Understanding of Relationship between Phospholipid Membrane

Article pubs.acs.org/molecularpharmaceutics

Understanding of Relationship between Phospholipid Membrane Permeability and Self-Diffusion Coefficients of Some Drugs and Biologically Active Compounds in Model Solvents Svetlana V. Blokhina, Tatyana V. Volkova,* Vasiliy A. Golubev, and German L. Perlovich Institute of Solution Chemistry, Russian Academy of Sciences, 153045 Ivanovo, Russia S Supporting Information *

ABSTRACT: In this work we measured self-diffusion coefficients of 5 drugs (aspirin, caffeine, ethionamide, salicylic acid, and paracetamol) and 11 biologically active compounds of similar structure in deuterated water and 1-octanol by NMR. It has been found that an increase in the van der Waals volume of the molecules of the studied substances result in reduction of their diffusion mobility in both solvents. The analysis of the experimental data showed the influence of chemical nature and structural isomerization of the molecules on the diffusion mobility. Apparent permeability coefficients of the studied compounds were determined using an artificial phospholipid membrane made of egg lecithin as a model of in vivo absorption. Distribution coefficients in 1-octanol/buffer pH 7.4 system were measured. For the first time the model of the passive diffusion through the phospholipid membrane was validated based on the experimental data. To this end, the passive diffusion was considered as an additive process of molecule passage through the aqueous boundary layer before the membrane and 1-octanol barrier simulating the lipid layer of the membrane. KEYWORDS: membrane permeability, lipophilicity, self-diffusion coefficients, partition/distribution

1. INTRODUCTION The biological membrane is an element of a living cell performing barrier and transport functions between the intracellular and extracellular environments.1 Different mechanisms of membrane permeation (passive diffusion, active uptake, endocytosis, efflux, and paracellular pathways) take place. The most significant permeability mechanism for drug discovery is passive diffusion.2 Mass transfer is an important phenomenon in pharmaceutical sciences, drug synthesis, preformulation investigations, dosage form design and manufacture, and ADME (adsorption, distribution, metabolism, and excretion) studies.3 However, due to the complex structure of biological membranes, which consist of many kinds of lipids as well as proteins and carbohydrates, the mechanism of such transport of substances, including drugs, is not sufficiently understood and difficult to be determined by computer modeling.4,5 When studying such biological objects, it is of special interest to imitate the structure of the cell membranes and the transfer process of the substances through them. Among the techniques currently used for screening membrane permeability are Caco-2 monolayer model,6 the PAMPA (parallel artificial membrane permeation assay),7 the ILC (immobilized liposome chromatography), immobilized artificial membrane (IAM)8 methods, and ex-vivo animal tissue model.9 We have used a novel approach to measure drug permeability based on a permeation barrier made of a tight layer of phospholipid vesicles, developed by Flaten et al.10 The method © 2017 American Chemical Society

has been validated using a series of drugs covering a broad range of physicochemical properties and absorption properties upon oral administration in humans. Previously, Higuchi11 and Flynn and Yalkowsky12 described drug permeation through the lipophilic membrane as a series of individual and additive layers. They based their work on earlier observations emphasizing that the diffusive boundary layer before the membrane must be treated as a part of the total barrier.13 A quantitative characteristic of membrane permeability is the permeability coefficient (P, cm·s−1) which is determined by the compound diffusion coefficient (D, m2·s−1), the partition coefficient (K) of the substance in the system membrane/ isotropic environment, and the membrane thickness (h, cm) and described by the equation derived from Fick’s law of diffusion.14 P=

D·K h

(1)

Determination of the partition and the diffusion coefficient, especially in view of membrane heterogeneity is a separate and rather complicated experimental task. Consideration of the ternary system “water−octanol−drug” provides an insight into Received: Revised: Accepted: Published: 3381

May 13, 2017 August 24, 2017 August 29, 2017 August 29, 2017 DOI: 10.1021/acs.molpharmaceut.7b00401 Mol. Pharmaceutics 2017, 14, 3381−3390

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Molecular Pharmaceutics

compounds was performed using the pulse sequence of double stimulated echo (DSTE) in order to eliminate the effects of convection in the sample.19 The diffusion time in the pulse sequence DSTE was constant in all the experiments and equal to 0.1 s. The duration of the gradient pulses varied depending on the tested compound, and was chosen in such a way that the attenuation was about 95% at the maximum value of the gradient and on the average equaled 2000 μs for D2O solutions and 7000 μs for 1-octanol solutions. The measurements were performed at 298 ± 0.05 K. The solutions were prepared gravimetrically on the scales Sartorius Genius ME235S (with the weighing accuracy of 10−5 g). The concentration of the tested substances did not exceed 10−3 mole fraction. The diffusion coefficient determination was performed in duplicates or triplicates. 2.3. Permeation Study. Permeability of the investigated substances was determined using phospholipid vesicle-based barriers. The preparation procedure was published before.10 In brief, phosphatidyl choline from egg was dissolved in a chloroform−methanol mixture with the successive removal of the organic solvent under vacuum. As a result, a lipid film was obtained and dissolved in phosphate buffer containing 10% (v/ v) ethanol to obtain a 6% (w/v) liposomal dispersion. The suspensions of small and large liposomes were prepared by the filtration through 0.4 and 0.8 μm pore size polycarbonate membrane filters, respectively. The liposomal suspensions were placed sequentially on filter supports (0.65 μm DAWP mixed cellulose ester, MILLIPORE, Ireland) using a centrifuge. First, the small liposomes were disposed in the pores of the support and then the large ones on its surface. As a result, there are no aqueous channels going through the whole barrier.20 Freeze− thaw cycling was also used to promote liposome fusion to produce a tight barrier. The barriers preserved their integrity during the experiment in the pH 7.4 buffer solution. Stock solutions of the compounds under study were prepared by dissolving the drug in buffer pH 7.4 and the volumes of 100 μL were placed in the donor chambers of the inserts (see Abstract graphic). The acceptor compartments contained phosphate buffer solution (600 μL). The permeation experiments were carried out at room temperature without agitation, and the insets were moved to the wells containing equal quantities of the fresh buffer solution once every hour during 3 h and then every 30 min until the end of the permeation experiment (6 h). The samples of 200 μL from each acceptor compartment were transferred into 96-well UV black plates (Costar) and drug concentrations were measured spectrophotometrically (Cary-50 (USA)) at the most appropriate wavelength for each drug. Absorption characteristics of the compounds used are given in Table A.1 of the Supporting Information. The electrical resistance of the lipid barriers was measured (Millicell-ERS, Millipore, USA) immediately after completing the permeation studies in order to check the barrier integrity. To find out the resistance of the lipid barrier itself, a value of 119 Ω resulting from the filter characteristics was subtracted from the observed resistance meaning. Such a difference was then multiplied by the surface area (0.45 cm2) to be normalized for the dimensions of the insert. An electrical resistance between 3000 and 1000 Ω is what has been normally observed when using the model.10 The inserts with insufficient resistances were excluded. The experiments were performed at least in triplicate with three inserts in each parallel for every compound. The r2-values were always higher than 0.99. The repeatability corresponded

the mechanisms and driving forces of passive transport processes of drug compounds through the real media of the human body.15 The process of membrane permeability is modeled in this case by replacing a cell membrane lipid layer with 1-octanol and the outer medium with aqueous solution. Due to its amphiphilic nature and the ability to form hydrogen bonds, 1-octanol is often used for imitating the features of the phospholipids of biological membranes.16 Since the determination of the drug permeability through the biological membranes is a labor-consuming process, mathematical models aimed at the estimation of this parameter is of considerable interest. The aim of the present work was, first, to experimentally determine the permeability coefficients of several drugs and biologically active compounds of similar structure using an artificial phospholipid membrane; second, to measure their diffusion coefficients in water and octanol, which simulate water and lipid layers of the biological membrane; and third, to investigate the possibility of using a diffusion model17 to evaluate the permeability coefficients of these substances based on diffusion mobility and lipophilicity data. It is quite difficult to measure the diffusion coefficients in the biological membranes experimentally,18 due to this fact, the proposed approach can be useful for modeling the permeability processes through biological water/lipid barriers.

2. MATERIALS AND METHODS 2.1. Materials. The following compounds were purchased from Sigma-Aldrich, and are listed in Table 1. The structural formulas are illustrated in Figure 1. Table 1. Chemical Name and Purity Data of the Compounds Studied in This Worka compound number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

chemical name benzoic acid 2-hydroxybenzoic acid (salicylic acid) 3-hydrohybenzoic acid 4-hydrohybenzoic acid 2-acetamidobenzoic acid 3-acetamidobenzoic acid 4-acetamidobenzoic acid 2-acetamidophenol 4-acetamidophenol (paracetamol, acetaminophen) 2-methoxybenzoic acid (o-anisic acid) 2-acetoxybenzoic acid (acetylsalicylic acid, aspirin) 4-methylbenzoic acid (p-toluic acid) 2-pyridinecarbothioamide 4-pyridinecarbothioamide 2-ethylpyridine-4-carbothioamide (ethionamide) caffeine

purity ≥99.5% ≥99.0% ≥99.0% ≥99.0% ≥99.5% ≥98.0% 98.0% 97.0% 98.0% 99.0% ≥99.0% 98.0% 97.0% 97.0% 97.0% 99.0%

a

Deuterated water D2O (the mass fraction of D2O is 99.95%) was obtained from Astrachem, St. Petersburg. 1-Octanol, CAS number 111-87-5 was purchased from Sigma Chemical Co, the purity is 99%.

2.2. Diffusion Study. Self-diffusion coefficients of the compounds in D2O and n-octanol were measured separately by the pulsed field gradient NMR method using the Bruker Avance III 500 BioSpin spectrophotometer at 1H −500.13 MHz with a 5 mm TBI probe. Diffusion attenuation of the intensities of the NMR signals in the spectra of the tested 3382

DOI: 10.1021/acs.molpharmaceut.7b00401 Mol. Pharmaceutics 2017, 14, 3381−3390

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Figure 1. Structures of the studied compounds.

octanol/buffer (pH 7.4) system.22−24 In brief: buffer (pH 7.4) and 1-octanol were mixed vigorously for 24 h at 298 ± 0.1 K to promote mutual saturation in both buffer and 1-octanol phases. The solvents were left to stand long enough to allow the phases to separate, and the studied compound was dissolved in the 1octanol phase to obtain a stock solution. The buffer saturated 1octanol phase with the dissolved substance, and 1-octanol saturated buffer phase were placed in glass vials and mixed for 24 h at 298 K. The substance concentrations were determined spectrophotometrically using calibration curves. The distribution coefficient was calculated by the following equation:

to 3.9−20.1%, and an intermediate precision of 2.7−29.4% was observed for different drugs using such a novel method. The linear part of the curve slope of the standard time dependence of the cumulative amount represented the steady state flux rate. If a lag time was observed before the steady-state conditions were attained, and the saturation conditions were achieved at the end of the experiments, only the middle points of the cumulative plot were used for the calculation procedure, based on the steady-state flux. The obtained flux rates were used to calculate the apparent permeability coefficient (Papp, cm·s−1) using the following equation: Papp =

K oct/buf = C oct/buf /C buf/oct

J A ·(Cd − Ca)

(2)

(3)

where Coct/buf and Cbuf/oct are the molar concentrations of the solute in the mutually saturated phases of 1-octanol and buffer. The accuracy of the distribution coefficient value was verified by checking the mass balance of the starting amount of compound i compared to the total amount of the compound partitioned between the two phases:

where J is the observed flux rate at steady-state (nmol·s−1), A is the surface area of the insert (cm2), and Cd and Ca are the concentrations of the solutions in the donor and acceptor chambers (nmol·ml−1), respectively. The experiments were performed under sink conditions, i.e., the drug concentration in the acceptor chamber did not exceed 10% of the drug concentration in the donor chamber at any time.21 2.4. Distribution Studies. The shake flask method was used to determine the distribution coefficients (Koct/buf) in 1-

mi = moct/buf + m buf/oct

(4)

where mi = Ci · Vi is the starting mass (in moles) of the compound, moct/buf = Coct/buf · Voct/buf is the mass of the 3383


0.02 0.02 0.01 0.03 0.03 0.01 0.02 0.02h 0.01 0.02 0.04 0.04 0.04 0.02 0.01 0.02h

6.76·10 3.60·10−2f 9.46·10−2 1.79 7.16·10−1 9.81·10−2 9.66·10−2 5.24 3.236e 6.87·10−1 7.1·10−2i 3.66·10−1 13.8 4.07 5.50 4.27·10−1e

−1e

Koct/buf −6

(4.13 ± 0.10)·10 (0.59 ± 0.03)·10−6g (1.34 ± 0.02)·10−6 (1.38 ± 0.03)·10−6 (2.37 ± 0.01)·10−6 (8.59 ± 0.31)·10−7 (7.64 ± 0.43)·10−7 (6.47 ± 0.13)·10−6 (3.51 ± 0.03)·10−6 (3.56 ± 0.02)·10−6 (1.49 ± 0.23)·10−6g (6.41 ± 0.21)·10−6 (9.66 ± 0.30)·10−6 (7.26 ± 0.09)·10−6 (7.34 ± 0.09)·10−6 (12.54 ± 0.89)·10−6g

Papp cm·s−1 8.64·10 1.44·10−2 2.81·10−3 9.35·10−4 1.93·10−2 2.21·10−3 1.03·10−3 5.83·10−4 4.07·10−4 3.81·10−3 1.24·10−2 6.88·10−3 9.66·10−6 7.26·10−6 7.34·10−6 1.25·10−5

−3

Pintr(0)b cm·s−1 5.786·10 5.450·10−5 4.875·10−5 5.086·10−5 4.143·10−5 4.550·10−5 4.379·10−5 4.833·10−5 4.714·10−5 5.278·10−5 4.657·10−5 5.679·10−5 6.000·10−5 5.571·10−5 4.821·10−5 4.429·10−5

−5

PABL(cal) 8.79·10 4.36·10−6 6.96·10−6 1.34·10−4 6.51·10−5 6.48·10−6 6.18·10−6 5.24·10−4 1.94·10−4 8.24·10−5 8.61·10−6 4.39·10−5 2.87·10−3 4.24·10−4 5.28·10−4 3.93·10−5

−5

Poct(cal) 3.49·10 1.01·10−5 6.09·10−6 3.65·10−5 2.53·10−5 5.67·10−6 5.42·10−6 4.43·10−5 3.79·10−5 3.22·10−5 7.27·10−6 2.48·10−5 5.88·10−5 4.92·10−5 4.42·10−5 2.08·10−5

−5

Papp(cal) 4.08 3.01 4.08 4.57 3.49 3.99 4.27 9.35 9.46 4.37 3.48 4.37 1.86 3.71 4.34 0.52

pKac

0.557 0.701 0.824 0.832 0.695 0.698 0.649 0.572 0.542 0.545 0.578 0.486 0.417 0.449 0.349 0.399

∑(Cad/α)d

Calculated using Bondi tables.25 bIntrinsic permeability coefficients (Pintr(0)) characterize the membrane permeability of neutral molecular forms of the compounds. cCalculated using Advanced Chemistry Development (ACD/Laboratories) Software V11.02. dPhysicochemical descriptors which characterize the molecular capacity for donor−acceptor interactions were calculated by the program package HYBOT-PLUS (version of 2003) in Windows.28 eTaken from Markopoulou et al.32 fTaken from Zhu et al.33 gMeasured in Flaten et al.10 hCalculated from the Stokes−Einstein equation.26 iTaken from Lewis et al.34

a

1.30 1.21 0.74 0.75 0.91 0.66 0.64 1.00 0.60 1.20 1.09 1.20 2.08 1.04 0.96 0.92

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

0.05 0.04 0.02 0.02 0.01 0.02 0.03 0.02 0.04 0.02 0.03 0.04 0.06 0.03 0.03 0.03

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

108.9 122.3 122.3 122.3 164.5 164.5 164.5 145.1 145.1 140.3 159.7 131.6 120.7 120.7 160.4 166.1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

8.10 7.63 6.83 7.12 5.80 6.37 6.13 6.77 6.60 7.39 6.52 7.95 8.40 7.80 6.75 6.20

Doct·1010 m2·s−1

Dw·1010 m2·s−1

Vvdw Å3a

compound

Table 2. van der Waals Volumes (Vvdw), Self-Diffusion Coefficients in Water (Dw) and 1-Octanol (Doct), Distribution Coefficient in 1-Octanol/Buffer pH 7.4 System (Koct/buf), Apparent (Papp) and Intrinsic (Pintr(0)) Permeability Coefficients, Calculated Permeability Coefficients through Water Layer (PABL(cal)) and 1-Octanol Layer (Poct(cal)), Total Permeability Coefficient through Both Layers (Papp(cal)), pKa,, and ∑(Cad/α) for the Studied Compounds

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partition coefficient of the substance in water is equal to 1. The thickness of the 1-octanol layer (hoct) equal to the thickness of the artificial phospholipid membrane used for the permeability measurements was 100 μm.20 As stated by Loftsson et al.,17 the thickness of the structured water layer before the membrane in in vitro conditions exceeds 1000 μm. In accordance with the experiment conditions the thickness of the water layer (hABL) used for the calculations was 1400 μm (as the maximum possible). As it follows from eqs 7 and 8, the basic physicochemical parameters determining the permeation of the substance through the aqueous and lipid barriers are the diffusion coefficients in water and 1-octanol. At low concentrations of the solutes the diffusion coefficients in eqs 7 and 8 can be replaced by the self-diffusion coefficients. In the present study deuterated water was used to measure the self-diffusion coefficients because the NMR technique requires it. However, we applied ordinary water in partitioning studies and permeability measurement. Since the viscosity of D2O is higher than that of H2O,29 the diffusion coefficients of the organic compounds in deuterated water, according to eq 5, are slightly lower when compared to ordinary water.30 Meanwhile, the data of Longeville and Lechner31 show that the dynamics of D2O and H2O molecules can be described by the same model with similar diffusion coefficients. This fact proves the validity and applicability of self-diffusion coefficients measured in D2O instead of H2O when searching for correlations using these parameters.

substance dissolved in the buffer-saturated 1-octanol phase, and mbuf/oct = Cbuf/oct · Vbuf/oct is the mass of the substance dissolved in the 1-octanol saturated buffer phase. Taking into account the considerable difference in lipophilicity of the test compounds, a wide range of the volume ratios was used, namely the ratio of Vbuf/oct/Voct/buf ranged from 1/15 to 10/1. Three shake-flask determinations were done for each volume ratio. 2.5. Calculation Procedure. van der Waals volumes of the investigated compounds were calculated using Bondi tables.25 The experimental diffusion values of compounds (16) and (8) in 1-octanol were not measured due to the poor solubility of these compounds in 1-octanol. These self-diffusion coefficients were calculated from the Stokes−Einstein equation:

D=

kT 6πrη

(5)

where k is the Boltzmann constant, T is the absolute temperature, η is the dynamic viscosity of 1-octanol,26 and r is the solute hydrodynamic radius. The values of solute hydrodynamic radius were estimated based on the experimental self-diffusion coefficient values in D2O for the substance and dynamic viscosity of D2O.27 Physicochemical descriptors were calculated by the program package HYBOT-PLUS (version of 2003) in Windows.28 2.6. Theoretical Background. One of the factors determining drug compound bioavailability is membrane permeability, which shows the ability of the molecules to permeate through cell membranes for interaction with the receptor. As it has been mentioned above, the permeability coefficient is determined by the value of the diffusion coefficient, partition coefficient membrane/environment, and membrane thickness.17 Passive diffusion through the phospholipid membrane can be considered as an additive process of molecule passage through the structured water barrier before the membrane and the lipid layer of the cell membrane: 1 1 1 = ABL + lip Papp P P

3. RESULTS AND DISCUSSION 3.1. Diffusion Study. Self-diffusion coefficients in deuterated water and 1-octanol have been obtained for five drugs and 11 biologically active substances of similar structure, including ortho-, para-, and meta-isomers of benzene and pyridine derivatives (Table 2). The values of the self-diffusion coefficients of the studied compounds are in the range of (8.4−5.8)·10−10 m2·s−1 and (2.0−0.6)·10−10 m2·s−1 in water and 1-octanol, respectively. Poorer diffusion mobility of the molecules in 1-octanol as compared to that in water is attributed to the greater viscosity of 1-octanol. A general tendency has been revealed for the selfdiffusion coefficients to decrease in water with an increase in the molecular van der Waals volume of the dissolved substances (Figure 2). A similar diffusion behavior of the compounds in 1-octanol was observed indicating a similarity of hydration and solvation processes in both investigated solvents. It is of interest that the same tendency of self-diffusion coefficient decreasing with the increase in the molecular volume of the nonassociated solutes in methanol has been revealed earlier by Lu et al.35 Benzoic acid (1) was shown to have the greatest diffusion mobility among the studied benzene derivatives. Introduction of a methyl(compound 12), methoxy- (10), and hydroxyl- (2, 3, and 4) groups into the benzene ring lowers the self-diffusion coefficients. A more significant decrease in the self-diffusion coefficient was observed in acetyloxy- (compound 11), and acetamido- (6, 7, and 5) derivatives of benzoic acid. Pyridinecarbotioamides (compounds 13 and 14) have the highest self-diffusion coefficients among the studied heterocyclic compounds. It is important to note that introduction of an ethyl-substituent into the ortho-position of the parapyridinecarbotioamide molecule (compound 14), producing ethionamide as a result (15), decreases the self-diffusion

(6)

where Papp is the total permeability coefficient through the water and the lipid layers, PABL and Plip are the permeability coefficients through the water and the lipid layer, respectively. It should be noted that the resistance of the water layer after the membrane is assumed to be negligible due to relatively rapid removal of drug molecules from the receptor side of the membrane.17 We assume that the permeability coefficient through the octanol layer is given by the equation:12−14,17 P oct(cal) =

Doct ·K oct/buf hoct

(7)

oct

where D is the self-diffusion coefficient of the investigated substance in 1-octanol, Koct/buf is the distribution coefficient in the 1-octanol/buffer pH 7.4 system, hoct is the thickness of the 1-octanol layer assigned to be equal to the thickness of the artificial membrane used in the permeability experiment. The water layer permeability coefficient can be calculated by the equation:

P ABL(cal) =

Dw hABL

(8)

w

where D is the self-diffusion coefficient of the investigated substance in D2O; hABL is the water layer thickness; and the 3385


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lower values of log(Doct) at a given log(Dw). It is important to note that monosubstituted compound 1 (benzoic acid) belongs to the second group, while compound 16 with a bicyclic structure (caffeine) belongs to the first group. The following decreasing order of the self-diffusion coefficients in water and 1-octanol for the structural isomers of hydroxybenzoic acids (compounds 2−4), pyridinecarbothioamides (compounds 13 and 14), and acetamidophenoles (compounds 8 and 9) was found: ortho- > para- > meta(Figure 4a and b, respectively).

Figure 2. Dependence of self-diffusion coefficients of studied compounds measured experimentally using NMR in water (Dw) on the molecule van der Waals volume calculated using Bondi tables (Vvdw), R = 0.8563. The compounds numbering corresponds to Figure 1.

coefficient in water and has almost no effect on the molecule mobility in 1-octanol. Logarithmic dependences of the self-diffusion coefficients of the studied compounds in water and 1-octanol are represented in Figure 3.

Figure 4. Histogram depicting the order of decreasing of diffusion coefficients of structural isomers (o-, ortho-isomer; p-, para-isomer; mmeta-isomer) in water (a) and 1-octanol (b). The compounds numbering corresponds to Figure 1.

Figure 3. Correlation between the logarithms of diffusion coefficients of the substances measured experimentally using NMR in water (log Dw) and 1-octanol (log Doct) (values for compounds 8 and 16 in 1octanol were calculated from the Stokes−Einstein equation). The compounds numbering corresponds to Figure 1.

Interestingly, the melting temperature of the solids and the boiling point of the liquids are lower for ortho-isomers than for para- and meta- ones. This fact is attributed to the effect of steric factors on the intermolecular interactions involving isomers.36 As a result, hydrogen bonding plays a more important role in para- and meta-isomers reducing their diffusion mobility than in the ortho-isomer. Figure 4 shows that the acetamidobenzoic acid isomers (compounds 5−7) have a different order of changing self-diffusion coefficients in water and 1-octanol than the other studied substances. The

According to the values of the self-diffusion coefficients all the investigated substances can be divided structurally into two groups (Figure 3). The first group of the compounds with higher values of log(Doct) at a given value of log(Dw) includes the benzene and pyridine derivatives with the substituents in the ortho-position (2, 5, 8, 10, 11, 13, and 15). The second group includes all the remaining compounds (3, 4, 6, 7, 9, 12, and 14) characterized by the presence of a substituent in the meta- or para-position of the molecular cyclic moiety with 3386


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The apparent permeability coefficients of the studied compounds through the water barrier (PABL(cal)) and octanol barrier (Poct(cal)) were calculated by eqs 7 and 8 (Table 1) using the experimental values of the diffusion coefficients in water (Dw) and 1-octanol (Doct) and the distribution coefficients in the 1-octanol/buffer pH 7.4 system. Comparative analysis of the calculated values of the permeability coefficients through an aqueous layer and a layer of 1-octanol leads to the conclusion that for compounds 2, 3, 6, 7, 11, 12, and 16, with relatively low lipophilicity (Koct/buf < 0.5) the inequality PABL(cal) > Poct(cal) is realized and, consequently, the limiting permeability stage is the passing of the substances through the 1-octanol layer. In contrast, for compounds 1, 4, 5, 8, 9, 10, 13−15 with relatively high lipophilicity (Koct/buf > 0.5) the inequality PABL(cal) < Poct(cal) is satisfied and the rate-limiting step is the substance passing through the aqueous layer. Such division of the studied compounds is arbitrary, since the values of PABL(cal) and Poct(cal) are rather close and the total membrane permeability is influenced both by the water and 1octanol layers. The total membrane permeability coefficient (Papp(cal)) was calculated using the PABL(cal), and Poct(cal) values by eq 6. The experiments carried out on the membrane permeability and distribution of the studied substances have shown that there is a linear correlation between the parameters log(Papp(cal)) and logKoct/buf (Figure 5), which can be described by the equation:

diffusion mobility estimated for the other isomers is probably explained by the formation of an intramolecular hydrogen bond in the ortho-acetamidobenzoic acid, as well as the mutual influence of the substituents of a rather large volume on the solvation processes. Figure 4 shows that the replacement of the hydroxyl group in acetamidophenoles (compounds 8 and 9) and the transition to the acetamidobenzoic acid structure (compounds 5 and 7) does not only reduce the self-diffusion coefficient in water but also changes the order of this parameter decrease in structural isomers. 3.2. Membrane Permeability Studies. Membrane permeability investigation into drugs and biologically active structurally related compounds was conducted using an artificial phospholipid membrane based on a liposomal bilayer simulating in vivo absorption.10 Molecules of the studied compounds have ionizable functional groups and can exist in solution in the form of charged and neutral species. The apparent permeability coefficient (Papp) experimentally determined in the present study characterizes the ability of all the molecular forms of the compound in the solution to permeate the membrane. The apparent permeability coefficients were determined at room temperature in pH 7.4 aqueous buffer solution simulating the human blood plasma medium, and are listed in Table 1. As the experiment has shown, the Papp-values of the compounds under study vary within a range from 0.59· 10−6 to 12.34·10−6 cm·s−1. An analysis of physicochemical characteristics of the compounds with high and low membrane permeability was carried out. Benzoic acid (compound 1) was selected as a reference substance because (unlike all the other compounds) it has only one substituent in the aromatic ring. The substances with an arbitrarily low membrane permeability (less than that of benzoic acid) include hydroxy- (compounds 2−4), acetamido(compounds 5−7), methoxy-(compound 10), and acetoxy(compound 11) benzoic acids, as well as 4-acetamidophenol (compound 9), with hydroxyl and acetamide groups in the structure. Compounds with membrane permeability higher than that of benzoic acid are 2-acetamidophenol (compound 8), 4-methylbenzoic acid (compound 12), and pyridinecarbothioamides (compounds 13 and 14). Caffeine demonstrated the highest value of membrane permeability consistent with the literature.8 The obtained results indicate that not only the chemical nature of the substituents but also the structural isomerism of the compounds has an effect on membrane permeability. This fact is confirmed by the data for benzoic acid derivatives with a hydroxyl- (compounds 2, 3, and 4) and acetamide- (compounds 5, 6, and 7) groups with different membrane permeability values depending on the substituent position. The main physicochemical property determining the permeation of the substances through the cell phospholipid membrane is lipophilicity which is commonly characterized by the logarithm of the distribution/partition coefficient in 1octanol/buffer pH 7.4 system. The distribution coefficients (logKoct/buf) of compounds 1, 2, 9, 11, 16 in 1-octanol/buffer pH 7.4 system were reported in the literature32−34 and compounds 3−8, 10, 12−15 were measured by us in this work. The distribution coefficients values are summarized in Table 1. It should be emphasized that the well-known drugs caffeine (16) and ethionamide (15) are the most lipophilic ones among the studied substances. Salicylic acid (2) and ptoluidine (12) were found to have relatively low distribution coefficients.

log(Papp(cal)) = −(4.60 ± 0.04) + (0.43 ± 0.05) · log(K oct/buf )

R = 0.9143; σ = 0.1552; n = 16

(9)

Figure 5. Logarithmic dependence of total permeability coefficients (log(Papp(cal)) calculated according to eq 6 versus distribution coefficients log(Koct/buf) in 1-octanol/buffer pH 7.4 system. The compounds numbering corresponds to Figure 1.

This correlation is expectable and was clearly showed by Flaten et al. in 2006.20 Figure 6 shows the correlation between the experimental values of apparent permeability coefficients of the studied substances and the total permeability coefficients calculated by eq 6, including PABL(cal) and Poct(cal) (caffeine and 4hydroxybenzoic acid are excluded from the correlation). The correlation equation can be presented as 3387


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these substances in order to determine the role of specific interactions in the process of the permeability through the lipophilic membrane. The calculated values of the descriptors for the molecules of the studied compounds are summarized in Table 2. The molecular capacity for donor−acceptor interactions was evaluated using the descriptor ∑(Cad/α), where Cad is H-bond acceptor and donor factor; α is the molecular polarizability (mainly the bulk effect descriptor). The descriptors were taken from the correlation equations, built on H-bonding Gibbs energies analysis of various compounds.38 The choice of the descriptor was driven by the fact that the equation had the best statistical criteria when the descriptor ∑(Cad/α) was used as the independent variable. The correlation between the logarithm of the experimental apparent membrane permeability coefficient of the studied substances and ∑(Cad/α) descriptor is shown in Figure A.1 of the Supporting Information. The correlations of logPapp with some other HYBOT descriptors are given in Figure A.2, A.3, A.4 of the Supporting Information. The presented data show that the substances membrane permeability tends to decrease when the capacity for donor−acceptor interactions increases.37 Since the formation of hydrogen bonds is accompanied by substance ionization, it is quite reasonable that the compounds with higher ∑(Cad/α) have lower membrane permeability.

Figure 6. Correlation between the experimental values of apparent permeability coefficients (logPapp) of the studied substances and the total permeability coefficients (log(Papp(cal)) calculated according to eq 6. The compounds numbering corresponds to Figure 1.

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log(Papp) = − (1.2 ± 0.4) + (0.91 ± 0.08)· log(Papp(cal)) R = 0.9525; σ = 0.13; n = 14

CONCLUSION Self-diffusion coefficients of 5 drugs (aspirin, caffeine, ethionamide, salicylic acid, and paracetamol) and 11 biologically active compounds of similar structure were measured in deuterated water and 1-octanol by NMR. A correlation of poorer diffusion mobility and bigger van der Waals volume of the molecules of the studied substances has been found in both solvents. The values of the self-diffusion coefficients of the compounds with similar molecular volumes were shown to be influenced by the chemical nature of the cyclic moiety and substituents as well as the structural isomerization. All the investigated substances can be divided structurally into two groups: compounds with the substituents in the ortho-position of the benzene or pyridine ring and compounds with the substituents in the meta- and para- positions. The following order of the self-diffusion coefficients decrease in both solvents for the structural isomers of hydroxybenzoic acids, pyridinecarbothioamides, and acetamidophenoles was found: ortho- > para- > meta-. Apparent permeability coefficients were measured using an artificial phospholipid membrane made of egg lecithin as a model of in vivo absorption. The measured permeability coefficients of the studied compounds varied between 0.59·10−6 and 12.34·10−6 cm·s−1cm/s. It has been shown that both the chemical nature of the substituents and structure of the compounds have an effect on the membrane permeability. A model conception considering the passive diffusion through the phospholipid membrane as an additive process of the molecule passing through the structured water barrier before the membrane and the 1-octanol barrier simulating the lipid layer of the membrane has been suggested. The apparent permeability coefficients of the studied compounds through water layer and 1-octanol layer have been calculated using the values of the diffusion coefficients for the studied substances in deuterated water and 1-octanol and distribution coefficients in the 1-octanol/buffer pH7.4 system. We found a linear correlation between the total permeability coefficients of the investigated compounds through the water and 1-octanol

(10)

Statistical analysis of the equation confirmed the possibility of evaluating membrane permeability for structurally similar benzene and pyridine derivatives based on the distribution in 1octanol/buffer pH 7.4 system and self-diffusion coefficients of the compounds in water and 1-octanol. 3.3. Membrane Permeability of Un-ionized Forms of the Molecules. Since membrane permeability through a lipophilic membrane depends on molecule ionization state, we calculated the intrinsic permeability coefficients (Pintr(0)) characterizing the membrane permeability of neutral molecular forms of the compounds (Table 2). The calculations were carried out according to the approach proposed by Avdeef37 which relates the intrinsic permeability coefficients with the experimentally determined apparent permeability coefficients, the concentration of hydrogen ions in solution and the dissociation constants of the compounds. In this article the calculations were performed for acids and bases by the following equations:37 Pintr(0) = Papp(1 + 10−pKa + pH) (weak acid)

(11)

Pintr(0) = Papp(1 + 10 pKa − pH) (weak base)

(12)

The calculated dissociation constants of the substances studied are summarized in Table 2. As expected, the inequality Pintr(0) > Papp is realized. And the experimentally determined Pintr(0) is equal to Papp for compounds 13, 14, 15, and 16 which are uncharged in the used buffer solution pH 7.4. It should be mentioned that the ABL permeability was not taken into account. The highest permeability of the un-ionized molecular forms of compounds (2, 1, and 12) through the phospholipid membrane (see Table 2) can be attributed to their high lipophilicity. 3.4. Permeability Correlations with HYBOT Descriptors. All the compounds investigated in this study have functional groups (hydroxyl, carboxyl, and amide) capable of hydrogen bonding. We calculated HYBOT descriptors28 for 3388


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additive barrier and the experimental values of the apparent permeability coefficients through an artificial phospholipid membrane. The intrinsic membrane permeability coefficients for neutral forms of the molecules have been calculated. The reduction of the membrane permeability with an increase in the substances capacity for donor−acceptor interactions has been confirmed. For the first time the additive diffusion model of membrane permeability has been validated based on the experimental data and applied to forecasting the permeability coefficients of the structurally similar benzene and pyridine derivatives based on the self-diffusion coefficients in water and 1-octanol and the distribution coefficients in 1-octanol/buffer system. Our ability to predict the permeability coefficients will be useful for future modeling of the permeability process.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.molpharmaceut.7b00401. Absorption characteristics and correlation between the logarithms of apparent membrane permeability coefficients and ∑(Cad/α) descriptors, ∑(Cad) descriptors, ∑(Cd) descriptors, and ∑(Cd/α) descriptors (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +7 4932 533784; Fax.: +7 4932 336237; E-mail: [email protected]. ORCID

Tatyana V. Volkova: 0000-0002-5841-2724 German L. Perlovich: 0000-0002-6267-5244 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by grant No. 14-13-00640 of the Russian Science Foundation. REFERENCES

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Understanding of Relationship between Phospholipid Membrane

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