Sulpiride, Amisulpride, Thioridazine, and Olanzapine - ACS Publications

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Sulpiride, amisulpride, thioridazine and olanzapine: interaction with model membranes. Thermodynamic and structural aspects Piotr Skrobecki, Anna Chmieli#ska, Piotr Bonarek, Piotr Stepien, Anna Wisniewska-Becker, Marta Dziedzicka-Wasylewska, and Agnieszka Polit ACS Chem. Neurosci., Just Accepted Manuscript • Publication Date (Web): 04 Apr 2017 Downloaded from http://pubs.acs.org on April 5, 2017

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ACS Chemical Neuroscience

Sulpiride, amisulpride, thioridazine and olanzapine: interaction with model membranes. Thermodynamic and structural aspects

Piotr Skrobecki,†,§ Anna Chmielińska,†,‡ Piotr Bonarek,‡ Piotr Stepien,‡ Anna Wisniewska-Becker,‡ Marta Dziedzicka-Wasylewska,‡,§ and Agnieszka Polit*,‡

‡

Faculty of Biochemistry Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland §

Department of Pharmacology, Institute of Pharmacology, Polish Academy of Sciences, Smetna 12, 31-343 Krakow, Poland

†

Both authors contributed equally to this work.

*

Correspondence to: A. Polit, Faculty of Biochemistry Biophysics and Biotechnology, Jagiellonian University, Gronostajowa 7, 30-387 Kraków, Poland, Tel.: +48 12 6646156, e-mail: [email protected]

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ABSTRACT Neuroleptic drugs are widely applied in effective treatment of schizophrenia and related disorders. The lipophilic character of neuroleptics, means that they tend to accumulate in the lipid membranes, impacting their functioning and processing. In this paper, the effect of four drugs, namely thioridazine, olanzapine, sulpiride and amisulpride on neutral and negatively charged lipid bilayers was examined. The interaction of neuroleptics with lipids and the subsequent changes in the membrane physical properties was assessed using several complementary

biophysical

approaches

(isothermal

titration

calorimetry,

electron

paramagnetic resonance spectroscopy, dynamic light scattering and Zeta potential measurements). We have determined the thermodynamic parameters, i.e., the enthalpy of interaction and the binding constant to describe the interactions of the investigated drugs with model membranes. Unlike thioridazine and olanzapine, which bind to both neutral and negatively charged membranes, amisulpride interact with only the negatively charged one, while sulpiride does not bind to any of them. The mechanism of olanzapine and thioridazine insertion into the bilayer membrane cannot be described merely by a simple molecule partition between two different phases (the aqueous and the lipid phase). We have estimated the number of protons transferred in the course of drug binding to determine which of its form, ionized or neutral, binds more strongly to the membrane. Finally, electron paramagnetic resonance results indicated that the drugs are localized near the water membrane interface of the bilayer and presence of a negative charge promotes their burying deeper into the membrane.

KEYWORDS: neuroleptics, drug-membrane interactions, liposomes, calorimetry, Zeta potential, EPR


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INTRODUCTION Neuroleptic drugs are widely applied in the treatment of neuropsychiatric diseases. They are used primarily in disorders associated with psychotic symptoms, especially in schizophrenia, which is characterized by a broad range of symptoms, divided into the so-called positive (hallucinations, delusions, movement disorders) and negative ones (depression, indifference, blunted affect). The commonly used neuroleptics are effective in the treatment of positive symptoms, while therapy of negative symptoms is less effective and depends, on the kind of medication applied. A more straightforward explanation of this discrepancy has been usually linked with neurotransmitter receptor affinity of these drugs since neuroleptics bind to numerous receptors, i.e. dopaminergic, serotoninergic, muscarinic, adrenergic and histaminergic receptors with various affinity1,2. The basic pharmacological activity of neuroleptics is connected with their affinity to dopamine receptors, especially the antagonism toward the D2 receptor3–6, and the serotonin 5-HT2A receptors7,8. However, this does not explain all clinical or side effects associated with neuroleptic therapy4–6,9. An important aspect schizophrenia pathophysiology apart from receptor disregulation has been raised by Schmitt and colleagues, who have revealed alterations in the lipid composition of thalamus membranes in a post mortem study of schizophrenic patient10. Since the receptor proteins, which are targets for neuroleptics, are situated within the lipid bilayer, together they form an integral biological system – both at structural and functional levels. Therefore, the influence of neuroleptics on the lipid bilayer should also be taken into account in order to fully understand the mechanism of action of these drugs. Changes in the physicochemical properties of the lipid bilayer as the result of an interaction with neuroleptics may impact the physiological activity of the membrane as a whole – by changing its permeability, fluidity, and therefore affecting functioning of membrane proteins. Neuroleptics may alter lateral pressure of lipids in the membrane, which in turn may change protein conformation and consequently their biological activity in a manner similar to anesthetics11–13. For all these reasons, the issue of neuroleptics' impact on the lipid bilayer may be fundamental for comprehensive understanding of these drugs’ mechanism of action. A number of studies have indeed confirmed the impact of neuroleptic drugs on the lipid bilayer properties and function. Haloperidol, risperidone and 9-OH-risperidone interact with liposomes made of phosphatidylcholine, sphingomyelin and cholesterol, and induce membrane reorganization14. Also, chlorpromazine has been shown to have considerable affinity to the POPC and POPC/POPS liposomes15. The interaction depends on the charge of a lipid polar group – chlorpromazine binds more strongly to acidic lipids than to neutral


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ones16. Once in the membrane, chlorpromazine acts as factor affecting membrane permeability and fluidity17. In short, numerous studies conducted for different drugs clearly indicate that their interaction with biological membranes plays a significant role both in therapeutic and in side effects 18–20. In general, an interaction between small molecules and the lipid membrane can proceed in two basic stages: firstly, electrostatic attraction to the external membrane area consisting of polar head groups, and secondly, incorporation into the hydrophobic bilayer core mainly as a result of van der Waals forces. Furthermore, other processes in the interfacial area are possible, depending on the interacting molecule setup − for instance ionization of particular groups and formation of hydrogen bonds21. Therefore, the chemical structure of a molecule determines the thermodynamics of its interaction with a given lipid membrane. In the present study we have focused on the interaction of four neuroleptics with the lipid bilayer; three atypical ones: sulpiride (subclass: benzenesulfonamides), amisulpride (subclass: benzoic acids and derivatives), olanzapine (subclass: thienobenzodiazepines) and a typical one: thioridazine (subclass: phenothiazines). Their structures have been shown in Figure 1. These compounds belong to distinct chemical subclasses and vary in therapeutic profile. Since the interaction between chlorpromazine (structurally related to thioridazine) and lipids depends on its charge, we performed our research using unilamellar liposomes made of POPC (neutral charge) as well as POPC/POPG at molar ratio 3:1 (negative net charge). Both POPC and POPG differ only in the structure of polar headgroups. POPC should mimic well the properties of synaptic plasma membranes – phosphatidylcholines constitute the major lipid class of synaptic plasma membranes and within this class 16:0 and 18:1 are the predominant acyl chains22,23. Phosphatidylserine is the most abundant anionic lipid class of synaptic plasma membranes23, but we have chosen POPG as anionic lipid instead – both POPS and POPG possess negative net charge, but in contrast to POPG, POPS bears two negative charges and one positive charge, while POPG is a monoanion which facilitates the interpretation of our results. We have investigated the thermodynamic parameters of neuroleptics interaction with model membranes and the effects induced by these molecules on the membrane organization. We have utilized titration calorimetry (ITC) to characterize the thermodynamics of interaction (in particular the apparent equilibrium constant and enthalpy of interaction) and electron paramagnetic resonance (EPR) spectroscopy for in-depth investigation of the physical structure of the lipid bilayer. Additionally, we have used complimentary techniques: dynamic light scattering (DLS) and Zeta potential measurements to study the interaction in more detail.


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The obtained results provide evidence that sulpiride does not bind to the POPC and POPC/POPG bilayers, while amisulpride binds only to the negatively charged POPC/POPG bilayers. Thioridazine and olanzapine interact with both types of bilayers. To our best knowledge, this is the first detailed report of the impact of thioridazine and olanzapine on the lipid bilayers. Our studies have shed light on the differences of neuroleptic molecule interaction with lipids which might be correlated with their distinct biological response.

Figure 1. Chemical structures of thioridazine (TH), olanzapine (OLP), sulpiride (SU) and amisulpride (AMS). The structures were obtained from DrugBank database. Groups changing their protonation state are highlighted in blue.


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RESULTS AND DISCUSSION Isothermal titration calorimetry experiments

The enthalpies ∆H of neuroleptic – membrane interaction Isothermal titration calorimetry experiments can assess whether an interaction between the studied neuroleptics and the model membrane exists (only in the case when the interaction is correlated with heat emission/absorption, but these situations occur predominantly in biological systems). They also permit to estimate the interaction strength determining two crucial thermodynamic parameters: the enthalpy, ∆H, and the interaction constant, K. In variant I of the ITC experiment, the liposomes were titrated with a neuroleptic solution. The concentrations were selected so that the liposomes in the measurement cell were in significant excess over the titrated drug portion. This approach enables to investigate whether complete binding of the drug to the membrane (in equilibrium) takes place. Representative titration curves of the studied neuroleptics have been shown in the Supporting Information (see Figures S1 and S2). The obtained curves indicate a different heat effect in an interaction of the studied neuroleptics with the membrane. The molar enthalpy of interaction, ∆H, has been calculated by averaging all the measured points of enthalpograms (taking into account all the performed titrations for one set drug–lipid and subtraction of the corresponding reference data). The obtained values have been presented in Table 1 (see variant I). Only the results for the neuroleptics which exhibited thermal effects have been presented. The enthalpies in TRIS and phosphate buffers are different which suggests that the interaction is correlated with proton exchange/transfer (discussed below).

Effect of proton transfer For each neuroleptic, ITC experiments of variant I were performed in two buffers with the same pH=7.5 (T=25ºC) but with different ionization enthalpy: a TRIS I buffer (20mM, ionic strength=50 mM, KCl) – high ionization enthalpy (∆HJ=47.45 kJ/mol32) and a phosphate buffer (20 mM KH2PO4, ionic strength=50mM) – low ionization enthalpy (∆HJ= 3.60 kJ/mol32). The selected buffers of different ionization enthalpies allow to determine the contribution of possible ionization processes to the detected heat effects. Therefore, if the observed interaction is correlated with a proton transfer between the interacting system and a buffer, we can observe differences in enthalpies between these two buffers.


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Table 1. Molar enthalpies (∆H) of thioridazine (TH), olanzapine (OLP) and amisulpride (AMS) interaction with POPC and POPC/POPG liposomes, obtained from ITC experiments POPC POPC/POPG Drug

TH

Variant of experiment*

variant I variant II

OLP


AMS


Buffer

∆H (kJ/mol)

TRIS I

-21.5 ± 1.3

-30.8 ± 2.6

phosphate

-19.9 ± 4.5

-25.5 ± 2.3

TRIS I

-48.0 ± 2.0

-69.3 ± 4.5

phosphate

—

—

TRIS I

-3.7 ± 0.9

-0.2 ± 1.1

phosphate

8.2 ± 0.7

-8.2 ± 2.2

TRIS I

-2.4 ± 0.5

—

phosphate

—

-1.6 ± 0.1

TRIS I

—

-1.4 ± 0.1

phosphate

—

-1.9 ± 0.4

TRIS I

—

—

phosphate

—

-0.4 ± 0.1

∆H (kJ/mol)

variant I: neuroleptic to liposomes, variant II: liposomes to neuroleptic.

As already mentioned, the obtained enthalpies in both buffers are different (Table 1), which confirms a proton transfer during the interaction. The amount of protons transferred in the course of binding a mole of drug was calculated from the following equation: ∆ H= ∆ H p +n ∆ H J

(1)

where:

∆H – the molar enthalpy of interaction, determined by ITC experiment in a particular buffer; ∆Hp – the molar enthalpy of interaction independent from the buffer used; n – the amount of protons transferred during binding a mole of a drug. We have assumed that the studied drugs bind to the lipid bilayer according to the following model (adapted from33):

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where subscripts M, I and B denote the membrane, the membrane interface and bulk solution regions, respectively. K1 and K2 are binding constants of the deprotonated and protonated form, respectively and K is the acid dissociation constant. The proton transfer n may be associated with K1/K2 via Equations 2 and 333:

= 1 +

10

= + 0.43ψ

− 1 + 10

(2)

(3)

where ψ0 is the bilayer surface potential, F is Faraday constant, R is the gas constant and T is the temperature. The ψ0 potential of the POPC bilayer was assumed to equal 0 mV, whilst for the POPC/POPG bilayers, the ψ0 potential was calculated according to Guy-Chapmann theory as -75 mV for liposomes in TRIS I buffer (lipid area 68 A2; K+ binding to POPG lipid with binding constant 0.15 M-l , both taken from34). The ∆Hp, n and K1/K2 values of TH, OLP and AMS – the neuroleptics, which interact with the bilayer have been presented in Table 2. The obtained enthalpies indicate an exothermic effect for all the studied combinations, except for OLP with POPC. Lower enthalpy values are associated with the interaction of TH – both for neutral vesicles made of POPC and negatively charged vesicles made of POPC/POPG (molar ratio 3:1). Endothermic binding enthalpy of OLP with the POPC bilayers suggests that this process is driven by an increase in entropy. Table 2. The molar enthalpy of interaction independent of the buffer used (∆Hp), the amount of protons transferred in the course of binding a mole of drug (n), and the binding constants ratio for the deprotonated and protonated form (K1/K2) for thioridazine (TH), olanzapine (OLP) and amisulpride (AMS) † Drug-bilayer combination ∆Hp (kJ/mol) n (mol/mol) * K1/K2 TH to POPC

-19.79 ± 1.50

-0.04 ± 0.05

n. d.

TH to POPC/POPG

-25.10 ± 0.91

-0.12 ± 0.03

78.3 ± 17.2a

OLP to POPC

9.18 ± 0.23

-0.27 ± 0.01

10.8 ± 0.5b

OLP to POPC/POPG

-8.84 ± 0.77

0.18 ± 0.02

1.3 ± 0.2c

AMS to POPC/POPG

-1.93 ± 0.18

0.01 ± 0.01

n. d.

* negative n sign means H+ transfer direction from interacting system to buffer, positive n sign from buffer to interacting system. †

Calculated according to Equations 2 and 3 assuming ψ0=0 mV and ψ0=-75 mV for POPC and POPC/POPG bilayers, respectively; pKa values: a 8.85, b 8.88, c 5.76.


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For TH with POPC and AMS with POPC/POPG n is close to zero and given the magnitude of its uncertainties, the K1/K2 cannot be reliably determined for those combinations. For the POPC bilayers we can assume that the pH in the interface region equals the pH of the bulk solution (pH=7.5) and it is reasonable to consider only the first protonation step of OLP (with pKa 8.88). Then the observed proton transfer n can be explained by preferential binding of a non-protonated form of OLP. For the POPC/POPG bilayers, the pH in the interface region is lower than the pH of the bulk solution since the protons are drawn to the negatively charged bilayers − for the liposomes with ψ0=-75 mV at 25ºC, the pH in the interface drops to 6.24. In this case we can consider only the second protonation step of OLP (with pKa 5.76) and neglect the binding of non-protonated form of OLP. The observed proton transfer n in the course of OLP binding to the POPC/POPG bilayers is thus caused mainly by a decrease of pH near the bilayer – the mono- and di-protonated forms bind with comparable strength.

Figure 2. Normalized heat titration of liposome into TH versus lipid concentration in cell. POPC liposomes (PC); POPC/POPG liposomes (PC/PG). 3.5 mM liposome dispersion titrated into 50 µM TH solution. Insets: raw data of normalized heat flow versus time after baseline correction; reference measurements in blue shifted below sample measurements.


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TH and OLP absorption spectra measurements (see Figure S4 in the Supporting Information) confirm calorimetric results − both drugs bind to lipid bilayer. The spectra change in presence of liposomes as compared to the spectra obtained solely for the drugs, which points out a change of environment after binding to the lipid bilayer. Particularly visible is a bathochromic shift of the TH absorption peak (see Figure S4 in the Supporting Information). The binding constants K of neuroleptic – membrane interactions Variant II of titration was based on adding liposomes to a neuroleptic solution. The representative titration curves of the studied drugs have been presented in Figures 2−4.This experiment variant was intended to determinate the interaction constant, K (detailed analysis below). The values of enthalpy, ∆H, obtained from variant II, have been presented in Table 1. This experiment variant has been limited to TH, OLP and AMS which exhibit interaction with the lipid membrane as was shown in variant I of the ITC experiments. Variant II of the ITC experiments was performed in a buffer where stronger signal in variant I was observed.

Figure 3. Normalized heat titration of liposome into OLP versus lipid concentration in cell. POPC liposomes (PC); POPC/POPG liposomes (PC/PG). 5 mM liposome dispersion titrated into 40 µM OLP solution. Insets: raw data of normalized heat flow versus time after baseline correction; reference measurements in blue shifted below sample measurements. ACS Paragon Plus Environment

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In order to determine the reaction enthalpy, ∆H, and the binding constant, K, we have taken two different approaches. The first one was based on cumulative reaction heats21 and the second one − on finite concentration differences of the bound solute28. Evaluation of K from cumulative reaction heats requires plotting the binding isotherm, Xb(cf), which is the dependence of binding degree, Xb, on free drug concentration cf. They can both be calculated from the measured reaction heats upon titration, provided that the heat of the last injection is zero. If a particular drug binds to the bilayer according to a simple partition only, the binding degree Xb is linearly dependent on free drug concentration cf with K as the slope coefficient (for details and results see Supporting Information). In the second approach, the theoretical heat of each titration, Q, is calculated from the reaction enthalpy, ∆H, and the amount of the bound solute, which in turn, is based on the binding constant, K. The values of ∆H and K are plugged-in by the solver until the calculated Q converges with the measured one (for details see paper by Tsamaloukas28). The results of the fitting have been shown in Figures 2−4; the obtained ∆H and the K values have been presented in Table 1 (see variant II) and Table 3, respectively. Table 3. Apparent binding constants Kapp of thioridazine (TH), olanzapine (OLP) and amisulpride (AMS) interaction with POPC and POPC/POPG liposomes POPC POPC/POPG Drug

Kapp (mM-1)

Kapp (mM-1)

TH

6.38 ± 0.32

8.10 ± 0.62

OLP

2.87 ± 0.66

3.56 ± 1.38

AMS

—

0.69 ± 0.22

In both approaches, with the binding model describing only a simple drug partition into the bilayer, the fitted functions did not reflect the data perfectly. In the first approach, the binding isotherms showed a non-linear relationship (Figure S3 in the Supporting Information). This behavior indicates that, apart from simple partition and Van deer Waals interactions, other effects are involved. Under our experimental conditions the majority of both TH and OLP are charged. Thus, we took into account the electrostatic effects in the applied model and derived the binding isotherms where the concentration of the un-bound drug near the bilayer surface was corrected. However, after this correction, the binding isotherms still remained non-linear. This may indicate that assumptions about electrostatics do not hold. Especially important is the assumption concerning homogeneous charge density on the bilayer surface −


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if drug charges were buried in low-dielectric environment of bilayer interior, the surface charge density should be calculated considering distribution of discrete charges (for comprehensive discussion see Mclaughlin review35). Furthermore, it was shown that OLP interacts electrostatically with the POPS headgroup36. Taking into account that both lipids and drugs studied here possess charged moieties, it is likely that electrostatic interactions also contribute to deviation from simple partition model. The failure of the used model to describe the experimental data is also the main reason why the ∆H values obtained from variant II and variant I experiments are not consistent with each other. Furthermore, in case of variant I experiments the provided conditions may be insufficient for complete binding of a molecule to the lipid bilayer leading in consequence to underestimated enthalpies37. The Kapp values of binding to the lipid bilayer determined here coincide with the reported Kapp range for anesthetics (4−20 mM-1)38 and neuroleptics (0.26−15 mM-1)15,39. Phenothiazines (chlorpromazine, thioridazine) in pH 5.9 bind to DMPC bilayers with Kapp in range 0.26−0.51 mM-1 39. For chlorpromazine, Moreno and colleagues report Kapp in pH 7.4: 13 mM-1 (binding with pure POPC) and 15 mM-1 (binding with POPC/POPS mixture)15.

Figure 4. Normalized heat titration of liposome into AMS versus lipid concentration in cell. POPC/POPG liposomes (PC/PG). 15 mM liposome dispersion titrated into 350 µM AMS solution. Inset: raw data of normalized heat flow versus time after baseline correction; reference measurement in blue shifted below sample measurement. It is worth noting that for many drugs the main mechanism of transport across the blood-brain barrier (BBB) is passive diffusion through the lipid bilayer which cannot occur without drug binding to the bilayer in the first place. Despite the fact that both AMS and SU have pharmacological impact on the central nervous system, they both permeate poorly via passive diffusion across in vitro BBB models, with AMS showing almost no


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permeation40. Neuroleptics may have diverse pharmacological efficacy and clinical effects based solely on differences in the potency to penetrate BBB. It is especially evident for AMS in comparison to SU. Although in vitro AMS has higher affinity to dopamine receptors than SU, in vivo it is less efficient than SU. Furthermore, AMS is considered the most potent neuroleptic in elevating prolactin blood level41. Both issues can be explained by AMS highly restricted permeation through BBB41–43. Based on permeability through in vitro BBB models, the drugs can be put in following order: AMS < SU

Sulpiride, Amisulpride, Thioridazine, and Olanzapine - ACS Publications

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