polymer is lower than 351.1 µg/mL under these conditions. The addition of DMSO to the buffer causes the thermodynamic solubility to increase by more than 300%. The change in solubility is directly proportional to the amount of DMSO added.

3.2.2. Effect of co-solvent and polymer additives on pKa Table 4. Predicted, measured and extrapolated pKa values of Meloxicam and its formulations pKa,1

pKa,2

number of replicates

0.47

4.47

-

pure Meloxicam

1.07 ± 0.025

4.09 ± 0.021

3

ES_PVP K90

1.11 ± 0.035

4.09 ± 0.022

3

1.11 ± 0.018

4.09 ± 0.015

ES_PVP K30

1.09 ± 0.033

4.08 ± 0.026

9

ES_PVP VA64

1.11 ± 0.037

4.09 ± 0.015

9

ES_Soluplus

1.09 ± 0.078

4.09 ± 0.027

5

Predicted* pure Meloxicam Measured

ES_PVP HPBCD

K90

9

Extrapolated from measured pKa in presence of DMSO ** 0 % DMSO

1.19±0.02

4.13±0.01

19

* prediction was performed by MarvinSketch v.15.10.12.0 Software ** see results of pKa measurement and extrapolation in supporting information (Table 1 and Figure 2)

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The knowledge of pKa values is essential when investigating dissolution-permeation characteristics, since not only the solubility of the API, but also the permeability depends on its molecular form(s) present under the studied circumstances. Therefore, determination of the pKa values of pure MEL and its formulations were performed. According to the prediction software only one pKa (assigned to the weak acidic hydroxyl group) is expected in the pH range of 3-12. The pKa results of UV-pH titration for pure MEL in aqueous media (Table 4) show pKa,2 with the value of 4.09, which can be assigned to the weak acidic hydroxyl group (as predicted) considering the measured and ionization- logP profile of the compound in aqueous media (see in supporting information Figure 3). Based on the measured results the distribution of species in aqueous media can be seen in Figure 5. For determining the effect of polymer additives on pKa, the measurement was carried out using the solutions of formulations. From Table 4 it can be concluded, that polymer additives do not influence pKa when compared to the values measured without co-solvent or additives. In case of HP-ß-CD the results show no effect either, which indicates that the cyclodextrin and the API does not form strongly-bound inclusion complex. This conclusion is supported by the thermodynamic solubility results (Table 3), where also no solubilizing effect could be detected at pH 6.8. While as Table 4 showed polymers did not affect the ionization constant, in case of using cosolvents it is expected to influence the measured pKa based on the literature

49 56–58

,

.Thus, the

extrapolated pKa value using Yasuda-Shedlovsky method was in agreement with aqueous measurements (4.13), the slope in the psKa,2- DMSO (%) diagram (see in supporting information Figure 2) was negative meaning that increasing the content of co-solvent (DMSO) increased the acidity and so the pKa of the acid was decreasing. This is an unusual behavior for a weak acid,

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because addition of organic solvent usually results in increased apparent psKa49. The decrease in apparent psKa and the extreme increase in apparent solubility in presence of DMSO can be explained with the DMSO binding to the drug molecule49.

Figure 5. Distribution of Species of pure MEL in aqueous media

3.3. In vitro dissolution-permeability study on µFLUX™ platform

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k30

Figure 6. API concentration in the donor (left) and the acceptor (right) compartments ( (a) 62 µg/mL (b) 104 µg/mL (c) 134 µg/mL API load, 0.05 mol/dm3 KH2PO4 buffer in the donor compartment, GIT membrane, sink buffer as acceptor (ASB), 150 rpm, 37 °C) For every formulation 3 different loads were applied with the theoretical maximum concentration of 62 µg/mL, 104 µg/mL, 134 µg/mL in the donor chamber. In case of pure MEL only 62 µg/mL load was applied, since higher concentration could not be achieved without supersaturation. From dissolution profiles in the donor chambers (Figure 6) it is evident that for all formulations the dissolved amount of drug was over 90% for all studied loads meaning a significant improvement in the dissolution rate compared to the crystalline API. Among the five

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dissolution profiles of ES formulations there are only slight differences. From the Soluplus and PVP VA 64 containing formulations the drug dissolves slightly faster than from HP-ß-CD containing formulation. In case of 62 µg/mL load, the solutions in the donor chambers are slightly subsaturated (see solubility in Table 3). However the dissolution of the API from all formulations improved with more than 100% compared to the crystalline MEL, the flux through the membrane did not change significantly from supersaturated solutions obtained from formulations, except for the Soluplus containing formulation. When applying 104 µg/mL or 134 µg/mL loads in the donor chamber from all formulations supersaturated solutions are created. The flux through the membrane in case of Soluplus containing formulation is nearly twice as much as from formulations made of PVP derivatives, which is in agreement with the degree of supersaturation created in donor chambers from different formulations. However based on the solubility results (Table 3) from DMSO stock solutions only subsaturated solutions are formed, the flux from these solution are just slightly lower than from PVP containing supersaturated solutions.

4. DISCUSSION 4.1. Concentration gradient and supersaturation: the effect of polymer additives Transport model for pure API’s depicting two aqueous cells separated by a membrane barrier can be described by Eq.1: J = Pe* (cd-ca) (Eq.1.), 49 where J is the flux, cd is the concentration in the donor solution, ca is the concentration in the acceptor solution, Pe is the effective permeability of the API From Eq. 1 the flux is directly proportional to the difference in concentrations, and the coefficient of proportionality is effective permeability. Effective permeability can be defined by considering the total resistance (1/Pe)

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formed by the membrane barrier, which can be described as the sum of the resistance of the membrane (1/Pm) and the resistance of unstirred water layer on the surface of the membrane (1/PUWL):  



 



(Eq.2)49,



In case of ionized drug with efficient stirring on both side of the membrane, the rate determining step is permeation through membrane, rather than permeation through unstrirred water layer. For this reason Pm can be considered as the total effective permeability of the drug. At the very beginning of the dissolution-permeation experiments there is no API in the acceptor chamber, this is the reason why an assumption can be made, that the concentration of the API is zero in the acceptor chamber, meaning the flux only depends on the concentration in the donor compartment: J= Pe*cd (Eq.3.) Based on Eq.3. the flux results of MEL containing ASDs are shown in Figure 7. as a function of concentration reached in the donor chamber. The slope of lines are equal to effective permeability.

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Figure 7. Diffusive flux of pure MEL and MEL containing ASDs at 37°C as a function of concentration in the donor chamber Table 5. The equation and R2 of lines from flux-c diagram shown in Figure 7. Additive

equation

R2

PVP K90

f=0.0008c + 0.00126

0.9992

PVP K90 f=0.0008c HPBCD + 0.0089

0.9999

PVP K30

f=0.0006c + 0.0206

0.9941

PVP VA64

f=0.0008c - 0.0044

0.9950

Soluplus

f=0.0021c - 0.0437

0.9985

DMSO stock

f=0.0005c + 0.0208

0.9352

* f stands for flux, c stands for concentration measured in the donor chamber The flux is directly proportional to the API concentration in case of every formulation (see R2 in Table 5). From Figure 7 it can be seen, that linear fit lines of PVP derivative containing formulations are very similar. According to homogeneity of slopes test, the slopes of these lines are not significantly different meaning that the coefficient of proportionality, i.e. effective permeability does not differ in these cases. In contrast the line of Soluplus containing ASD is drastically different from other ones. The slope of this outstanding formulation is almost three times greater than the others, meaning the effective permeability is three times higher in that case, than in case of PVP derivatives. This means that Eq.3 is valid for each formulation separately, but not universally applicable for all formulations, because the effective permeability is not a constant. For this phenomenon there is two possible explanation. The first one is, that

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Soluplus has an effect on membrane integrity or is able to make the unstirred water layer (UWL) thinner. For the investigation of this effect Parallel Artificial Membrane Permeability Assay was carried out with 3 different model drugs (see in supporting information Table S3). The results of these tests showed that simply adding Soluplus in the donor buffer does not change the membrane integrity or UWL thickness. The other possibility, for the explanation of the increased apparent permeability of MEL in presence of Soluplus, is the so called solubility-permeability interplay. Namely the decrease in solubility (Table 3) results in the increase of the partition coefficient, which causes the increase of apparent permeability as well 11.

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Figure 8. Transport model diagram, depicting two aqueous chambers separated by a membrane barrier. The drug molecules are introduced in the donor chamber in formulation.

To find the universal driving force of membrane transport, independent from the type of formulation, mathematical description is needed, which takes into account the solubilitypermeability trade-off as well. For that reason the driving force is derived from Fick’s first law without any assumption on solubility or permeability being constant. J=Dm*





=Dm*

   





(Eq.4)

where c0m and chm are the concentrations of the uncharged form of the solute within the membrane at the two water–membrane boundaries (Figure 8) and where Dm is the diffusivity of the solute within the membrane. The partition coefficient between donor compartment and membrane for steady state: ∗

Kd1=  ∗ 

 



(Eq.5)

where Kd1 is the partition coefficient between donor compartment and membrane for steady state, cD is the concentration in the donor chamber, * is the property at saturation. Kd2=

∗ 

∗ 



 



(Eq.6)

where Kd2 is the partition coefficient between acceptor compartment and membrane for steady state, cA is the concentration in the donor chamber, * is the property at saturation.  From Eq.5  can be expressed as:  

∗ 

∗ 

∗  (Eq.7)

 From Eq.6  can be expressed as:

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∗ 

∗  (Eq.8.)

∗ 

If Eq.7 and Eq.8 is substituted to Eq.4: J=





∗(

∗ 

∗ 

∗  −

∗ 

∗ 

∗  ∗



∗  )=

*( ∗ 







 ∗ 

) (Eq.9)

From Eq.9 it can be concluded, that the real driving force of membrane transport is the difference between the relative concentrations in the donor and acceptor compartment. This difference is directly proportional to the flux, and the coefficient of proportionality is

 ∗ ∗





. This coefficient

of proportionality is a constant for a specific API, when a specific membrane is used. This constant (B) can be defined for each API separately, like permeability, except B’s (Eq.10) value does not depend on the solubility of the API in donor and acceptor compartment.  ∗ ∗

!=





(Eq.10)

Considering that solubility in the donor could be influenced by formulation additives, Eq.11 could be used for expressing flux, when measuring formulations and not just the pure API. 



J=B*(∗ − ∗ ) (Eq.11) 



When using sink condition in the acceptor chamber the relative concentration in the acceptor becomes negligible compared to

formulations

 ∗ 

 ∗ 

. In case of every flux measurements from MEL containing

was found less than 0.01, which results in less than 1% error when neglecting it.

For that reason the flux results of MEL containing ASDs are shown in Figure 9 as a function of only cD/cD*.

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Figure 9. Diffusive flux of pure MEL and MEL containing ASDs at 37°C as a function of cD/cD* (cD* is the measured thermodynamic solubility in pH 6.8 buffer in the presence of additives) Table 6. The equation and R2 of lines from flux-cD/cD* diagram shown in Figure 9 Additive

equation

R2

PVP K90

f=0.0521cD/cD* + 0.0126

0.9992

PVP K90 f=0.0539cD/cD* HPBCD + 0.0089

0.9999

PVP K30

f=0.0455cD/cD* + 0.0143

0.9941

PVP VA64

f=0.0472cD/cD* - 0.0044

0.9950

Soluplus

f=0.0522cD/cD* - 0.0437

0.9985

DMSO stock

f=0.1574cD/cD* + 0.0013

0.8933

* f stands for flux, cD/cD* stands for supersaturation ratio in the donor chamber

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For the calculation of cD/cD* values the thermodynamic solubility data (Table 3) was used as cD*. Figure 9 shows that, as expected flux is directly proportional to cD/cD* in case of every formulation (see R2 in Table 6). Comparing the slopes of lines shown in Figure 9 with homogeneity of slopes test, it can be concluded, that the slopes of lines in case of Soluplus and PVP derivative containing formulations do not differ significantly. This means that the coefficient of proportionality is

 ∗ ∗





,

is truly constant for Soluplus and PVP derivative

containing formulations. The difference between the formulations is in only the supersaturation ratio, which is superior in case of Soluplus containing formulation. This can be explained by the fact, that in contrast with other formulation additives which does not alter solubility, Soluplus decreases the solubility of MEL (Table 3), leading to supersaturated solution in the donor chamber even in case of 62 µg/mL dose.

4.2. Concentration gradient and supersaturation: the effect of co-solvent Although considering the driving force of membrane transport as the cD/cD* seem to work for polymer containing solutions, in case of DMSO stock solution the slope of line in Figure 9 appeared to be quite different according to homogeneity of slope test. This may lead first to the conclusion that DMSO has an effect on either diffusivity, the solubility in the apolar membrane or thickness of membrane. This seems unlikely considering the amount of DMSO in the donor compartment, and after considering the results shown in Figure 7, where the line parameters of DMSO stock solution was not different from the line parameters of PVP derivatives. This data inconsistency can be solved by understanding the mechanism of solubilization caused by DMSO. For that reason not only the result of thermodynamic solubility measurements, but also the result of pKa measurements need to be considered. The fact that the addition of only

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2.68 % DMSO to the buffer causes the thermodynamic solubility to increase by more than 300 % (Table 3) suggests that DMSO not only act as a co-solvent, but may also be in molecular interaction with the API. The existence of this interaction is supported by the result of pKa measurement as well. These results point to the conclusion that DMSO may have an interaction with the hydroxyl group of MEL. More than 99% of the hydroxyl groups is deprotonated on pH 6.8, meaning that this molecular interaction, which causes the extreme increase in solubility of the API, is between the anion form of MEL and DMSO. To support this theory thermodynamic solubility measurement was carried out on pH=1.0 (see in supporting information Table S4), where the anion form of the API is not present (Figure 5). The results show no significant increase in thermodynamic solubility compared to the pure API, which means that DMSO is only able to interact with the anion form of MEL, with the non-ionic and cation form it only acts as a co-solvent. While DMSO acts as a solubilizing agent for the anion form, increasing the total API concentration in the donor chamber, this effect does not increase the number of molecules able to partition into the membrane, because only the non-ionic form is capable of dissolving into the apolar membrane

49

. For that reason the thermodynamic solubility measured in presence of

DMSO cannot be used as cD*, when calculating the ratio of supersaturation. For calculation of the supersaturation ratio in case of DMSO stock solution, the co-solvent effect on solubility of the non-ionic form has to be measured. While the increase in solubility of non-ionic form (S0) caused by DMSO is hard to measure directly, because of the low aqueous solubility of the API, it can be easily calculated from the shift DMSO caused in pKa 49. log S0= log SAPP±∆ (Eq.12.),

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where S0 is the aqueous solubility of the non-ionic form of the API (i.e. intrinsic solubility), SAPP is the aqueous solubility of the non-ionic form of the API in presence of co-solvent, and ∆ is the pKa shift caused by the co-solvent. The results of this calculation showed that in case of 2.68 % DMSO content the increase in solubility was only 9 % compared the pure API. Using these results the supersaturation ratio was calculated, and the flux as the function of supersaturation ratio was plotted in Figure 10. As seen from Table 7 the slope of fit line in case of DMSO stock solution becomes close to the other slopes. Based on homogeneity of slopes test the values of slopes do not differ significantly, meaning that the coefficient of proportionality (Eq.11), the B is constant for the API regardless the presence of additives or co-solvents.

Figure 10. Diffusive flux of pure MEL and MEL containing ASDs at 37°C as a function of supersaturation ratio Table 7. The equation and R2 of lines from flux-supersaturation ratio diagram shown in Figure 10 Additive

equation

R2

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f=0.0521*SSR 0.9992 + 0.0126

PVP K90 f=0.0539*SSR 0.9999 HPBCD + 0.0089 PVP K30

f=0.0455*SSR 0.9941 + 0.0143

PVP VA64

f=0.0472*SSR 0.9950 - 0.0044

Soluplus

f=0.0522*SSR 0.9985 - 0.0437

DMSO stock

f=0.0397*SSR 0.8999 + 0.0174

*f stands for flux, SSR stands for supersaturation ratio 5. CONCLUSION For the dissolution enhancement of MEL three PVP derivatives (K30, K90 and VA 64), Soluplus and (2-Hydroxypropyl)-β-cyclodextrin were used to create five different amorphous solid dispersions of the poorly water-soluble API. The dissolution-permeation results of the five ASDs, having the same ratio of API to formulation additives, were compared to the dissolution of pure API and MEL from DMSO stock solution. The dissolution profiles of the five formulations were found to be very similar to each other and the DMSO stock, showing great improvement in dissolution compared to the pure API. Although based on the similarity in the dissolution profiles similar fluxes were expected, in case of Soluplus containing formulation the flux was found superior, showing that the driving force of membrane transport cannot be simplified to the concentration gradient. The degree of supersaturation (defined as the ratio of dissolved amount of neutral drug to its thermodynamic solubility) was found to be the real driving force of membrane transport. It was mathematically derived from Fick’s first law, and experimentally proved to be universal on several meloxicam containing ASDs and DMSO stock solution.

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In the case of DMSO stock solution, defining supersaturation as the ratio of dissolved amount of API to its measured thermodynamic solubility was found to be misleading because of the molecular interaction of DMSO and the anion form of MEL. Namely, measuring the total concentration of all the API forms, when determining the degree of supersaturation was not conclusive, knowing that the non-ionized form of the API is easily able to partition into the membrane, while the partition of the ionized form into the apolar membrane hampered. Due to the molecular interaction the solubility of anion form of MEL was highly increased by the presence of DMSO, while the solubility of the non-ionized form only increased by ~9%. It follows the degree of supersaturation has to be defined as the ratio of dissolved amount of uncharged API to its thermodynamic solubility. The results of dissolution-permeation studies exemplify well that formulation additives can have an effect on solubility-permeability interplay in a way that only by physicochemical characterization can be fully understood. Although the mechanism of action is difficult to determine in case of every excipient, the flux measurements reflect the combination of all these effects. This emphasizes importance of the flux measurements during formulation development.

ASSOCIATED CONTENT Figure S1. Scanning electron microscopic images of electrospun samples Table S1.a) Measured psKa values of Meloxicam in DMSO-water system Table S1.b) Calculated pKa values based on Yasuda-Shedlovsky extrapolation Figure S2. Illustriation for Yesuda-Shedlovsky extrapolation

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Figure S3. Theoretical titration curves of aqueous pKa and poKa and measured titration curves of poKa from the logP measurements. Table S3. The result of parallel artificial membrane permeability assay (PAMPA) Table S4. The results of thermodynamic solubility measurements in pH 1.0 This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *Zsombor K. Nagy * (Postal address: 1111 Budapest, Műegyetem rkp. 3, Hungary; Phone: +36-1463-1424; Email: [email protected]) Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. Funding Sources This work was financially supported by the New Széchenyi Development Plan (TÁMOP4.2.1/B-09/1/KMR-2010-0002), OTKA research fund (grant numbers K112644 and PD108975), MedInProt Synergy Program and the János Bolyai Research Scholarship of the Hungarian Academy of Sciences. ABBREVIATIONS

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Table of Contents Graphic and Synopsis

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