Painkiller Isoxicam and Its Copper Complex Can ... - ACS Publications

Article pubs.acs.org/JPCB

Painkiller Isoxicam and Its Copper Complex Can Form Inclusion Complexes with Different Cyclodextrins: A Fluorescence, Fourier Transform Infrared Spectroscopy, and Nuclear Magnetic Resonance Study Sathi Goswami, Anupa Majumdar,‡ and Munna Sarkar* Chemical Sciences Division, Saha Institute of Nuclear Physics, 1/AF Bidhannagar, Kolkata - 700064, India S Supporting Information *

ABSTRACT: The interaction of a painkiller Isoxicam, belonging to the oxicam group of nonsteroidal anti-inflammatory drugs (NSAIDs) and its copper complex with different cyclodextrins (β-CD, γ-CD, HPβCD, and HPγCD), has been investigated in both solution and the solid state. Steady state and time-resolved fluorescence spectroscopy, fluorescence anisotropy, 1 H NMR, and FTIR spectroscopy are used. Both the drug and its copper complex form a host−guest inclusion complex with all CDs. Fluorescence spectroscopy is used to determine binding constants and stoichiometries of the host−guest complex. The strongest binding is seen for γ-CD. 1H NMR study showed that Isoxicam penetrates into the CD cavity from the more accessible wider side. For β- and γ-CD, Isoxicam showed one type of binding, i.e., formation of an inclusion complex, whereas, for HPβCD and HPγCD, it showed two types of binding, i.e., inclusion in the CD cavities and interaction with the outer surface of the CD molecules mainly near the hydroxy propyl group. Deeper penetration occurred into the larger diameter cavity of γ-CD and HPγCD compared to β-CD and HPβCD. From FTIR and 1H NMR study, it is seen that predominantly the π-electron-rich benzene part of the drug and its complex penetrate into the host cavity.

1. INTRODUCTION Cyclodextrins (CDs) act as a good vehicle for drug delivery, but to be such a vehicle, they need to form a host−guest inclusion complex with the drugs to be delivered as the guest. Cyclodextrins are a class of cyclic oligosaccharides made up of 6−12 α-D-glucopyranose monomers, which are connected at 1 or 4 carbon atoms. They are characterized by an inverted bucketlike structure or a truncated cone.1 From X-ray structures of CDs, it appears that the secondary hydroxyl groups (attached on C2 and C3) are located on the wider edge of the ring, while the primary hydroxyl groups (attached on C6) are on the other edge, i.e., on the narrower side of the torus.2 The −CH groups carrying the H-1, H-2, and H-4 protons are located on the exterior of the molecule, and the hydroxyl groups are oriented to the cone exterior, which makes the external faces of CDs decidedly hydrophilic and soluble in water. However, the interior of the cavity is relatively hydrophobic and is lined by two rings of −CH groups (H-3 and H-5), a ring of glucosidic oxygens (O-4), with H-6 located externally near the cavity3 (Figure 1). The size of the cavity increases with increasing number of α-D-glucopyranose units. CDs with six, seven, and eight α-D-glucopyranose units are known as α-, β-, and γ-CD, respectively. The cavities of CDs allow them to form inclusion complexes with several molecules with varying sizes. Encapsulation of different guest molecules in CD hosts, particularly drugs, serves as a good tool for delivering them to their targets and is also © 2017 American Chemical Society

Figure 1. General structure of cyclodextrins.

known to increase their efficacies, solubility, dissolution rate, etc. CDs forming inclusion complexes with some drugs enhance their stability, decrease toxicity, control drug release, and improve bioavailability.4,5 For these reasons, CDs are used as a good drug delivery agent.6,7 Received: June 9, 2017 Revised: August 14, 2017 Published: August 14, 2017 8454

DOI: 10.1021/acs.jpcb.7b05649 J. Phys. Chem. B 2017, 121, 8454−8466

Article

The Journal of Physical Chemistry B

transform infrared (FTIR) spectroscopy, and 1D 1H NMR were used to study the inclusion of Isx and [Cu(II)−(Isx)2] in CDs.

The driving forces for complexation have been attributed to hydrophobic interactions, van der Waals−London dispersion forces, steric factors, hydrogen bonds, etc.8−10 In aqueous solution, the relatively apolar CD cavity is occupied by water molecules which is energetically unfavorable, and therefore can be readily substituted by appropriate guest molecules which are less polar than water. Among the different types of CDs, α-CD is not suitable for many drugs because of its small cavity size, whereas β- and γ-CDs having wider cavity size are suitable for a wide range of guest molecules. Inclusion complexes of some alkylated derivatives of β- and γ-CD such as 2-hydroxypropyl-β-CD (HPβCD) and 2-hydroxypropyl-γ-CD (HPγCD) have attracted growing interest due to their improved complexing ability, greater water solubility, and less toxicity than β- and γ-CD.11 These inclusion complexes can be formed either in solution or in the crystalline state. The formation of host/guest complexes in the solid state effectively protects the compounds against some type of reactions (e.g., oxidation, hydrolysis) and decreases their sublimation and volatility. In this work, we have probed whether a painkiller Isoxicam (Isx) (Figure 2a) belonging to the oxicam group of nonsteroidal

2. MATERIALS AND METHODS Isoxicam (4-hydroxy-2-methyl-N-[5-methyl-3-isoxolyl-2H-1,2benzothiazine-3-carboxamide-1,1-dioxide]) was purchased from Sigma chemicals and was used without any further purification. As the drug is sparingly soluble in aqueous solutions, 0.5 mM stock solution was prepared using DMSO (Spectrochem, India), and was further diluted by the 10 mM Tris−HCl buffer at pH 7.4 (Merck, Germany) to achieve the desired concentration of 30 μM which was kept constant for all experiments. In all working solutions, the concentration of DMSO did not exceed 3% v/v. All CDs were purchased from Sigma-Aldrich. Stock solutions of all CDs (15 mM) were prepared with the same buffer. Ethanol was purchased from Spectrochem, India. All other solvents (methanol, n-propanol, and n-butanol) were purchased from Merck, Germany. All of the solvents were spectroscopic grade and were used without further distillation. 2.1. Synthesis of the Cu(II)−NSAID Complex and Its Characterization. The synthesis of the copper complex of Isx was performed according to the following literature.17 To synthesize this complex, copper acetate was bought from SRL (Sisco Research Laboratories Private Limited, India) and spectroscopic grade ethanol was purchased from Spectrochem (India) and both were used without further purification. At first, NSAID and copper acetate were dissolved in hot ethanol (60 °C) in two separate beakers such that the concentration of copper acetate:NSAIDs is 1:2. When both NSAID and copper acetate fully dissolved in ethanol, they were mixed in a round bottomed flask and stirred under reflux for 2 h to synthesize [Cu(II)− (Isx)2]. A pale green solid was precipitated which was filtered off and then washed thrice with hot ethanol (60 °C). These complexes were then dried in vacuum desiccators on anhydrous calcium chloride. Theses complexes were characterized by several methods before further use. 2.1.1. Electron Paramagnetic Resonance (EPR). For the characterization of the [Cu(II)−(Isx)2] complex, EPR spectra

Figure 2. Chemical structures of Isx and [Cu(II)−(Isx)2].

anti-inflammatory drugs (NSAIDs) and its copper complex [Cu(II)−(Isx)2] (Figure 2b) can form inclusion complexes with a variety of CDs (β-CD, γ-CD, HPβCD, and HPγCD) at a physiological pH of 7.4. The drugs belonging to NSAIDs are widely used as anti-inflammatory, antipyretic, and analgesic agents. Besides their principal functions, they also show several alternate functions. Many of them are found to exhibit chemoprevention and chemo-suppression effects on different cancer cell lines such as breast cancer,12 lung cancer,13 colon cancer,14 etc. Also, it is already shown that the coordination of copper with some NSAIDs greatly enhances their activities. The Cu(II)−NSAID complexes as compared to the free drugs show lesser toxicity with reduced side effects. Some Cu(II)−NSAID complexes show enhanced anticancer effects over the bare drugs which has been attributed to their DNA binding ability15 that leads to their epigenomic/genomic level interactions.16 Hence, encapsulating them in a suitable host could pave the way to better delivery and enhanced efficacy. The inclusion complexes of various drugs including different NSAIDs with host CDs are strongly dependent on the pH of the environment. We have kept the working pH at 7.4 which makes the results of this study physiologically relevant such that these host−guest systems can be used for drug delivery. It should be mentioned that, while Isx is an anion at this pH, its copper complex as well as the host CDs remain unaffected. It should be mentioned that [Cu(II)−(Isx)2] is in a square planar geometry. This could facilitate the formation of inclusion complexes with CDs as hosts. Steady state and time-resolved fluorescence, fluorescence anisotropy, Fourier

Table 1. Nature of Transition of Isx and [Cu(II)−(Isx)2] at pH 7.4 Isoxicam solvent (pH 7.4)

ET(30)

absorption maximum

H2O methanol ethanol n-propanol n-butanol

63.1 55.5 51.9 50.7 50.2

346.5 352 354.5 355 357

[Cu(II)(Isx)2]

nature of transition

absorption maximum

nature of transition

n−π*

341.5 348.5 349.5 353.5 355

n−π*

Table 2. Fluorescence Quantum Yields (Φf) of Isx and [Cu(II)−(Isx)2] at pH 7.4 quantum yield (Φf) ET(30) H2O methanol ethanol n-propanol n-butanol 8455

63.1 55.5 51.9 50.7 50.2

Isx (pH 7.4) −3

0.16 × 10 0.35 × 10−3 0.37 × 10−3 0.38 × 10−3 0.39 × 10−3

[Cu(II)−(Isx)2] (pH 7.4) 0.17 × 10−3 0.30 × 10−3 0.31 × 10−3 0.32 × 10−3 0.34 × 10−3 DOI: 10.1021/acs.jpcb.7b05649 J. Phys. Chem. B 2017, 121, 8454−8466

Article


Figure 3. Fluorescence emission spectra of 30 μM Isx with increasing concentration of (a) HPβCD and (b) HPγCD. Fluorescence emission spectra of 30 μM [Cu(II)−(Isx)2] with increasing concentration of (c) HPβCD and (d) HPγCD. The concentrations of both CDs were varied from 0 to 14 mM. The spectra were taken after 1 h of incubation of drug and complex with corresponding CDs. All experiments were done at pH 7.4.

2.3. Absorption Spectroscopy. Absorption spectra were recorded by a JASCO V-650 spectrophotometer. A pair of 0.4 cm × 1 cm path length quartz cuvettes was used for the measurements. Nonaqueous solutions using different solvents were made alkaline by adding an equal volume of alkali (dilute NaOH) which is required to change the pH of the same volume of aqueous solution to pH 7.4. A series of aliquots were prepared which contained a constant concentration (30 μM) of guest molecule, i.e., drug or metal−drug complex with different solvents (alkaline in nature). Baseline correction was done using the corresponding solvents. All measurements were done at room temperature, i.e., at 298 K. 2.4. Fluorescence Spectroscopy. Fluorescence spectra were recorded using a Hitachi F-7000 spectrofluorimeter with a 0.4 cm × 1 cm path length quartz cell to avoid any blue edge distortion of the spectrum due to the inner filter effect.18 To study the inclusion complex formation, different aliquots were prepared containing a constant concentration of guest with increasing concentration of host, i.e., CD (0−14 μM) at pH 7.4. Each aliquot was incubated for 1 h before their spectra were recorded. This was done to allow sufficient time for inclusion complex formation. All measurements were recorded at room temperature, i.e., at 298 K. 2.5. Determination of Binding Parameters. The binding constants of guest (Isx and [Cu(II)−(Isx)2]) with the host (βCD, γCD, HPβCD, and HPγCD) due to the formation of inclusion complexes have been determined using a modified

were recorded in a JEOI JES-FA 200 ESR spectrophotometer. [Cu(II)−(Isx)2] (0.5 mM) was dissolved in DMSO, and the spectrum was taken at 298 K. The EPR spectrum (Figure S1a) suggests the presence of mononuclear Cu(II) and also the transgeometry of the [Cu(II)−(Isx)2]. This matches with the EPR spectrum of our previously synthesized batches.15 2.1.2. Fourier Transform Infrared Spectroscopy (FTIR). For further characterization of the Cu(II)−NSAID complex, infrared spectra of both Isx and [Cu(II)−(Isx)2] were recorded by Fourier transform infrared (FTIR) spectrometer (PerkinElmer model Spectrum 100). The spectra were found to match with the FTIR spectra of previously synthesized batches.15 The FTIR spectra of Isx and [Cu(II)−(Isx)2] are shown in Figure S1b. In the free Isx, a band appeared at 1546 cm−1 due to N−H bending vibration of the secondary amide (−CONH−) group. It shows a slight shift (11 cm−1) to higher wavenumber, i.e., 1557 cm−1 in the [Cu(II)−(Isx)2] complex. Also, a strong absorption peak appeared at 1624 cm−1 in the free Isx due to C−O stretching vibration of the −CONH− group. This band is shifted to 1557 cm−1 in the spectra of [Cu(II)−(Isx)2]. The above observations indicate the participation of the carbonyl oxygen atom in coordination. 2.2. Stability of the Cu(II)−NSAID Complex. Absorption spectra of the [Cu(II)−(Isx)2] complex were recorded in the working buffer (10 mM Tris−HCl buffer at pH 7.4) at different time intervals up to 180 min to see whether the complex is stable. The spectra were overlaid, and no significant changes were observed in the spectrum (Figure S2). 8456


Article


Figure 4. Benesi−Hildebrand plot for a 1:1 host−guest complex of (a) Isx and HPβCD, (b) Isx and HPγCD, (c) [Cu(II)−(Isx)2] and HPβCD, and (d) [Cu(II)−(Isx)2] and HPγCD. The concentrations of drug and complex were taken as 30 μM, and both CDs were varied from 0 to 14 mM. All experiments were done at pH 7.4.

Since the concentration of the fluorophore, i.e., the guest, is kept very low, we can reasonably assume that the fluorescence intensity is proportional to the concentration of the fluorophore. Then, the fluorescence intensity of the inclusion complex is given by

Table 3. Binding Constant Values of Isx and [Cu(II)−(Isx)2] with Different Cyclodextrins at pH 7.4 drug

pH

β-CD

γ-CD

HPβCD

HPγCD

Isx [Cu(II)−(Isx)2]

7.4 7.4

41.70 19.45

90.80 55.56

47.26 48.50

21.43 22.44

FG − CDn = Q [G−CDn]k1 19

Benesi−Hildebrand equation. The reaction between a guest (G) molecule with CD (CD) is given below

where Q is the quantum yield of the complex and k1 is an instrumental constant. Substituting eq 4 into eq 3 and rearranging eq 3 gives

G + nCD → G−CDn

C0/FG − CDn = [(1/CCDn) × (1/Kk1Q )] + (1/k1Q )

where n is the stoichiometry. The binding constant of the above association reaction is given by k = [G−CDn]/[G][CD]n

(5)

With increasing concentration of the host, there was an increase in fluorescence emission intensities despite the constant concentration of the guests. If F is the fluorescence intensity at a particular concentration of CD and F0 is the intensity in the absence of CD, then FG−CDn = F − F0 and eq 5 can be written as

(1)

Equation 1 can be rewritten as k = [G−CDn]/(C0 − [G−CDn])(CCD − [G−CDn])n (2)

C0/F − F0 = [(1/CCDn) × (1/Kk1Q )] + (1/k1Q )

where C0 and CCD are the initial concentrations of the guest molecule and CD, respectively. We have used Isx and [Cu(II)−(Isx)2] as the guest molecules. In all of our experiments, cylodextrin concentration was kept in a large excess (0−14 mM) with respect to the guest molecule (30 μM) such that CCD ≫ [G−CDn] and eq 2 can then be written as k = [G−CDn]/CCDn(C0 − [G−CDn])

(4)

(6)

Then, C0/F − F0 versus was plotted for n = 1, 2, 3, etc. The binding stoichiometry n corresponds to a linear plot, and the binding constant K was calculated as the ratio of the intercept to the slope. 2.6. Anisotropy Measurement. Fluorescence anisotropy measurements give information about the rigidity of the environment around a fluorophore. The anisotropy (r) was determined 1/CCDn

(3) 8457


Article


Figure 5. Changes in anisotropy of 30 μM Isx with increasing concentration of (a) HPβCD and (b) HPγCD. Changes in anisotropy of 30 μM [Cu(II)−(Isx)2] with increasing concentration of (c) HPβCD and (d) HPγCD. The concentrations of both CDs were varied from 0 to 14 mM. The spectra were taken after 1 h of incubation of drug and complex with corresponding CDs. All experiments were done at pH 7.4.

Table 4. Time-Resolved Fluorescence of Isx in the Presence and Absence of Different Concentrations of Cyclodextrins (HPβCD and HPγCD) sample (pH 7.4)

τ1 (ns)

Isx Isx + 6 mM HPβCD Isx + 6 mM HPγCD

B1 (%)

τ2 (ns)

B2 (%)

τ3 (ns)

B3 (%)

χ2

τav

15.50

0.3 0.4 0.1

90.64 77.74 66.21

3.7 3.4 4.6

9.36 22.26 18.29

1.02 1.09 1.03

2.20 2.53 3.72

1.4

Table 5. Time-Resolved Fluorescence of [Cu(II)−(Isx)2] in the Presence and Absence of Different Concentrations of Cyclodextrins (HPβCD and HPγCD) sample (pH 7.4)

τ1 (ns)

B1 (%)

τ2 (ns)

B2 (%)

[Cu(II)−(Isx)2] + 6 mM HPβCD [Cu(II)−(Isx)2] + 6 mM HPγCD

3.0 0.7

10.58 18.19

0.4 0.1

89.42 61.49

(IVV − G × IVH) (IVV + 2 × G × IVH)

3.7

B3 (%)

χ2

τav

20.32

1.01 1.09

1.62 3.06

was done to allow sufficient time for inclusion complex formation. In our experiments, we have monitored the emission anisotropy of the drug−CD complex at pH 7.4 to see the extent of encapsulation of the drug/complex in CD. 2.7. Time-Resolved Fluorescence Measurements. The fluorescence lifetime measurements of Isx, [Cu(II)−(Isx)2], and their inclusion complexes with different CDs have been recorded using a Jobin Yvon Horiba picosecond time-correlated singlephoton counting (TCSPC) instrument. For every ligand, two different aliquots were prepared where the concentration of the drug and complex was taken as 60 μM and the CD concentration was varied as 0 and 6 mM. All aliquots were prepared at pH 7.4. The fluorescence intensity decay measurements for Isx and [Cu(II)−(Isx)2] at pH 7.4 were performed using a pulsed diode

using an inbuilt matched set of polarizer and analyzer using the following equation18 r=

τ3 (ns)

(7)

where IVV, IVH, IHV, and IHH are the emitted light intensities with vertical (V) and horizontal (H) polarizer/analyzer arrangements. Here, the first and second subscripts refer to the orientation of the polarizer and analyzer, respectively. G is the grating efficiency factor that measures the sensitivities of the detection system which is the ratio of IHV/IHH. A series of aliquots were prepared where the drug/complex concentration is kept constant with gradual increase of CD concentration (0 to 14 μM). Each aliquot was incubated for 1 h before their spectra were recorded. This 8458


Article


Figure 6. 1H NMR spectra of (a) βCD and (b) inclusion complex of Isx and βCD in DMSOd6.

dissolved, then the two solutions were mixed up and stirred for 24 h. Any undissolved amount of drug/complex was separated by filtration. Then, the solution was evaporated under reduced pressure to remove the solvent and dried in a vacuum to give the host−guest complex. 2.9. NMR Study of the Drug−CD Inclusion Complex. All 1 H NMR experiments were recorded on a Bruker Avance III 500 MHz spectrometer, equipped with a 5 mm SMART probe and in a shigemi tube (SHIGEMI Inc.) at 298 K using deuterated dimethyl sulfoxide (DMSO-d6) as solvent and tetramethylsilane (TMS) as an internal reference. The chemical shifts are reported in ppm relative to TMS. 2.10. FTIR Study of the Host−Guest Inclusion Complex. Infrared spectra of the solid inclusion complexes of drug or metal−drug complex with different CDs were recorded by a Fourier transform infrared (FTIR) spectrometer (PerkinElmer model Spectrum 100) by the conventional KBr pellet method. The samples were ground gently with anhydrous KBr in a mortar and pestle and then compressed to form a pellet. Each spectrum was the signal average of 10 scans in the range 400−4000 cm−1 at room temperature of 298 K.

laser with excitation at 340 nm. All experiments were performed at 20 °C. Ludox was used as a reference (τ = 0.0 ns). The instrument response functions (IRFs) of the 340 nm Nano-LEDs have a full width at half-maximum (fwhm) value of 1 ns. Depending on the ligand, the decay profiles were fitted to either biexponential or triexponential decay functions using IBH DAS 6.2 data analysis software using the following equation: 2or3

I(t ) = B0 +

⎡

t⎤ ⎥ ⎣ τn ⎦

∑ Bn exp⎢− n=1

(8)

B0 is the constant, Bn are the pre-exponential factors, and τn are the corresponding fluorescence lifetimes. The goodness of fit was determined by the reduced χ2 value. For each case, the reduced χ2 value is close to 1. Average lifetime values are calculated from the following equation τav =

B1τ12 + B2 τ2 2 + B3τ32 τ1 + τ2 + τ3

(9)

where τav is the average lifetime. 2.8. Preparation of the Drug−CD Inclusion Complex for NMR and FTIR Study. For FTIR and NMR studies, the inclusion complexes were formed in the solid state by mixing the guest and the host at 1:1 molar ratio. Equimolar CD and drug/ complex were dissolved in 1:3 water:ethanol and ethanol in two separate beakers. When both CD and drug/complex were fully

3. RESULTS AND DISCUSSION 3.1. Determination of the Nature of Electronic Transition and Fluorescence Quantum Yield of Isoxicam and Its Copper Complex. To identify the nature of electronic 8459


Article


Figure 7. 1H NMR spectra of (a) γCD and (b) inclusion complex of Isx and γCD in DMSOd6.

different CDs (βCD, γCD, HPβCD, and HPγCD) at pH 7.4. Figure S3 shows the fluorescence spectra of a constant concentration of 30 μM each of Isx and [Cu(II)−(Isx)2] with increasing concentration of βCD and γCD. Figure 3 shows the fluorescence spectra of the same guests with increasing concentration of HPβCD and HPγCD. The fluorescence intensities of both Isx and [Cu(II)−(Isx)2] increase gradually with increasing concentration of CDs, which implies that the drug and its copper complex move from the polar aqueous phase to a relatively nonpolar medium, as is expected from their quantum yield values. It indicates that both the drug and the complex get encapsulated as guests in the cavity of host CDs upon formation of inclusion complexes.21,22 From the spectra of Figure 3, plots of C0/F − F0 versus 1/CCDn were plotted for n = 1, 2, 3, etc. Only for n = 1, i.e., host−guest stoichiometry of 1:1, linear plots are obtained, as shown in Figure 4. The binding constants of host−guest inclusion complexes are determined using the modified Benesi−Hildebrand equation (eq 6), and the binding constant K is calculated as the ratio of the intercept to the slope. The corresponding plots of Isx and [Cu(II)−(Isx)2] with βCD and γCD as hosts are shown in Figure S4. The binding constant values for Isx and [Cu(II)−(Isx)2] with different CDs are given in Table 3. Both Isx and [Cu(II)−(Isx)2] show the highest binding constants with γCD but relatively

transition of both drug and its copper complex at physiological pH 7.4, we have used the shift in the absorption maxima in solvents with decreasing polarity. It is reported that the pKa value for the enolic −OH of Isx is 3.93.20 Hence, at pH 7.4, the population of the drug mainly exists in the anionic form. On the other hand, [Cu(II)−(Isx)2] exists in its neutral form as both the enolic −OH groups from the two Isx are involved in covalent bond formation with the metal. Table 1 shows the shifts in the absorption maxima of both Isx and [Cu(II)−(Isx)2] in alkaline solvents having different polarity as expressed by ET(30). It is seen that the absorption maxima of both Isx and [Cu(II)− (Isx)2] increase with decreasing polarity of the solvents, which is characteristic of n−π* transition. The fluorescence quantum yields of Isx and [Cu(II)−(Isx)2] in different solvents are calculated, and the values are given in Table 2. From the table, it is seen that the fluorescence quantum yield of both the drug and its complex increases with decreasing polarity of the solvents. However, in the case of water, the quantum yield is very low with respect to that seen in the primary alcohols. The above information is important to characterize the inclusion complexes with Isx and [Cu(II)−(Isx)2] as guest and different CDs as host. 3.2. Characterization of Inclusion Complexes by Fluorescence Spectroscopy. We have recorded the fluorescence spectra of Isx and [Cu(II)−(Isx)2] in the presence of 8460


Article


Figure 8. 1H NMR spectra of (a) HPβCD and (b) inclusion complex of Isx and HPβCD in DMSOd6.

weaker binding with HPγCD. This could be due to the larger cavity of γCD, which helps it to form an inclusion complex with guest molecules, but the bulky hydroxy propyl group of HPγCD could prevent the guests from approaching the host cavities to form inclusion complexes. The smaller cavities of both βCD and HPβCD result in weaker binding with the guests, as seen from Table 3. 3.3. Characterization of the Drug−CD Inclusion Complex by Fluorescence Anisotropy. Fluorescence anisotropy measurements were carried out with increasing CD concentration at pH 7.4, at which the drug mostly exists in the anionic form. From the figures (Figures 5 and S5), it is seen that there is an increase in anisotropy values of drug and complex with increasing CD concentration. After a certain CD concentration, the anisotropy value reaches saturation. Our results indicate that, for all host−guest partners, binding leads to inhibition of rotational mobility of the guest fluorophores. However, fluorescence anisotropy cannot differentiate between different modes of binding, viz., inclusion in the host cavity or binding to the host outer surface. 3.4. Time-Resolved Fluorescence Study of the Drug− CD Inclusion Complex. Time-resolved fluorescence decay profiles were recorded with Isx as guest and with HPβCD and HPγCD as host, and the lifetime values of fitted decay traces are shown in Table 4. The goodness of fits is given by chi square values. Free Isx shows biexponential decay with a short lifetime

component (τ = 0.3) having major contribution to the decay profile and a long lifetime component (τ = 3.7) are obtained. When Isx is added to 6 mM HPβCD, the short lifetime increases, whereas the long lifetime decreases. The average lifetime increases compared to free Isx, as is expected when binding occurs. In the case of 6 mM HPγCD, three lifetime components are obtained and the average lifetime is greater than the free drug. Table 5 shows the lifetime of [Cu(II)− (Isx)2] in the presence of both HPβCD and HPγCD. Timeresolved decay could not be recorded for free [Cu(II)−(Isx)2] due to its very weak fluorescence. However, here too biexponential decay is seen for HPβCD and triexponential decay for HPγCD. 3.5. Characterization of the Host−Guest Inclusion Complex by 1H NMR Spectroscopy. The different techniques that we have used to study the host−guest complexes cannot provide a clear answer about the type of complex. To further characterize the inclusion complexes and to identify the different modes of binding, viz., inclusion within the host cavity or external surface binding, we have used 1H NMR chemical shifts which give a clear distinction between inclusion and other possible external interactions. CD has six identifiable protons in the NMR spectrum: H1, H2, and H4 protons located at the outer surface of the CD cavity and H3 and H5 protons located in the inner surface of the cavity. If a guest molecule is incorporated into the CD cavity, the hydrogen atoms located in the inner surface of the 8461


Article


Figure 9. 1H NMR spectra of (a) HPγCD and (b) inclusion complex of Isx and HPγCD in DMSOd6.

cavity (H-3 and H-5) will be considerably shielded by the guest molecule and show a significant upfield chemical shift, whereas the chemical shifts of the hydrogen atoms on the outer surface (H-1, H-2, H-4, and H-6) will be unaffected or experience only a minimum shift.4,23 The NMR spectrum of free Isx is given in Figure S6. The NMR spectra of free CD and drug−CD inclusion complexes are given in Figures 6−9. To know the possible inclusion mode of drug−CD complexes, we compare the 1H NMR spectra of CDs in the presence and absence of Isx and the chemical shifts are listed in Table 6. Table 7 shows the chemical shifts of Isx protons in the presence and absence of different CDs. The protons located inside the β-CD and γ-CD cavities (H-3 and H-5) clearly undergo significant drug-induced chemical shift changes compared with those which are on the exterior of the torus. The chemical shift values for H-1, H-2, H-4, and H-6 are very small, which confirm that the drug only interacts with the inside of the cavity as is expected upon formation of an inclusion complex. In addition, for β-CD, the upfield shift of the H3 protons lying on the inner surface of the cavity at the secondary OH group side is most prominent followed by H5 protons located on the inner surface at the primary OH group side. The higher shielding effect seen on H3 proton with respect to H5 indicates that Isx preferentially

penetrates the torus from the more accessible wider side of the cavity where the secondary OH groups are located.24,25 Unlike β-CD, for γ-CD, similar upfield shifts of both H3 and H5 are seen. This indicates that, due to the wider cavity of γ-CD, deeper penetration of the drug affects H5 equally. In the case of HPβCD and HPγCD, all 1H’s show significant changes of chemical shift value after interaction with drugs. The clear upfield shift (shielding effect) of the signals of H-3 and H-5 protons has been attributed to the magnetic anisotropy effects in the CD cavity due to the inclusion of a π-electron-rich group of the drug into the host cavity.26 The only large group with π-electrons in the Isx is the benzene ring. The magnetic anisotropy of an aromatic nucleus results in an upfield 1H-chemical shift located above or below the π-electron cloud.3,25 In fact, these shifts could be the result of anisotropic shielding induced by the “ringcurrent” effect produced by the aromatic ring inside the CD macrocycle.25,27,28 Our results indicate that the benzene ring of Isx gets inserted into the host cavities. This will further be confirmed by the 1H NMR results of the drug. The outer protons (H-2, H-4, H-6, and −CH3) of both HPβCD and HPγCD show significant upfield changes of chemical shift, suggesting some partial interaction of the drug molecules with the outer surface of these CDs. Also, for HPβCD and HPγCD, of all the outer protons, H-6 and −CH3 shows maximum changes of chemical 8462


Article

The Journal of Physical Chemistry B Table 6. 1H-Chemical Shift (δ) Values Corresponding to Cyclodextrins (βCD, γCD, HPβCD, and HPγCD) in the Presence and Absence of Isx

a

CD protons

δCD (free)

δCD+Isx (complex)

Δδa

H-1 of β-CD H-2 of β-CD H-3 of β-CD H-4 of β-CD H-5 of β-CD H-6 of β-CD H-1 of γ-CD H-2 of γ-CD H-3 of γ-CD H-4 of γ-CD H-5 of γ-CD H-6 of γ-CD H-1 of HPβCD H-2 of HPβCD H-3 of HPβCD H-4 of HPβCD H-5 of HPβCD H-6 of HPβCD CH3 of HPβCD H-1 of HPγCD H-2 of HPγCD H-3 of HPγCD H-4 of HPγCD H-5 of HPγCD H-6 of HPγCD CH3 of HPγCD

4.762 (d) 3.363 (dd) 3.566 (t) 3.282 (t) 3.506 (m) 3.482 4.820 (d) 3.363 (dd) 3.559 (t) 3.282 (t) 3.479 (m) 3.459 5.001 (d) 3.425 (dd) 3.744 (dd) 3.327 (dd) 3.590 (m) 3.590 (dd) 1.042 (dd) 5.006 (d) 3.432 (dd) 3.749 (dd) 3.329 (dd) 3.627 (m) 3.533 (dd) 1.042 (dd)

4.763 (d) 3.363 (dd) 3.548 (t) 3.282 (t) 3.503 (m) 3.483 4.820 (d) 3.363 (dd) 3.547 (t) 3.282 (t) 3.468 (m) 3.460 5.005 (d) 3.391 (dd) 3.679 (dd) 3.320 (dd) 3.540 (m) 3.540 (dd) 0.999 (dd) 5.051 (d) 3.391 (dd) 3.683 (dd) 3.319 (dd) 3.563 (m) 3.468 (dd) 0.999 (dd)

+0.001 0.000 −0.018 0.000 −0.003 +0.001 0.000 0.000 −0.012 0.000 −0.011 +0.001 +0.004 −0.034 −0.065 −0.007 −0.050 −0.050 −0.043 −0.045 −0.041 −0.066 −0.010 −0.064 −0.065 −0.043

xazole CH located at 6.596 (s), and N−CH3 located at 2.750 (s). In the presence of CD, the chemical shift values due to the benzene H’s of Isx were changed and split into two groups, one shifted upfield and the other downfield. The upfield shift is probably due to a variation in local polarity when the protons are inside the CD cavity3,26,30 and indicates weaker interactions with hydrogen atoms (shielding effect due to van der Waals forces between the drug and carbohydrate chains).3 A downfield displacement of the drug protons indicates that they are close to an electronegative atom, like oxygen.31 These results further establish that the drug enters the CD cavities by the benzene ring. From the table, it is also seen that the signals due to isoxazole CH and N−CH3 group are not affected after the complex formation with CDs, indicating that the isoxazole ring of Isx is not incorporated into the CD cavity and ruling out the possibility of this ring being incorporated in another CD molecule to make the host:guest stoichiometry 2:1. This is consistent with our fluorescence data. Thus, from overall 1H NMR studies, it is concluded that Isx forms inclusion complexes with all of the CDs by incorporating its benzene part into the CD cavity from the wider side. For β- and γ-CD, Isx has only one binding mode, i.e., formation of an inclusion complex, whereas, for HPβCD and HPγCD, the drug shows two types of binding modes, viz., inclusion in the CD cavities and interaction with the outer surface of the CD molecules mainly near the hydroxy propyl group. 3.6. Characterization of the Host−Guest Inclusion Complex by FTIR Spectroscopy. From NMR study, we have already concluded that Isx forms an inclusion complex with different CDs and have identified different binding modes. However, 1H NMR is not applicable for [Cu(II)−(Isx)2] as Cu(II) is paramagnetic. Therefore, to confirm the formation of an inclusion complex for [Cu(II)−(Isx)2], we have done FTIR spectroscopy and have compared the results with Isx−CD complexes. The FTIR spectra of β-CD, γ-CD, HPβCD, HPγCD, and their corresponding conjugated complexes with Isx, [Cu(II)−(Isx)2] (1:1 molar ratio), are shown in Figure 10. The FTIR spectra of the free CDs (all four) show intense broad absorption bands around 3412−3400 cm−1 corresponding to the free −OH stretching vibration (for β-CD, 3400 cm−1; γ-CD, 3400 cm−1; HPβCD, 3412 cm−1; HPγCD, 3401 cm−1). This band of CD was found to be narrowed upon formation of the inclusion complex along with a shift to the higher wavenumbers which is a good indicator for the formation of a host− guest inclusion complex. These changes are general characteristics of host−guest inclusion complexes with CDs as hosts.9,32 The vibration of the −CH and −CH2 groups appeared in the region 2934−2921 cm−1 (for β-CD, 2921 cm−1; γ-CD, 2932 cm−1; HPβCD, 2932 cm−1; HPγCD, 2934 cm−1). Another large band assigned to the C−O−C stretching vibration occurred between 1159 and 1154 cm−1 (for β-CD, 1158 cm−1; γ-CD, 1159 cm−1; HPβCD, 1154 cm−1; HPγCD, 1157 cm−1). There are significant changes in these bands when CDs form complexes with drug and its copper complex. Table 8 shows some changes (increase and decrease) of frequency values between free CD and the host− guest complexes. It is seen that, in some cases, the frequency of host (CD)−guest (drug or complex) complex increases and in some cases decreases. The increment is due to the insertion of an electron-rich group into the CD cavity which increases the electron density of the cavity, leading to an increase in frequency.9,33 Such an electron-rich group in the Isx and [Cu(II)−(Isx)2] is the benzene ring. Therefore, the increment of frequency may be explained by the fact that both Isx and

Δδ = δCD+Isx (complex) − δCD (free).

Table 7. 1H-Chemical Shift (δ) Values of Isx in the Presence and Absence of Different Cyclodextrins (βCD, γCD, HPβCD, and HPγCD) δ (ppm) drug/drug−CD

CH of isoxazole

N−CH3

4H (4 aromatic H’s of benzene ring)

Isx βCD + Isx γCD + Isx HPβCD + Isx HPγCD + Isx

6.596 (s) 6.597 (s) 6.596 (s) 6.596 (s) 6.598 (s)

2.750 (s) 2.746 (s) 2.747 (s) 2.747 (s) 2.744 (s)

7.799−7.984 7.809−7.983 7.811−7.983 7.813−7.983 7.813−7.984

shift values. These results indicate that the outside interactions particularly occur near the H-6/−CH3 groups of the large hydroxy propyl group at the C-6 position. Even for the hydroxy propyl derivatives, the changes in the chemical shift of H-3 are larger than H-5, indicating that in the case of an inclusion complex the drug enters the host cavity from the wider side.25,29 For HPγCD, the values for H-3 and H-5 are close due to deeper penetration of Isx into the host because of the wider diameter of HPγCD. From the above results, we can clearly say that, for β-CD and γ-CD, the drug only forms an inclusion complex, whereas, for HPβCD and HPγCD, two types of binding modes, viz., inclusion complex and surface binding near the hydroxy propyl group, were observed. The 1H NMR chemical shifts of drug were also investigated in the presence and absence of different CDs, and the chemical shifts are listed in Table 7. The NMR spectrum of free Isx comes at δ = 7.799−7.984 (m, 4 aromatic H’s of benzene ring), iso8463


Article


Figure 10. FTIR spectra of (a) β-CD (black), inclusion complex of Isx with β-CD (red), and inclusion complex of [Cu(II)−(Isx)2] with β-CD (blue); (b) γ-CD (black), inclusion complex of Isx with γ-CD (red), and inclusion complex of [Cu(II)−(Isx)2] with γ-CD (blue); (c) HPβCD (black), inclusion complex of Isx with HPβCD (red), and inclusion complex of [Cu(II)−(Isx)2] with HPβCD (blue); and (d) HPγCD (black), inclusion complex of Isx with HPγCD (red), and inclusion complex of [Cu(II)−(Isx)2] with HPγCD (blue). All of the spectra were taken at 298 K.

Table 8. Changes in Band Positions when Isx and [Cu(II)−(Isx)2] Form Inclusion Complexes with Different Cyclodextrins β-CD β-CD + Isx β-CD + [Cu(II)−(Isx)2] γ-CD γ-CD + Isx γ-CD + [Cu(II)−(Isx)2] HPβCD HPβCD + Isx HPβCD + [Cu(II)−(Isx)2] HPγCD HPγCD + Isx HPγCD + [Cu(II)−(Isx)2]

−OH str. (cm−1)

−CH str. (cm−1)

C−O−C str. (cm−1)

−OH bending (cm−1)

3400 3413 3447 3400 3413 3435 3412 3436 3435 3401 3435 3436

2921 2933 2920 2932 2932 2974 2932 2973 2973 2934 2973 2973

1158 1157 1121 1159 1160 1169 1154 1161 1169 1157 1166 1169

1028 1031 1040 1028 1028 1032 1039 1048 1034 1032 1031 1032

A sharp band appeared due to −OH bending vibration (for β-CD, 1028 cm−1; γ-CD, 1028 cm−1; HPβCD, 1039 cm−1; HPγCD, 1032 cm−1). The changes in frequency of −OH bending vibration between uncomplexed γ-CD/HPγCD and their inclusion complexes with drug/complex are insignificant compared to the β-CD/HPβCD. This is because, due to the wider torus diameter of γ-CD/HPγCD, the −OH groups at the

[Cu(II)−(Isx)2] form an inclusion complex with CDs by incorporating their benzene ring into the CD cavity. The decrease in the frequency between the inclusion complex and its constituent molecule is due to the changes in the microenvironment which lead to the formation of hydrogen bonding and the presence of van der Waals forces during their interaction to form the inclusion complex.9,10 8464


Article

The Journal of Physical Chemistry B ORCID

rim of the torus do not strongly interact with the guest like in the case of host β-CD/HPβCD. Thus, changes in the band positions in the FTIR spectra clearly prove the formation of the host− guest inclusion complexes for both Isx and its copper complex as guests with all types of CDs as hosts. From fluorescence spectroscopy and anisotropy study, it is seen that both Isx and [Cu(II)−(Isx)2] form an inclusion complex with four different cyclodextrins with a 1:1 stoichiometry (host:guest). The strongest binding occurs with γ-CD, due to its higher cavity diameter compared to β-CD. In spite of the higher cavity diameter, HPγCD shows weak binding with the guests. Changes in fluorescence intensity can sense only inclusion of guest molecules in the host CD cavity which is more apolar than the aqueous environment. Binding of the guests to the hosts, which do not change the microenvironment around the guests, cannot be sensed by this technique. Changes in fluorescence anisotropy give information on the total binding of the guests to the hosts, irrespective of the binding modes. From the 1H NMR study, it is seen that guest Isx mainly penetrates into the torus of the host molecules from the more accessible wider side of the cavity. The drug shows deeper penetration into the wider cavity of γ-CD and HPγCD with respect to β-CD and HPβCD. For β- and γ-CD, the drug shows one type of binding, i.e., formation of an inclusion complex, whereas, for HPβCD and HPγCD, it shows two types of binding, i.e., inclusion in the CD cavities and interaction with the outer surface of the CD molecules mainly near the hydroxy propyl group. FTIR results showed that [Cu(II)−(Isx)2] can form inclusion complexes with all CDs. From FTIR and 1H NMR study, we conclude that, for Isx and [Cu(II)−(Isx)2], the benzene part penetrates into the CD cavity.

Munna Sarkar: 0000-0003-4061-7163 Present Address ‡

A.M.: Department of Biological Sciences, Indian Institute of Science Education and Research (IISER), Mohali, Sector 81, S.A.S Nagar, P.O. Manauli, Mohali-140306, Punjab, India. Notes

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This work was financially supported by BARD (Biomolecular Assembly, Recognition and Dynamics) project of Saha Institute of Nuclear Physics (SINP), Kolkata, funded by the Department of Atomic Energy, Government of India (12-R&D-SIN-5.04-0103). S.G. acknowledges SINP for her fellowship. We acknowledge Mr. Barun Majumder of Bose Institute for his help in operating the NMR. The EPR study of the copper complex was done at the central facility of the Inorganic Chemistry Department of Indian Association for the Cultivation of Sciences, Kolkata, India.

■

4. CONCLUSIONS Isx and [Cu(II)−(Isx)2] form inclusion complexes with the four CDs, viz., β-CD, γ-CD, HPβCD, and HPγCD, with a 1:1 stoichiometry. For both Isx and [Cu(II)−(Isx)2], the benzene part penetrates into the CD cavity. Deeper penetration occurred for Isoxicam into the larger diameter cavity of γ-CD and HPγCD compared to β-CD and HPβCD. The drug shows different modes of binding with the CDs. For β- and γ-CD, the drug shows one mode of binding, i.e., formation of an inclusion complex, whereas, for HPβCD and HPγCD, it shows two modes of binding, i.e., inclusion in the CD cavities and interaction with the outer surface of the CD molecules mainly near the hydroxy propyl group.

■

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.7b05649. EPR (Figure S1a) and FTIR spectra (Figure S1b) for the characterization of the complex; absorption spectra to check the stability of the complex (Figure S2); fluorescence spectra (Figure S3) and Benesi−Hildebrand plot (Figure S4) to calculate the binding constant; fluorescence anisotropy spectra (Figure S5) and NMR (Figure S6) to know the mode of binding (PDF)

■

REFERENCES

(1) Sambasevam, K. P.; Mohamad, S.; Sarih, N. M.; Ismail, N. A. Synthesis and Characterization of the Inclusion Complex of βCyclodextrin and Azomethine. Int. J. Mol. Sci. 2013, 14, 3671−3682. (2) Abarca, R. L.; Rodríguez, F. J.; Guarda, A.; Galotto, M. J.; Bruna, J. E. Characterization of Beta-Cyclodextrin Inclusion Complexes Containing an Essential Oil Component. Food Chem. 2016, 196, 968−975. (3) Veiga, F. J. B.; Fernandes, C. M.; Carvalho, R. A.; Geraldes, C. F. G. C. Molecular Modelling and 1H-NMR: Ultimate Tools for the Investigation of Tolbutamide: β-Cyclodextrin and Tolbutamide: Hydroxypropyl-β-Cyclodextrin Complexes. Chem. Pharm. Bull. 2001, 49, 1251−1256. (4) Szejtli, J. Cyclodextrin Technology; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1988; pp 1−78. (5) Uekama, K.; Hirayama, F.; Irie, T. Cyclodextrin Drug Carrier Systems. Chem. Rev. 1998, 98, 2045−2076. (6) Maciollek, A.; Munteanu, M.; Ritter, H. New Generation of Polymeric Drugs: Copolymer from NIPAAM and Cyclodextrin Methacrylate Containing Supramolecular-Attached Antitumor Derivative. Macromol. Chem. Phys. 2010, 211, 245−249. (7) Maatz, G.; Maciollek, A.; Ritter, H. Cyclodextrin-Induced HostGuest Effects of Classically Prepared Poly (NIPAM) Bearing Azo-Dye End Groups. Beilstein J. Org. Chem. 2012, 8, 1929−1935. (8) Szejtli, J. Introduction and General Overview of Cyclodextrin Chemistry. Chem. Rev. 1998, 98, 1743−1754. (9) Sambasevam, K. P.; Mohamad, S.; Sarih, N. M.; Ismail, N. A. Synthesis and Characterization of the Inclusion Complex of βCyclodextrin and Azomethine. Int. J. Mol. Sci. 2013, 14, 3671−3682. (10) Hamdi, H.; Abderrahim, R.; Meganem, F. Spectroscopic Studies of Inclusion Complex of β-Cyclodextrin and Benzidine Diammonium Dipicrate. Spectrochim. Acta, Part A 2010, 75, 32−36. (11) Loftsson, T.; Duchêne, D. Cyclodextrins and their Pharmaceutical Applications. Int. J. Pharm. 2007, 329, 1−11. (12) Retsky, M.; Rogers, R.; Demicheli, R.; Hrushesky, W. J.; Gukas, I.; Vaidya, J. S.; Baum, M. Forget, P.; DeKock, M.; Pachmann, K. NSAID Analgesic Ketorolac used Perioperatively may Suppress Early Breast Cancer Relapse: Particular Relevance to Triple Negative Subgroup. Breast Cancer Res. Treat. 2012, 134, 881−888. (13) Ogawa, F.; Amano, H.; Ito, Y.; Matsui, Y.; Hosono, K.; Kitasato, H.; Satoh, Y.; Majima, M. Aspirin Reduces Lung Cancer Metastasis to Regional Lymph Nodes. Biomed. Pharmacother. 2014, 68, 79−86. (14) Rigas, B.; Tsioulias, G. J. The Evolving Role of Nonsteroidal AntiInflammatory Drugs in Colon Cancer Prevention: a Cause for Optimism. J. Pharmacol. Exp. Ther. 2015, 353, 2−8.

AUTHOR INFORMATION

Corresponding Author

*Phone: +91-33-2337-5345 to 49. Fax: +91-33-2337-4637. E-mail: [email protected]. 8465


Article


trofluorimetric Determination of Trace Gallium. Talanta 2006, 68, 575−580.

(15) Goswami, S.; Ray, S.; Sarkar, M. Spectroscopic Studies on the Interaction of DNA with the Copper Complexes of NSAIDs Lornoxicam and Isoxicam. Int. J. Biol. Macromol. 2016, 93, 47−56. (16) Goswami, S.; Sanyal, S.; Chakraborty, P.; Das, C.; Sarkar, M. Interaction of a Common Painkiller Piroxicam and Copper-Piroxicam with Chromatin Causes Structural Alterations Accompanied by Modulation at the Epigenomic/Genomic Level. Biochim. Biophys. Acta, Gen. Subj. 2017, 1861, 2048−2059. (17) Cini, R.; Tamasi, G.; Defazio, S.; Hursthouse, M. B. Unusual Coordinating Behavior by Three Non-Steroidal Anti-Inflammatory Drugs from the Oxicam Family Towards Copper (II). Synthesis, X-ray Structure for Copper (II)−Isoxicam, − Meloxicam and − CinnoxicamDerivative Complexes and Cytotoxic Activity for a Copper (II)− Piroxicam Complex. J. Inorg. Biochem. 2007, 101, 1140−1152. (18) Lakowicz, J. R. Principles of Fluorescence Spectroscopy, 3rd ed.; Springer: New York, 2006. (19) Banerjee, R.; Chakraborty, H.; Sarkar, M. Host-Guest Complexation of Oxicam NSAIDs with β-Cyclodextrin. Biopolymers 2004, 75, 355−365. (20) Tsai, R. S.; Carrupt, P. A.; Tayar, N. E.; Giroud, Y.; Andrade, P.; Testa, B. Physicochemical and Structural Properties of Non-Steroidal Anti-Inflammatory Oxicams. Helv. Chim. Acta 1993, 76, 842−854. (21) Haiyun, D.; Jianbin, C.; Guomei, Z.; Shaomin, S.; Jinhao, P. Preparation and Spectral Investigation on Inclusion Complex of βCyclodextrin with Rutin. Spectrochim. Acta, Part A 2003, 59, 3421− 3429. (22) Wang, H. Y.; Han, J.; Feng, X. G. Spectroscopic Study of Orange G−β-Cyclodextrin Complex and its Analytical Application. Spectrochim. Acta, Part A 2007, 66, 578−585. (23) Pinto, L. M.; Fraceto, L. F.; Santana, M. H. A.; Pertinhez, T. A.; Junior, S. O.; de Paula, E. Physico-Chemical Characterization of Benzocaine-β-Cyclodextrin Inclusion Complexes. J. Pharm. Biomed. Anal. 2005, 39, 956−963. (24) Ventura, C.; Puglisi, G.; Zappala, M.; Mazzone, G. A PhysicoChemical Study on the Interaction Between Papaverine and Natural and Modified β-Cyclodextrins. Int. J. Pharm. 1998, 160, 163−172. (25) Fernandes, C. M.; Carvalho, R. A.; da Costa, S. P.; Veiga, F. J. Multimodal Molecular Encapsulation of Nicardipine Hydrochloride by β-Cyclodextrin, Hydroxypropyl-β-Cyclodextrin and Triacetyl-β-Cyclodextrin in Solution. Structural Studies by 1H NMR and ROESY Experiments. Eur. J. Pharm. Sci. 2003, 18, 285−296. (26) Djedaïni, F.; Lin, S. Z.; Perly, B.; Wouessidjewe, D. High-Field Nuclear Magnetic Resonance Techniques for the Investigation of a βCyclodextrin: Indomethacin Inclusion Complex. J. Pharm. Sci. 1990, 79, 643−646. (27) Otagiri, M.; Uekama, K.; Ikeda, K. Inclusion Complexes of βCyclodextrin with Tranquilizing Drugs Phenothiazines in Aqueous Solution. Chem. Pharm. Bull. 1975, 23, 188−195. (28) Sun, D.-Z.; Li, L.; Qiu, X.-M.; Liu, F.; Yin, B.-L. Isothermal Titration Calorimetry and 1 H NMR Studies on Host-Guest Interaction of Paeonol and Two of its Isomers with β-Cyclodextrin. Int. J. Pharm. 2006, 316, 7−13. (29) Liu, Y.; Chen, G.-S.; Chen, Y.; Lin, J. Inclusion Complexes of Azadirachtin with Native and Methylated Cyclodextrins: Solubilization and Binding Ability. Bioorg. Med. Chem. 2005, 13, 4037−4042. (30) Zornoza, A.; Martın, C.; Sánchez, M.; Vélaz, I.; Piquer, A. Inclusion Complexation of Glisentide with α-, β-and γ-Cyclodextrins. Int. J. Pharm. 1998, 169, 239−244. (31) Ganza-Gonzalez, A.; Vila-Jato, J.; Anguiano-Igea, S.; OteroEspinar, F.; Blanco-Méndez, J. A Proton Nuclear Magnetic Resonance Study of the Inclusion Complex of Naproxen with β-Cyclodextrin. Int. J. Pharm. 1994, 106, 179−185. (32) Li, N.; Liu, J.; Zhao, X.; Gao, Y. a.; Zheng, L.; Zhang, J.; Yu, L. Complex Formation of Ionic Liquid Surfactant and β-Cyclodextrin. Colloids Surf., A 2007, 292, 196−201. (33) Tang, B.; Chen, Z.-Z.; Zhang, N.; Zhang, J.; Wang, Y. Synthesis and Characterization of a Novel Cross-Linking Complex of βCyclodextrin-o-Vanillin Furfuralhydrazone and Highly Selective Spec8466


Painkiller Isoxicam and Its Copper Complex Can ... - ACS Publications

Recommend Documents