key: cord-0683752-3lfljgys
authors: Aree, Thammarat
title: Advancing insights on β -cyclodextrin inclusion complexes with SSRIs through lens of X-ray diffraction and DFT calculation
date: 2021-09-20
journal: Int J Pharm
DOI: 10.1016/j.ijpharm.2021.121113
sha: dcb783c3793aa152ec0a18420793c112894c96be
doc_id: 683752
cord_uid: 3lfljgys

Depression—the global crisis hastened by the coronavirus outbreak, can be efficaciously treated by the selective serotonin reuptake inhibitors (SSRIs). Cyclodextrin (CD) inclusion complexation is a method of choice for reducing side effects and improving bioavailability of drugs. Here, we investigate in-depth the β -CD encapsulation of sertraline (STL) HCl (1) and fluoxetine (FXT) HCl (2) by single-crystal X-ray diffraction and DFT complete-geometry optimization, in comparison to the reported complex of paroxetine (PXT) base. X-ray analysis unveiled the 2:2 β -CD–STL/FXT complexes with two drug molecules inserting their halogen-containing aromatic ring in the β -CD dimeric cavity, which are stabilized by the interplay of intermolecular O2–H ⋯ N1–H ⋯ O3 H-bonds, C3/C5–H ⋯ π and halogen ⋯ halogen interactions. Similarly, the 1:1 β -CD–tricyclic-antidepressant (TCA) complexes have an exclusive inclusion mode of the aromatic ring, which is maintained by C3/C5–H ⋯ π interactions. By contrast, the 2:1 β -CD–PXT complex has a total inclusion that is stabilized by host–guest O6–H ⋯ N1–H ⋯ O5 H-bonds and C3–H ⋯ π interactions. The inherent stabilization energies of 1 and 2 evaluated using DFT calculation suggested that the improved thermodynamic stabilities via CD encapsulation facilitates the reduction of drug side effects. Moreover, the SSRI conformational flexibilities are thoroughly discussed for understanding of their pharmacoactivity.

Depression is a global mental illness as over 300 million people suffer from it and about 800,000 people die from suicide each year (WHO, 2017)the situation before the coronavirus disease 2019 pandemic. Recent data of the COVID-19 era frighten us : i) 1.5-year since the outbreak, the number of confirmed COVID-19 cases has gone beyond 170 million worldwide (Our World in Data, 2021) ; ii) about one-fourth of COVID-19 patients globally commonly experience depression (Rogers et al., 2021) ; and iii) one-half of the COVID-19 survivors suffer from depression (Perlis et al., 2021) . An efficacious treatment for depression is achievable by using antidepressants. Selective serotonin reuptake inhibitors (SSRIs) are first-line, second-generation antidepressants including for example, sertraline (STL; Zoloft), fluoxetine (FXT; Prozac) and paroxetine (PXT; Paxil), Scheme 1. SSRIs for the treatment of depression have been approved by the US Food and Drug Administration (FDA, 2014) . Prozac is one of the most commonly prescribed and popular SSRIs in the US (Stokes and Holtz, 1997) as it is the only FDA-approved drug for adolescents (McClanahan, 2009) . SSRIs have equivalent clinical efficacy in treating depression to tricyclic antidepressants (TCAs), firstgeneration drugs, and have fewer side effects due to their high selectivity in binding with serotonin transporters. Hence, SSRIs are safer in overdose than TCAs (Peretti et al., 2000) . SSRI drugs are rather less soluble in water and hence have limited oral bioavailability. To overcome the problem, the cyclodextrin encapsulation is a method of choice for enhancing aqueous solubility and reducing side effects of SSRIs (Passos et al., , 2012 Buko et al., 2017; Abouhussein et al., 2019) . Plus, light in the mist of CIVID-19 crisis, SSRIs with antiinflammatory properties can be used in treating COVID-19-associated inflammatory lung disease, thus reducing severity of COVID-19 (Lenze et al., 2020; Hoertel et al., 2021; Meikle et al., 2021) .

-, -, -Cyclodexrins (CDs) are versatile encapsulating agents comprising 6, 7, 8 Dglucose units, respectively. CDs adopt a shape of hollow, truncated cone and are amphiphilic with hydrophobic central cavity and hydrophilic rims (Scheme 1). They can accommodate a variety of guest molecules fitting partly or wholly to the ir apolar cavity, yielding inclusion complexes (Dodziuk, 2006) . The host-guest supramolecular complexes are stabilized by noncovalent intermolecular interactions, e.g., hydrogen bonds, hydrophobic and van der Waals interactions. The CD encapsulation has various practical applications including pharmaceutics, cosmetics, medicine, food, chromatography, biotechnology, and nanotechnology (Adeoye and Cabral-Marques, 2017; Bakshi and Londhe, 2020; Fourmentin et al., 2018; Liu et al., 2020; Morin-Crini et al., 2021) . In pharmaceutical technology, CD inclusion complexation improves the physicochemical and pharmacological properties of drugs; see a recent patent review for antidepressants (Diniz et al., 2018) . Here, we provide an insightful literature review of the CD-SSRI inclusion complexes, focusing on STL, FXT and PXT. These SSRIs are less structurally similar, but commonly contain halogens relating to their serotonin reuptake efficacy. Considerable research attention has been paid to the CD-STL/FXT/PXT inclusion complexes over the past 20 years. (Wong et al., 1985) .

The CD encapsulation of STL is the most studied among the SSRIs. The 2:1 and 1:1 -CD-STL HCl complexes are in equilibrium with the tetrahydronaphthalene (A-B-rings), dichlorophenyl (C-ring) and methylamine moieties embedded in the CD cavities, and the DFT-derived stabilization energies are −12.7 and −14.5 kcal mol −1 , respectively . The stable equimolar -CD-STL HCl complex with an association constant (K a ) of 4999±495 M −1 as determined by isothermal titration calorimetry (ITC) (Passos et al., , 2013 helped to improve the drug solubility and transport efficacy in vivo (Passos et al., 2012) . Thermodynamic properties of the 1:1 complex with the STL methylamine portion entering the -CD O2-H/O3-H-side as derived from DFT calculation in the explicit hydration water agree with experimental data (Lopes et al., 2015) . NMR, UV-vis, and ITC data revealed that both -CD and dimethyl--CD (DIMEB) form 1:1 complexes with STL HCl with K a 's in the respective ranges of 5300-5850±500 and 7960-8600±500 M −1 (Belica et al., 2014) . The greater K a 's of DIMEB complexes over those of -CD complexes are due to the better fit of STL aromatic portion to the more hydrophobic DIMEB cavity (Belica et al., 2014) . Moreover, the pharmacological activity of STL HCl is improved via equimolar complexation with hydroxypropyl--CD (HP--CD) with K a = 6530±54 M −1 from ITC (Buko et al., 2017) . In the solid state, NMR data indicated the stable 1:1 -CD-STL base complex with the A-B-rings partially entrapped in the -CD cavity and K a = 4300 M −1 , based on the vibrational spectra (IR, Raman) and phase solubility data, respectively (Ogawa et al., 2015) . Recently, sublingual orodispersible tablet formulated from the -CD-STL solid dispersion enhanced the STL dissolution and rapid absorption through the oral mucosa, thus improving the drug bioavailability (Abouhussein et al., 2019) .

The CD-FXT complexes draw attention to some extent. FXT HCl embedded in the -CD cavity increased the drug pharmacological efficacy (Géczy et al., 2000) . Ali et al. (2005) inferred from NMR data the 1:1 -CD-FXT HCl inclusion complex with rather low and underestimated K a of ~70 M −1 . The trifluorotoluene B-ring is enclosed in the -CD cavity and the CF 3 group is protruded from the O6-H side, establishing O6-HF H-bond and thus stabilizing the inclusion complex (Ali et al., 2005) . The host-guest stoichiometry and inclusion structure of the -CD-FXT HCl complex have been confirmed by NMR combined with DFT calculation, and the K a value deduced from ITC seems more reasonable, 6921±316 M −1 (de Sousa et al., 2008) .

The -CD encapsulation of PXT received less attention compared to other complexes.

In solution, NMR combined with molecular dynamics (MD) simulation suggested the 1:1 -CD-PXT base inclusion complex is quite stable with K a ~2000 M −1 (Bernini et al., 2004) .

The corresponding inclusion structure is that the benzodioxole (C-D-ring) moiety is deeply inserted in the -CD cavity and the fluorophenyl B-ring is outside the cavity nearby the O2-H/O3-H side (Bernini et al., 2004) . On the contrary, in the solid state where intermolecular interactions between CD molecules are present, the apparent host-guest molar ratio is 2:1 (Caira et al., 2003) . PXT base is totally entrapped in the head-to-head β-CD dimer such that the piperidine A-ring is nearby the O6-H side of one -CD monomer, whereas the B-ring and C-D-ring moieties respectively reside in the dimer interfacial region and the cavity of another -CD monomer (Caira et al., 2003) .

CDs are non-toxic, biodegradable, and biocompatible (Del Valle, 2004) . Among them, -CD is the most studied because of its lower price and more optimum cavity size for harboring various guest molecules containing aromatic ring(s). This is evidenced by the predominant number of reported -CD structures deposited to the Cambridge Crystallographic Data Centre (CCDC) (Groom et al., 2016) . As thoroughly reviewed above, the atomic-level characteristics of -CD-SSRI complexes (i.e., the inclusion structures and the host-guest stoichiometric ratios) remain rather controversial and deserve a comprehensive structural investigation. Because the SSRI drugs have varied structural moieties including the five-, six-membered rings and the side chain, hence this study has a twofold hypothesis. i) Among different SSRI structural moieties, the halogen-bearing aromatic ring provides better fit to the -CD nanocavity for thermodynamically favorable inclusion complexation that is stabilized by optimal host-guest interactions. ii) SSRIs conformational flexibility in different lattice environments (in free salt form, in the CD cavity and in complex with proteins), is correlated with their pharmaceutical activity. To prove the two hypotheses, we carry out single-crystal X-ray diffraction and DFT full-geometry optimization of the -CD encapsulation of STL and FXT. " Because PXT is rather bulkier than other SSRIs and it shares a structural motif of an aryloxypropylamine with FXT, we make detailed structural comparisons of the two complexes with the -CD-PXT complex (Caira et al., 2003) and of STL, FXT and PXT in three distinct circumstances.

-CD (≥ 95%) was purchased from Cyclolab, Budapest, Hungary (code CY-2001).

(1S,4S)-STL HCl (≥ 98%) and rac-FXT HCl (≥ 98%) were supplied by TCI Chemicals (codes S0507 and F0750). Absolute EtOH (≥ 99.8%) was provided by Liquor Distillery Organization, Excise Department, Thailand. All chemicals were used as received. The ultrapure water was obtained from a Milli-Q Water System.

Homogeneous, concentrated equimolar solutions of the -CD-STL HCl (1) and -CD-FXT HCl (2) inclusion complexes were prepared by dissolving the solid powders of -CD 50 mg (0.044 mmol), STL HCl 15.1 mg (0.044 mmol) and FXT HCl 15.2 mg (0.044 mmol) in 1 mL of 50% (v/v) EtOH-H 2 O at 323 K. The vials containing solutions of both complexes were left standing still in an air-conditioned room (298 K). After two weeks of slow solvent evaporation, single crystals for X-ray analysis were harvested.

Colorless rod-shaped single crystals of 1 and 2 were separately mounted in a thinwalled glass capillary (Hilgenberg, Germany). For both complexes, several crystals were checked for consistent unit cell constants and sufficient diffracting power. Different specimens crystallizing in the triclinic system with similar unit cell parameters, provide strong evidence of a given inclusion complex. The best diffracting crystals of 1 and 2 were chosen for X-ray data collection at 296 K to 0.83-Å atomic resolution on a Bruker APEXII CCD area-detector diffractometer (MoK radiation;  = 0.71073 Å). Data processing assisted by the APEX2 software suite (Bruker 2014) was accomplished according to standard procedures, i.e., began with integration using SAINT (Bruker, 2008) , followed by scaling and multi-scan absorption correction using SADABS (Bruker, 2014) , and completed by merging with XPREP (Bruker, 2008) . The total numbers of 30,769 and 33,629 independent reflections with R int of 0.0488 and 0.0518 for the respective complexes 1 and 2 were obtained.

The crystal structures of 1 and 2 were solved by intrinsic phasing method with SHELXTL XT (Bruker, 2014) , providing all non-H atoms of -CD dimers, STL, and most non-H atoms of FXT molecules. The missing non-H atoms of FXT were located by difference Fourier electron density maps graphically assisted by WinCoot (Emsley et al., 2010) . Then a number of restraints were applied to FXT during the refinement. In the -CD dimeric cavity of 1 and 2, the occupancy factors of two STL and two FXT sites, each was refined to unity, yielding 2:2 host-guest complexes. The protonated STL and FXT molecules were counterbalanced and indirectly coordinated by doubly disordered chlorides, which were previously observed in the crystals of eight -CD-TCA HCl inclusion complexes (Aree, 2020a (Aree, , 2020b (Aree, , 2020c (Aree, , 2021 .

The rest of non-H atoms including water O atoms and chloride ions were located by difference Fourier maps. Most of non-H atoms were refined anisotropically by full-matrix least-squares on F 2 using SHELXTL XLMP (Bruker, 2014) , except for some O6-H groups of -CD (2), two FXT (2) and disordered water molecules, which were refined isotropically. H atoms of rigid groups were positioned geometrically and treated with a riding model: C−H = 0.93 Å, U iso = 1.2U eq (C)(aromatic); C−H = 0.98 Å, U iso = 1.2U eq (C)(methine); C−H = 0.97 Å, U iso = 1.2U eq (C)(methylene); C−H = 0.96 Å, U iso = 1.5U eq (C)(methyl); and N−H = 0.89 Å, U iso = 1.2U eq (2° ammonium). The hydroxyl H-atoms initially located by difference Fourier maps were refined using 'AFIX 147' or 'AFIX 83' with restraints O−H = 0.84 Å, U iso = 1.5U eq (O). Water H-atoms could not be located by difference Fourier maps. To prevent short H•••H distances in the refinement, BUMP antibumping restraints were applied. The refinement of 1 converged to a final R 1 = 0.0855. For 2, 27.4 water molecules were spread over 50 sites outside the -CD dimeric cavity, filling in the intermolecular interstices. Most water sites were not in hydrogen bonding contact with -CD or FXT molecules. Therefore, all water sites as large solvent voids were removed from the structure model in the final refinement using the PLATON SQUEEZE procedure (Spek, 2015) , yielding R 1 = 0.1035, which was improved by 0.0252. For more details of data collection and refinement statistics , see Table S1 .

Accurately determined X-ray structures are good sources of initial atomic coordinates for DFT complete-geometry optimization of the supramolecular CD inclusion complexes, of which CDs are conformationally flexible. This has been successfully demonstrated in our previous works on the -CD inclusion complexes with TCAs (Aree, 2020a (Aree, , 2020b (Aree, , 2020c (Aree, , 2021 . As noted in Section 3.2, in the solid state the 2:2 -CD-STL HCl (1) and 2:2 -CD-FXT HCl (2) inclusion complexes comprised two pseudo-monomeric units of which the two -CD and two drug molecules were quite different. Therefore, the energy minimization by DFT method was carried out for each monomeric complex to evaluate the sole host-guest interactions necessary for the stabilization of the -CD-STL/FXT complexes. Before the calculation, the underestimated X-ray-derived X-H bond lengths in the 1:1 -CD-STL base and 1:1 -CD-FXT base inclusion complexes were normalized to neutron hydrogen distances: C-H, 1.083 Å; N-H, 1.009 Å; and O-H, 0.983 Å (Allen and Bruno, 2010) . The corrected structures were optimized by semiempirical PM3 method and then fully reoptimized by DFT calculation using the B3LYP functional in the gas phase with mixed basis sets 6-31+G* for H, N, O and 4-31G for C. All calculations were carried out using program GAUSSIAN09 (Frisch et al., 2009 ) on a DELL PowerEdge T430 server. Stabilization energy and interaction energy of the complex (E stb and E int ) were calculated using eqn (1) and (2).

where E cpx , E -CD_opt and E D_opt are the molecular energies from full-geometry optimization of complex, host -CD and drug STL/FXT, respectively; E -CD_sp and E D_sp are the corresponding single-point energies in the complexed states. Moreover, to assess on the extent of dimerization that helped to improve the thermodynamic stability, energy minimizations of the dimeric complexes were carried out, and the corresponding E stb , E int were evaluated; see Table S7 .

Here, we use the conventional carbohydrate nomenclature for CDs, i.e., atoms C63- 

In solution, CD inclusion complexation can take place from the uncomplexed CD hydrate, of which the water molecules residing in the CD cavity are repelled away and replaced with the hydrophobic guest. The CD macrocycle adapts its cavity to some extent to optimally accommodate partial or entire drug molecule, yielding mostly the 1:1 or 2:2 hostguest complex as frequently observed in the crystalline state (Groom et al., 2016) . Here, the complexation of 2:2 -CD-STL HCl (1) and 2:2 -CD-FXT HCl (2) is facilitated by three factors. i) The drug molecule is rather bulky to be totally enclosed by a single CD molecule.

ii) The drug molecules form dimer that is stabilized by intermolecular halogenhalogen interactions (see Sections 3.2 and 3.3). iii) The chemical nature of the least aqueous soluble native CD (β-CD) due to the more rigidity of its macrocycle and the β-CD dimer-based selfaggregates in water (Naidoo et al., 2004; Cai et al., 2008) . To quantify the degree of -CD structural adaptation upon the drug inclusion, the -CD·12H 2 O structure (Lindner and Saenger, 1982) is used as a reference structure. The CD inclusion complexation is a paradigm for the induced-fit process (Koshland, 1973) driven through weak non-covalent intermolecular interactions.

The root-mean-square (rms) deviation of structure superposition is a global parameter for quantification of the similarity of a structure pair. For CDs, the non-H atoms of CD skeleton excluding O6 are used for the calculation of rms fit. The three -CD dimers in complex with SSRIs are non-superimposable because 1 and 2 prefer a tail-to-tail motif while the PXT complex (Caira et al., 2003) favors a head-to-head structure (see Section 3.3). The corresponding O4-centroid-O4-centroid distances and O4 interplanar angles are 9.011 Å, 1.5°; 9.051 Å, 2.7°; and 7.000 Å, 1.2°, for 1, 2 and the PXT complexes, respectively (Table 1 and Figs. 4, 5). The rms fit of the -CD dimers in 1 and 2 is rather high, 1.633 Å. Therefore, the monomeric -CD-SSRI complexes are chosen for comparison. For the 2:1 -CD-PXT complex (Caira et al., 2003) , the main -CD #2 encapsulating the PXT piperidine moiety (Aring) is considered (labeled i in Fig. 3a 9.011 9.051 7.000 a All SSRIs are in HCl salt fo rm, except PXT base. For co mparison to other drugs in free HCl salt form and in complex with proteins, see Table S6 . b Non-planar ring puckering coordinates including radius Q, azimuthal angle , and meridian angle  (Cremer and Pople, 1975) . c For ato m nu mbering, see Scheme 1. d PXT base in co mplex with -CD (Caira et al., 2003) . All the piperidine A-rings of PXT molecules in various lattice environ ments adopt a normal chair form, hence the 1,3-dio xo le D-ring parameters are shown here. e When the -CD O6-side pointing upwards, the positive(negative) values indicate that the A/B/C-ring centroid of a drug molecule is above(beneath) the -CD O4 p lane. f Chiral centers of STL (C4), FXT (C13) and PXT (C4) are considered , see Scheme 1. g Channel structure built fro m the asymmetric unit of -CD dimer, tail-to-tail (T2T) and head-to-head (H2H). h Plane passing through C2-C3-C5-C6 o f PXT p iperidine ring. (2) and PXT base (Caira et al., 2003) . For comparison, data of the inclusion complexes of -CD-(-)-epicatechin(EC) (Aree and Jongrungruangchok, 2016) , -CD-clomipramine(CPM) (Aree, 2020c) and the uncomplexed -CD·12H 2 O (Lindner and Saenger, 1982) are also incorporated; see also Table S2 .

Angles and distances are in ° and Å. The origin of CD similarity is noteworthy. The CD structural parameters spring from its elemental components as follows. i) The O3(n)···O2(n + 1) distances describe the CD conformational rigidity, which is maintained by the "belt" of intramolecular, interglucose O3(n)···O2(n + 1) H-bonds. Consequently, the glucose inclination angles of a round CD structure fall into a small range. ii) The O4 glycosidic linkage related parameters include O4 deviations, O4(n)···O4(n -1), O4(n)···centroid distances, and torsional angles  [O5(n + 1)- Table S2 . iii) Furthermore, for the D-glucose units composing round CDs, an ideal chair form of cyclohexane has a puckering amplitude Q = 0.63 Å and a polar angle  = 0° (Cremer and Pople, 1975) .

Given examples of the -CD structural parameters are a) the glucose puckering parameters Q, ; b) the tilt angles; c) the O3(n)···O2(n + 1) distances; d) the average of O4(n)···O4(n -1)/O4(n)···centroid distance ratios (= 0.868 for a perfect heptagon) ; and e) the sum of averages of  and  (0 for closed CD structures (French and Johnson, 2007) ).

The corresponding values of the above parameters for 1, 2 and [uncomplexed -CD·12H 2 O] (Lindner and Saenger, 1982) Table S2 and Fig. 2a,b . On the contrary, for the -CD-EC complex (Aree and Jongrungruangchok, 2016), two opposed glucose units are tilted > 30°, giving rise to the broken belt of O3(n)···O2(n + 1) H-bonds with two O3(n)···O2(n + 1) distances > 3.2 Å.

SSRIs and TCAs do not share similar structural features, giving rises to their distinct pharmacological functions. Whereas TCAs possessing a [6-7-6]-tricyclic core linked with a propylamine side chain are inhibitors of both serotonin and norepinephrine, SSRIs having an aryloxypropylamine scaffold and 2-3 ring moieties are selective inhibitors of serotonin transporters (Nogrady and Weaver, 2005) . The three SSRI drugs are potent for serotonin uptake in the order of PXT > STL > FXT (Stahl and Stahl, 2013) . Because the -CD to the aromatic C-ring of PXT (Scheme 1 and Fig. 1) . Therefore, the SSRI structures can be described by a number of selected torsion angles, the distances between ring centroids, and the aromatic ring interplanar angles listed for SSRIs in complex with -CD (Table 1) , and for uncomplexed SSRIs and SSRIs in complex with proteins (Table S5 ).

The first crystal structure of -CD inclusion complex with SSRI (PXT base) has been reported 18 years ago (Caira et al., 2003) . Here, we re-scrutinize for further insights on the structure and inclusion characteristics of PXT base enclosed in the -CD cavity. Because PXT base is rather bulkier than other SSRIs and rather long to be hosted by a single CD, it exists in an elusive hairpin conformation stabilized by intramolecular face-to-face  interactions between the B-and C-rings and is encapsulated in the -CD dimeric cavity, forming the 2:1 -CD-PXT base complex (Caira et al., 2003) . The relevant structural parameters are the Cg(B)-Cg(C) distance of 3.637 Å and the interplanar angle of 9.0° (Table   1 ). The piperidine A-ring of PXT encapsulated in the -CD cavity (Caira et al., 2003 ) adopts a normal chair form similar to other PXT in different lattice environments. By contrast, the 1,3-dioxole D-ring exist as an envelope form for PXT embedded in the -CD cavity (Table 1) and as either a planar or an envelope structure for the PXT-protein complexes and the uncomplexed PXT (Table S5) . For detailed discussion on the SSRI flexibility, see Section 3.5.

Regarding the PXT arrangement in the -CD dimeric cavity, the PXT 1,3-benzodioxole (D-C-rings) and fluorophenyl (B-ring) moieties respectively occupy the cavity of -CD #1 and the intra-dimer interstices, and the piperidine A-ring is situated around the O6-H side of -CD #2 (Fig. 4c) . The A-ring (plane passing through C2-C3-C5-C6) and the B-ring make angles of 72.2 and 69.3° against the O4 plane of -CD #2; the chiral center C4 is 0.931 Å beneath the O4 plane (Fig. 4c) . Note that the 1,3-dioxole D-ring of PXT embedded in the -CD cavity is puckered in an envelope conformation (Caira et al., 2003) , whereas PXT in complex with the serotonin transporter (SERT) proteins has the 1,3-dioxole moiety in a planar geometry (Coleman and Gouaux, 2018) , Tables 1 and S5. The structural distinction can be attribute to the 1,3-benzodioxole moiety that has large amplitude out-of-plane vibrations, of which 1,3-dioxole can exist as either a puckered form or a planar geometry with a small energy barrier of 0.36 kcal mol −1 (Emanuele and Orlandi, 2005) . One P XT molecule is totally enclosed in the -CD dimeric cavity and is maintained in position by hostguest O63-HN1-HO53 hydrogen bonds and C3-H interactions ( Fig. 4c and Table 2 ). The head-to-head -CD dimer is stabilized by intermolecular O2-H_1/O3-H_1O2-H_2/O3-H_2 hydrogen bonds (Caira et al., 2003) .

The inclusion scenarios in the 2:2 -CD-STL (1) and 2:2 -CD-FXT (2) are somewhat different from what observed in the 2:1 -CD-PXT (Caira et al., 2003) . The two STL and two FXT molecules of each complex are similarly included in the -CD dimeric cavity (Fig.   4a,b) . The drug molecules insert their halogen-containing aromatic ring (STL C-ring and Table 2 ). Similar inclusion modes are observed for the -CD-TCA complexes (Aree, 2020a (Aree, , 2020b (Aree, , 2020c (Aree, , 2021 . The TCA aromatic A/B-rings are mostly vertically aligned in the -CD cavity (83.7-89.7°) and their centroids are 0.467-1.384 Å above the CD molecular plane. The STL and FXT halogen moieties are encapsulated in the -CD carrier cavity as they are bound to the transporter pocket (Zhou et al., 2009) , thus maintaining the SSRI specificity for SERT (Butler and Meegan, 2008) . The distinction of the two drug molecules in 1 and 2 deserves further discussion. The two drug molecules cannot be superimposed because their relative arrangements of the structural moieties are rather different. Therefore, the more rigid portions of STL (atoms C1-C16) and FXT (atoms C1-C13 and O1) are used for the calculation, giving the respective rms fits of 0.936 and 0.212 Å (Fig. 7a,b) . The greater difference between STL #1 and #2 (1) is due to the distinct B-rings existing in the half chair and boat conformations, respectively (Figs. 4a, 5a, 6a and Table 1 ). This gives rise to the differences of the B-centroid-C-centroid distance and torsion angle C12-C11-C4-C10: 3.783 Å, -76.7° for 1 and 4.289 Å, 0.7° for 2 (Table 1) . By contrast, FXT #1 and #2 (2) excluding the propan-1-amine side chain are more similar, as indicated by the A-centroid-B-centroid distance and torsion angle C12-C7-C13-O1: 4.743 Å, -40.9° for 1 and 4.586 Å, -67.6° for 2 ( Table 1 ). Note that starting from the racemic mixture of (S)-and (R)-FXT HCl, after the -CD encapsulation and crystallization, the final product is the 2:2 -CD-(S)-FXT inclusion complex, indicating the stronger intermolecular interactions and the enantioselective power of -CD with the (S)-over the (R)enantiomers. This result agrees with the enantioseparation by high performance liquid chromatography and capillary electrophoresis using -CD phenyl isocyanate (Yu et al., 2002) and trimethyl--CD (Cârcu-Dobrin et al., 2017) as chiral stationary phases, respectively.

TCAs adopt a butterfly-like shape and have a pseudo-reflection symmetry with a mirror plane bisecting the central C-ring, i.e., both wings (the aromatic A-and B-rings) are the same.

Crystallographic evidence revealed that the 1:1 -CD-TCA inclusion complexes with the TCA aromatic A/B-ring enclosed in the CD cavity exist exclusively in the orthorhombic, space group P2 1 2 1 2 1 ; -CDs are arranged in the head-to-tail column structure (Aree, 2020a (Aree, , 2020b (Aree, , 2020c (Aree, , 2021 . The crystal lattices of -CD complexes with six TCAs (nortriptyline, amitriptyline, desipramine, imipramine, doxepin and maprotiline) are stabilized by the unison of host-guest N-HO, C/O-H, host-host O-HO, and guest-guest edge-to-face  interactions (Aree, 2020a (Aree, , 2020b (Aree, , 2020c (Aree, , 2021 . On the contrary, without the guest-guest edge-to-face  interactions, the -CD-protriptyline complex in the monoclinic, space group P2 1 favors a herringbone structure (Aree, 2021) . Moreover, in the presence of chlorine in CPM, the crystal of -CD-CPM complex is further stabilized by host-guest O-HCl interactions (Aree, 2020c) .

The non-symmetric, more bulkier SSRIs induce the formation of -CD dimers to encapsulate their structures, attaining more energetically favorable 2:2 -CD-STL/FXT (1 and 2) and 2:1 -CD-PXT inclusion complexes and crystallizing in the less symmetric triclinic, P1 and monoclinic, P2 1 (Caira et al., 2003) , respectively. Note that the intermolecular  interactions between the adjacent A-rings of STL (1) and FXT (2) (Auffinger et al., 2004; Scholfield et al., 2013) .

In the 2:1 -CD-PXT complex (Caira et al., 2003) , the head-to-head (H2H) -CD dimers are stacked along twofold screw axis, parallel to the b-axis, giving rise to a screwchannel style stabilized by intermolecular O2/O3O2/O3 H-bonds (intradimer) and O6O6, N/O-HO/N H-bonds (interdimer), Fig. 5c and Table 2 . Note that the absence of intermolecular halogenhalogen interactions from the PXT complex is due to the entire inclusion of PXT fluorophenyl in the -CD dimeric cavity; fluorine atoms are nearby the O2-H/O3-H side and have no intermolecular interaction (Caira et al., 2003) . (2), both existing in the tail-to-tail (T2T) channel structure, and (c) 2:1 β-CD-PXT base in monoclinic, P2 1 , packing in the head-to-head (H2H) screw-channel structure (Caira et al., 2003) . β-CD macrocycles, STL, FXT and PXT molecules are shown with cyan wireframes, red, blue, and magenta space-filling models, respectively. Water molecules, chlorides and 

Weak intermolecular interactions including van der Waals, ••• and hydrogen bond interactions are vital for the formation and maintaining the thermodynamic stability of the CD inclusion complexes. Due to the high conformational flexibility and the varied structural components of SSRI drugs, it is worthwhile to evaluate the inherent thermodynamic stabilities of the -CD-SSRI inclusion complexes directly derived from X-ray analysis, as demonstrated here for both monomeric and dimeric -CD-STL/FXT complexes. The neutral SSRIs in complex with -CD in the gas phase are sufficient to give meaningful results as successfully established in the -CD-TCA inclusion complexes (Aree, 2020a (Aree, , 2020b (Aree, , 2020c (Aree, , 2021 . For the drugs without or with one H-bond donor and a few H-bond acceptors like SSRIs and TCAs, their complexes with -CD are primarily stabilized by host-guest C-H••• interactions. These weak intermolecular interactions contribute to the stabilization and interaction energies (E stb and E int ) of -7.99 to -17.37, -8.60 to -20.93 kcal mol -1 for -CD-SSRI (Table S7 ) and -4.22 to -8.37, -6.10 to -14.35 kcal mol -1 for -CD-TCA (Aree, 2020a (Aree, , 2020b (Aree, , 2020c (Aree, , 2021 .

Because the four monomeric -CD-STL and -CD-FXT #1, #2 inclusion complexes share the main moiety of drug (aromatic ring) that is embedded in the -CD cavity, they are commonly maintained in position by intermolecular C-H··· interactions (Fig. 6) . Further host-guest O3-H···N1X/N1Y interactions are established via the half-chair conformation of the B-ring of STL #1 and the propan-1-amine side chain of FXT #2. These intermolecular interactions yield the more energetically favorable complexes of -CD-STL #1 and -CD-FXT #2 with relative stabilization energies (E stb ) of 3.19 and 9.12 kcal mol -1 compared to the other monomeric complexes (Figs. 6, 1S and Tables S6, S7) . From monomeric to dimeric complexes, the 2:2 -CD-STL and 2:2 -CD-FXT complexes gain thermodynamic stability (E stb ) by -14.50 and -20.22 kcal mol -1 , respectively (Table S7 ). The dimeric -CD-FXT complex is 5.72 kcal mol -1 more energetically stable than the dimeric -CD-STL complex because the former is stabilized by intermolecular F

whereas the latter is maintained by intermolecular Cl•••Cl interactions (Table S7) . By contrast, the -CD-PXT complex has a different host-guest stoichiometric ratio of 2:1, allowing one -CD dimer to encapsulate one hairpin-like PXT molecule with self ··· interactions, without halogen···halogen interactions (Caira et al., 2003) . 

Previously, we point out that TCAs (particularly CPM) exhibit conformationally flexibility while they are bound to protein binding pockets for their pharmacological activity (Aree, 2020c) . Because SSRIs are much more flexible than TCAs, it is worthwhile to gain for (S)-FXT, and N1-C6, C18-C23 for (3S,4R)-PXT (Fig. 7a,b,c) . If all SSRI non-H atoms are included in the calculation, the rms fits are several times larger and increased to ~3 Å.

The structural comparisons described below indicate the SSRI conformational changes induced each other by SERT for optimal binding with the pocket therein (Tavoulari et al., 2009) . in Scheme 1). The corresponding rms fit for each structure pair is indicated by nearby distance. STL HCl #2 in the -CD cavity (1), STL HCl form I (Caruso et al., 1999) , form II (Ravikumar et al., 2006) , STL freebase (He et al., 2010) are similar with rms fits in the range 0.421-0.524 Å (Fig. 7a) . STL HCl #1 and #2 embedded in the -CD dimeric cavity (1) are rather different, rms fit 0.936 Å. This is due mainly to the distinct B-ring conformations, i.e., half chair vs. boat forms, as discussed in Section 3.2. On the contrary, a greater span of rms fits (0.491-0.854 Å) is observed when STL HCl #2 (1) is compared to other STL molecules in various protein binding sites including bacterial leucine transporter (LeuT; Zhou et al., 2009 ), human leucine biogenic leucine transporter (LeuBAT; Wang et al., 2013) , human serotonin transporter (SERT; Coleman and Gouaux, 2018) , and ebolavirus glycoprotein (Ren et al., 2018) , Fig. 7a . Obviously, STL in complex with SERT (Coleman and Gouaux, 2018 ) is most distinct from STL encapsulated in the CD cavity for its optimal binding to protein and pharmacological function. This is indicated by differences in structural parameters listed in Table S5a . The B-C ring centroid distance and torsion angle C11-C4-C10-C5 are 3.865 Å, -70.7° for the SERT-STL complex (Coleman and Gouaux, 2018), and 4.122-4.287 Å, -33.8° to -47.9° for the uncomplexed STL and STL in complex with other proteins.

For FXT disregarding the propan-1-amine side chain, FXT nitrate (Carvalho et al., 2016a) , FXT entrapped in carrier cavity (2) and FXT bound to LeuT protein (Zhou et al., 2009) , FXT molecules in varied lattice environments are similar with small rms fits, 0.212-0.325 Å (Fig. 7b) . The corresponding structural parameters including the A vs. B rings interplanar angle, the A-B ring centroid distance and torsion angle C12-C7-C13-O1 are: 78.0°, 4.665 Å, -54.3° for FXT--CD (average) and 83.8-86.0°, 5.015-5.081 Å, -16.6° to -46.8° for others (Tables 1 and S5b ).

Considering merely the PXT phenylpiperidine moiety (A-B-rings), one PXT base embedded in the -CD dimeric cavity (Caira et al., 2003) , two PXT in HBr salt form (Carvalho et al., 2016b) , and several PXT in complex with various proteins including LeuBAT (Wang et al., 2013) , SERT (Coleman et al., 2016; Coleman and Gouaux, 2018) , cytochrome P450 2B4 (Shah et al., 2013) , human G protein-coupled receptor kinase (Thal et al., 2012; Homan et al., 2014) , and ebolavirus glycoprotein (Ren et al., 2018) are all similar with rms fits of 0.072-0.326 Å (Fig. 7c) . Note that PXT HBr·0.5H 2 O (Carvalho et al., 2016b) is considered because it is isostructural to PXT HCl·0.5H 2 O (Ibers, 1999) and more accurately determined. The structural parameters including the B vs. C rings interplanar angle, the A-B, A-C ring centroid distances and torsion angle C7-O8-C9-C10 are: 9.0°, 4.347, 6.100 Å, -9.9° for the .5° for others (Tables 1 and S5c ). Note that the 1,3benzodioxole moiety (C-D-rings) connected to CH 2 -O group makes the difference among various PXT, in addition to the distinct D-ring conformations, planar vs. envelope forms (discussed in Section 3.2).

Depression, a global mental illness, became considerably worse due to the COVID-19 pandemic. Selective serotonin reuptake inhibitors (SSRIs) are first-line medications to effectively treat depression. SSRIs are equipotent to the classical drugs, tricyclic antidepressants (TCAs), but have fewer side effects. Cyclodextrins (CDs) are encapsulating agents for reducing side effects and improving drug bioavailability. Because SSRIs have variances in molecular constituents and structures without internal symmetry, their complexes with CDs and experimental findings concerning host-guest stoichiometric ratios, inclusion structures and intermolecular interactions are still rather controversial and lack the atomic details. We therefore complete an integrated structural chemistry study through single-crystal X-ray diffraction and DFT full-geometry optimization of the -CD-SSRI inclusion complexation. Here, we demonstrate the -CD encapsulation of sertraline (STL) HCl

(1) and fluoxetine (FXT) HCl (2), in comparison to the reported complex of paroxetine (PXT) base (Caira et al., 2003) .

Returning to the hypotheses posed in the introduction, it is now possible to state that Xray analysis revealed the 2:2 -CD-STL/FXT complexes with two drug molecules inserting their halogen-bearing aromatic ring in the -CD dimeric cavity. 1 and 2 are stabilized by the interplay of intermolecular O2-HN1-HO3 H-bonds, C3/C5-H and halogenhalogen interactions. On the other hand, the 1:1 -CD-TCA complexes having an exclusive inclusion mode of the aromatic ring (Aree, 2020a (Aree, , 2020b (Aree, , 2020c (Aree, , 2021 and the 2:1 -CD-PXT complex having a total inclusion (Caira et al., 2003) , they are commonly stabilized by hostguest C3/C5-H interactions. The inherent stabilization energies of both complexes evaluated using DFT energy-minimization in vacuum suggest the improved thermodynamic stabilities of drugs upon CD inclusion complexation. Moreover, the SSRI conformational flexibilities facilitate their pharmacological functions, as perceived from the insightful structural comparison of the drugs in free salt form, in carrier (CD) cavity and in complex with proteins. This work has gone a long way toward advancing our understanding at atomic level of the -CD encapsulation of antidepressants for an effective application of CDs in drug delivery system. The continuation study on other two key SSRIs, citalopram, and escitalopram (the most selective SSRI), is in progress, providing an integral picture of the -CD-SSRI inclusion complexation in the coming future.

Highlights  We investigate the -CD-SSRI complexes by X-ray analysis and DFT calculation  The -CD encapsulation of sertraline (STL) HCl [1] and fluoxetine (FXT) HCl [2]

 Host-guest molar ratios of 1, 2 and the paroxetine base complex are 2:2 and 2:1  In crystals, 1 and 2 are more stabilized by •••and halogen•••halogen interactions  SSRIs in the CD cavity are more stable and hence their bioavailability is improved

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Thammarat Aree: Funding acquisition, Conceptualization, Methodology, Formal analysis, Writing-Original draft preparation, Writing-Reviewing and Editing the manuscript

This research was made possible by a grant from the Ratchadapisek Sompoch Endowment Fund, Chulalongkorn University (RCU_H_64_031_23).

Supplementary data to this article can be found online at https://dx.doi.org/10.1016/ j.ijpharm.2021.xxxxxx.

☐ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.☒ The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Thammarat Aree reports financial support was provided by Chulalongkorn University Department of Chemistry.