Preparation of curcumin-piperazine co-amorphous phase and

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Preparation of curcumin-piperazine co-amorphous phase and fluorescence spectroscopic and DFT simulation studies on the interaction with bovine serum albumin Wenzhe Pang, Jie Lv, Shuang Du, Jiaojiao Wang, Jing Wang, and Yanli Zeng Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.7b00217 • Publication Date (Web): 13 Jul 2017 Downloaded from http://pubs.acs.org on July 18, 2017

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Molecular Pharmaceutics

Preparation of curcumin-piperazine co-amorphous phase and

fluorescence spectroscopic and DFT simulation studies on the interaction with bovine serum albumin Wenzhe Panga§, Jie Lva§, Shuang Dua, Jiaojiao Wangb, Jing Wanga∗∗, Yanli Zengb* a

College of Pharmaceutical Sciences, Hebei Medical University, Shijiazhuang, 050017, China

b

College of Chemistry and Material Science, Hebei Normal University, Shijiazhuang 050024,

China

Abstract In present study, new co-amorphous phase (CAP) of bioactive herbal ingredient curcumin (CUR) with high solubilitythe was screened with pharmaceutically acceptable co-formers. Besides, in order to provide basic information for the best practice of physiological and pharmaceutical preparations of CUR-based CAP, the interaction between CUR-based CAP and bovine serum albumin (BSA) was studied at the molecular level in this paper. CAP of CUR and piperazine with molar ratio of 1:2 was prepared by EtOH-assisted grinding. The as-prepared CAP was characterized by powder x-ray diffraction (PXRD), modulated temperature differential scanning calorimetry (MTDSC), thermogravimetric analysis(TGA), Fourier-transform infrared (FT-IR) and solid-state 13C nuclear magnetic resonance (ssNMR). The 1:2 CAP stoichioimetry was sustained by C=O…H hydrogen bonds between the N-H group of the piperazine and the C=O group of CUR; piperazine stabilized the di-keto structure of CUR in CAP. The dissolution rates §

Both authors have contributed equally.

*

Corresponding authors at: College of Pharmaceutical Sciences, Hebei Medical University, Shijiazhuang 050017, China. E-mail: [email protected] (J. Wang), Tel.: +86311-86265622 fax: +86311-86265622 b College of Chemistry and Material Science, Hebei Normal University, Shijiazhuang 050024, China, E-mail: [email protected] (Y.L. Zeng) a

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of CUR-piperazine CAP in 30% ethanol-water was faster than that of CUR; the t50 were 243.1 min for CUR and 4.378 min for CAP. Furthermore, interactions of CUR and CUR-piperazine CAP with BSA were investigated by fluorescence spectroscopy and density functional theory (DFT) calculation. The binding constants (Kb) of CUR and CUR-piperazine CAP with BSA were 10.0 and 9.1×103 L·mol-1 at 298K, respectively. Moreover, DFT simulation indicated that the interaction energy of hydrogen-bonded interaction in the tryptophan-CUR and tryptophan-CUR-piperazine complex were -26.1 and -17.9 kJ·mol-1, respectively. In a conclusion, after formation of CUR-piperazine CAP, the interaction forces between CUR and BSA became weaker. Keywords: Co-amorphous, fluorescence spectroscopy, DFT simulation, bovine serum albumin

1 Introduction At present, one of the main challenges of current drug development is some new synthetic drugs possessing poor water solubility [1]. Solubility, dissolution rates and even bioavailability are key factors for an active pharmaceutical ingredient (API) in the design of effective drugs. In recent years, some advanced technologies, such as combinatorial chemistry and high throughout screen, display great potential applications in improving the water solubility of insoluble API

[2]

. However, to improve the water solubility and

dissolution rate of lipid soluble drug molecules in parallel with the structures of API remained intact, will be very challenging for successfully developing new solid state pharmaceutical preparation. One of the mothods to surmount this obstacle is to transfer an intact API drug molecule into an API-small molecule excipient complex, thereby

2


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increasing solubility and dissolution rate

[3]

. Co-crystallization is a promising solid form

for improving the mechanical and physicochemical properties of commercialized APIs [4,5]

. Pharmaceutical co-crystals are formed from an API in the neutral or ionic forms and

a co-crystal former (CCF) that is a solid under ambient conditions

[6-8]

. Co-amorphous

phase (CAP), an amorphous complex formed by API and small molecular precursor, possesses special advantages in improving the solubility and dissolution rate of drugs and increasing the stability of amorphous drugs. For this reason, the coamorphous substance is considered to be an efficient replacement for cocrystal and drug-polymer molecular association

[9,10]

. This approach can also create solubility advantages and stabilize the

amorphous system through inter-molecular interactions, such as hydrogen bonds, and might overcome limitations associated with solid dispersions [11-13].

Figure 1. Chemical structures of CUR isomers and piperazine

Curcumin

(CUR,

1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-dione)

(Fig.1) , a most common spice in Indian cookings, is extracted from the rhizome of turmeric with special physiological activities, for example, anti-oxidation, anti-tumor, hypolipidemic, hypoglycemic, anti-ulcer, antibacterial, anti-inflammatory, antiviral and

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antifungal activities, which promote the CUR as a candidate in treating cancer, diabetes, coronary heart disease, arthritis, Alzheimer's disease, and other chronic diseases Although CUR has the safe dose for humans up to 12 g·day-1

[14]

.

[15]

, by virtue of its

extremely low water solubility, the therapeutic efficacy is greatly compromised. Meanwhile, the low absorption, quick metabolization and elimination in vivo, which are also caused by poorly soluble capacity (7.8 mg·L-1), results in its low bioavailability. Because of this, even the CUR possesses the characteristic of high-efficient and low-toxic, there is no related clinically available therapeutic agents or drugs. Novel drug complexes such as eutectic compositions through concomitant administration of curcumin with nicotinamide, ferulic acid, hydroquinone, p-hydroxybenzoic acid, and ltartaric acid were reported [16]. Other strategy adopted for solubility enhancement was to screen co-crystals through complexation with 4, 4′-bipyridine-N, N′-dioxide resorcinol and pyrogallol

[18]

, and phloroglucinol

[17]

[19]

. However, CUR-based CAP, which

is more promising to improve the solubility and dissolute rate than crystalline complex co-crystal, has not been reported. One of the objectives of this study is to screen new amorphous binary combination based on CUR possessing better physicochemical properties. In present study, the CAP of CUR and piperazine was developed and the solid-state properties of CAP were characterized by PXRD, FTIR spectroscopy, DSC, MTDSC, TGA, 13C SSNMR, and dissolution rate testing. Generally, the drug delivery in vivo is in the form of conjugates, which are reversibly combined with drugs and albumin via hydrogen bonds or Van der Waals forces, as a result, the research of interactions between drug and albumin is extremely important

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in drug development. Moreover, these interactions also affect the pharmacokinetic characteristics of drugs, for example, the the binding parameter of drug-protein concerns absorption, distribution, and even metabolism and elimination of drugs

[20,21]

. Take the

case of CUR, it exerts it’s pharmacological actions, metabolism and clearance only in the form of free molecules, that is, the drugs unconjugate with protein. For this reason, the binding degree of CUR to serum proteins directly affects its half-life in vivo. What is more, the bonding degree is related to the acidity or basicity of the drugs. For their alkalinity, the protein molecules preferentially bind with acidic or natural drug molecules. On this account, the binding constants of neutral CUR and serum albumin up to the orders of 104 L٠mol-1[21]. Further analysis confirms that the binding sites of CUR with protein are near the Trp134 and Trp212 amino acid residues. For API-based binary combination, including co-crystal and CAP, it is well known that API interacts with small molecule co-former mainly through weak force, such as hydrogen bond and van der Waals force. In the case of CUR-piperazine CAP, here an interesting question is risen: if the interaction between CUR and albumin are affected by the weak interaction force between CUR and piperazine, that is, if the binding mechanism and binding parameters for CUR change when piperazine is introduced. In order to provide basic information for best practice of physiological and pharmaceutical preparations of CUR-based CAP, the interactions between CUR-based CAP and BSA were studied at the molecular level in this paper. Moreover, we used density functional theory (DFT) simulation to investigate the molecular interactions in CAP on the basis of these characterizations.

2 Materials and methods 5



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2.1 Materials CUR

(purity≥65%)

was

purchased

from

Sigma

(ALDRICH,

Shanghai).

Hexahydrate piperazine was supplied by Damao Chemical Reagents Factory (Tianjin, China). Fatty-acid-free bovine serum albumin (BSA, 99% purity) lyophilized powder was purchased from Sigma Co.Ltd.(ALDRICH,Shanghai). Double-distilled water was used for all experiments.

2.2 Methods 2.2.1 Preparation of CAP of CUR-piperazine The preparation of CUR-piperazine CAP was attempted by neat powder grinding and EtOH-assisted grinding methods. The superiority of neat powder grinding method overcomes the shortcoming of possible solvent/water inclusion in the process of solution crystallization. With regard to the EtOH-assisted grinding, the small amount of EtOH dropped during the grinding progress increased the reaction interface between API and precursor molecules, which promote to form the final products. The mixtures of CUR and piperazine at different CUR/piperazine mole ratios of 2:1, 1:1 and 1:2 (total mass of 1000 mg) were ground in a laboratory-scale swinging ball mill (Mixer Mill GT200, Grindertech GmbH, Beijing, China) with 35mL of zirconia grinding jar and 1.5 cm of zirconia grinding ball at speed of 1800rpm. In EtOH-assisted grinding procedure, ethanol was added to the grinding jar to form the slurry in the grinding process. The slurry state of the mixture was maintained through dropping 0.2 mL of ethanol every 5 min. After 15 min of grinding, the collected solid product was dried at 313K in an vacuum oven for 4-5 h and stored in a vacuum desiccator protected from light at ambient conditions for 48

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hour to deleter the residual solvent.

2.2.2 Powder x-ray diffractometry (PXRD) PXRD patterns were collected utilizing an X ray diffractometer (D2 Phaser, Bruker Co., Germany) with Cu anode and Lynx eye detector over the interval 5 o to 40 o (2θ), with step size of 0.05 o (2θ) and time per step of 0.3s. The instrument was operated at 30 kV generator voltage and 10 mA current. The sample powder (about 50 mg) was placed inside glass groove. In order to avoid particle orientation, the surface was flattened softly. The date were collected and processed using suitable software.

2.2.3 Thermal analysis DSC and TMDSC were performed on DSC 214 (NETZSCH Company, Germany) equipped with a refrigerated cooling accessory (IC40) and a data analyzer (Proteus Analysis7.0) under the nitrogen atmosphere. An empty aluminum pan was applied as reference. Sample powder (5–7 mg) was placed in sealed aluminum pans with lids. DSC was scanned ranging from 298 to 573K at a scanning rate of 20 K· min-1. Samples in TMDSC were modulated at 0.5K every 60 s with heating rate of 2 K·min-1 from 273 to 473K. The moisture content of samples was measured using a TG 209 thermo gravimetric analysis instrument (NETZSCH company, Germany). TG experiment was carried out at a heating rate of 10 K·min-1, scanning from 298 to 723 K under nitrogen purge at a flow rate of 20 mL·min-1. Each sample was measured at least three times.

2.2.4 Fourier-transform infrared (FTIR) spectroscopy The FTIR spectrum of each sample was obtained in KBr diffuse reflectance mode using an Equinox 55(Bruker) FTIR spectrophotometer. The sample was scanned from

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4000 to 400 cm-1 at an interval of 2 cm-1. The total number of scans was 40 and the resolution was 2cm-1.

2.2.5 Solid-state 13C NMR spectroscopy (ssNMR) 13

C ssNMR characterizations of CUR, piperazine and CUR–piperazine CAP were

performed on a Bruker AVANCE III NMR spectrometer (Bruker, Germany) at 400 MHz by a combination of crosspolarization (CP) pulse sequence/high-power decoupling/magic angle spinning (MAS) (giving significant improvement in sensitivity). Samples were filled into a 4 mm rotor and spun with a rate of 10.0 KHz and the KBr method was used to calibrate the magic angle setting. The CP/MAS two-pulse phase modulation was exerted on heteronuclear decoupling in time of acquiring with a proton field H1H satisfying the equation: ω1H/2π = γHH1H = 60 Hz. The recycle delay was 2s. An adequate signal-to-noise ratio was obtained by multiple scans. The external secondary standard selected glycine (δglycine = 43.3 ppm) to calibrate the chemical shifts referenced to TMS. All ssNMR experiments were conducted at room temperature.

2.2.6 SEM The cryo-emission SEM system (Hitachi S-4800, Japan) was used to observe the morphologies of CUR and the as-prepared CUR-piperaznie CAP. Powder of crystalline CUR or CAP was soluble in absolute ethanol. Afterward, dropping the resulting ethanol suspension to the electric glass slice, the precipitate on the glass slice was analyzed by SEM after the solvent had volatilized.

2.2.7 Apparent solubility determination The apparent solubility of crystalline CUR and CAP in 0.1 mol·L-1 HCl and 30% EtOH-water after 48 hours air bath shaking were determined by shake-flask method. The 8


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calibration curves were obtained for CUR and CAP by plotting absorbance vs concentration using a UV-2450 UV-vis spectrometer (Shimadzu Co.Japan) for known concentration solutions in 0.1 mol·L-1 HCl and 30% EtOH-water, respectively. Added excess sample to the above solvents, the suspension was kept at different temperatures of 293 ± 0.5, 304 ± 0.5, 310 ± 0.5 and 315 ± 0.5 K and continuously shaken for 48 h using a SPH-200B air bath shaker (Shiping Tech. Co., Ltd., Shanghai,China). Afterwards, the concentration of CUR was determined at 430 nm on the UV spectrophotometer using above mentioned calibration curves after filtering the suspension via 0.22 µm microfiltration membrane.There was no interference to the CUR UVvis maximum at 430 nm by piperazine, because piperazine absorbed at 230 nm in the UV region. All tests were repeated thrice.

2.2.8 Powder dissolution rate determination The powder dissolution rate determination for the CUR and CUR-piperazine CAP were performed on a pharmaceutical RCZ-8M dissolution tester system(Tiandatianfa Technology, Co., Ltd., Tianjin, China) in two different dissolution media of 30% EtOH-water and 0.1 mol·L-1 HCl, respectively. Each test was repeated thrice. Approximately 30 mg of pure CUR or CAP equivalent to 30 mg of CUR was placed in the dissolution tester that contained 900 mL of dissolution media at 293 ± 0.5 and 310 ± 0.5K (paddle method, 100 rpm), respectively. At fixed intervals, suspensions of 5 mL volume were withdrawn with replacement with the same volume fresh medium. Solution concentrations were determined at 430 nm utilizing UV spectrophotometer. In order to analyze the drug dissolution mechanism from the CAP, dissolution kinetic models was

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fitted on the basis of non-linear regression.

2.3 Spectroscopic studies on the interaction of CUR and CUR-piperazine CAP with bovine serum albumin Bovine Serum Albumin (BSA, molecular weight assumed to be 66,400) was used without further purification. All BSA stock solutions were prepared in the pH 7.4 buffer solution (mixing 0.10 mol·L-1 Tris base and 0.10 mol·L-1 HCl, and adjusting the pH value to 7.4 by adding 0.1 mol·L-1 NaOH). By dissolving BSA in 0.05 mol·L-1 Tris-HCl, BSA solution was prepared with final concentration of 5×10-5 mol·L-1. As-prepared BSA stock solution was kept in the dark at 4oC. The stock solutions of CUR and CUR-piperazine CAP were prepared by dissolving the CUR or CUR-piperazine CAP with equivalent CUR concentration in ethanol with final CUR concentration of 1×10-3 mol·L-1. Fluorescence emission spectra were measured on a F-7000 spectrofluorophotometer (HITACHI, Japan) equipped with 1.0 cm quartz cells. 5×10-5 mol·L-1 BSA solution of 1.0 mL volume and various amount of CUR (or CAP) were added to the cell and the concentrations of CUR were ranged from 2 ×10-6 to 4 ×10-5 mol·L-1. Afterwards, all fluorescence emission spectra were measured from 220 to 450 nm at an excitation wavelength of 280 nm with 5 nm slit widths at three temperatures of 298, 310, and 315 K, respectively. Every case of all experiments was in the same buffer mentioned above. Based on the various concentrations of CUR, the synchronous fluorescence spectra of BSA-drug systems at different ∆λ values (15 and 60 nm) were obtained from 220 to 450 nm with the emission and excitation slit widths of 5/5 nm, respectively.

2.4 DFT simulation

10


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The Gaussian 09 package [22] was used to perform the DFT simulations. The initial structure of CUR was created from the crystal structure, and then optimized at the DFT(B3LYP) theoretical level combined with the 6-31G(d,p) basis set. The geometries of the piperazine, CUR-piperazine CAP were constructed and optimized at B3LYP/6-31G(d,p) computational level. The tryptophan was selected as the representative amino acid model from the BSA, and then the interactions of tryptophan with CUR and CUR-piperazine CAP were also constructed and investigated at the same computational level. The vibrational frequencies were computed to characterize the optimized geometries by verifying that there were no imaginary frequencies. The interaction energies were corrected by the basis set superposition error (BSSE) [23]. The analyses of electrostatic potentials were obtained on the contour of 0.001 electrons/bohr3 of electron density

[24]

using the WFA surface analysis suite

[25]

. The topological studies

based on the quantum theory of “atoms in molecules” (QTAIM)[26] were performed to give more insight to the interactions by the AIMALL professional software[27].

3. Results and discussion 3.1. Characterizations 3.1.1. PXRD Neat powder grinding and EtOH-assisted grinding methods were utilized to prepare CUR-piperazine CAP at various CUR/piperazine molar ratios of 2:1, 1:1 and 1:2. Fig.2 showed the PXRD patterns of the CUR-piperazine systems. When CUR was co-ground with piperazine at CUR /piperazine molar ratio of 1:2 by EtOH-assisted grinding method for 15 min, the PXRD pattern showed the typical halo patterns which indicated the

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formation of amorphous phase (Fig.2d), while, besides peaks assigned to CUR, no new peaks appeared in the XRD patterns of samples produced at the CUR/piperazine ratios of 1:1 and 2:1 (Fig.2c and e). This finding demonstrated the formation of CAP just occurred at the CUR/piperazine molar ratios of 1:2. Furthermore, prolonging grinding time could not include the transformation from amorphous form to crystalline form(Fig.2f). However, neither co-crystal nor CAP was produced by neat grinding method; there was no new peak appeared in the XRD patterns when neat grinding method was used (Fig.S1 in supporting materials). The EtOH-assisted grinding procedure could efficiently induce CAP formation via adding minor amounts of ethanol. In EtOH induced reaction slurry, CUR and piperazine molecules partially dissolved in ethanol, which dramatically increased the reaction interfaces, including liquid-liquid, solid-solid, and solid-liquid interfaces.

Fig.2 PXRD patterns of the CUR/piperazine system prepared using EtOH-assisted grinding method: (a) CUR;(b) piperazine; (c) CUR/piperazine 1:1; (d) CUR/piperazine 1:2 grinding for 15 min; (e) CUR/piperazine 2:1; (f) CUR/piperazine 1:2 grinding for 30 min.

12


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In present study, piperazine was used to screen the complex for CUR owing to the existance of two N-H groups as hydrogen donor in piperazine. CAP at molar ratio of 1:2 were achieved instead of the co-crystal in this study. Owing to higher free energy, the disordered CAP molecules had tendency to rearrange themselves into ordered crystalline phase, that meant, it was easy to form crystalline complex. However, the formation of CAP of CUR-piperazine in this case indicated that the energy barrier contributed by the entropic change ∆S for nucleation of crystalline phase was the main obstacle and hinders the formation of crystallinecomplex [28].

Fig. 3 Cryo-field emission SEM photographs of CUR (left) and CUR-piperazine CAP (right).

When the crystalline phase transformed into the amorphous phase, the crystalline structure is destroyed and image of the particles changes. The SEM micro-graphs were obtained to visually survey the differences in the CUR and CUR-piperazine CAP (Fig.3). Crystalline samples of pure CUR showed a block-shape, indicating the nature of the crystalline phase, while the image of CUR-piperazine CAP showed sphere-shape particles, indicating that crystal CUR transformed into the amorphous state in the CUR-piperazine complex. Sphere-shape particle is not special for CUR-based CAP. We

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have observed same sphere-shape in particles of azelnidipine-oxalic acid CAP

Page 14 of 41

[29]

. The

CAP powder should dissolve in ethanol solution during preparing sample for SEM observation. CAP with higher surface free energy and large surface area tended to arrange themselves into sphere-shape particle in solvent. Small sphere-shape particle is the true existing mode for CAP in solvent.

3.1.2 Thermal analysis DSC, TG and TMDSC experiments were utilized to analyze the thermal behavior of native CUR and its CAP, respectively. Native CUR and piperazine had melting point at 451.4K and 392.0K, respectively (Fig.S2 in supporting materials). TG curve (Fig S3) for CAP showed about 3.21% loss weight from 333 to 388 K. Although the CAP sample was dried at 313K in an vacuum oven for 4-5 h, samll amout of solvent, including ethanol and water molecules,existed in CAP. PXRD result showed the amorphous character for CAP, TMDSC was effective way to support that as-prepared CAP was truly amorphous but not microcrystallinity or mixture of amorphous and microcrystallinity. Generally, CAP formation indicates that orderliness of crystalline structure transforms to disorderliness of CAP complex, which can result in the disappearance of the melting points of crystalline phase and the appearance of Tg of amophous phase, that is, Tg is an true indicator of the amorphicity of a sample

[30]

. In our study, for CUR-piperazine CAP, the TMDSC was performed to

identify the Tg points (Fig.S2). In comparison, CUR-piperazine CAP showed obvious Tg at 306.2K, indicating the formation of a homogeneous phase. Generally, the Tg of the binary amorphous system values between the two Tgs of the two starting components [31].

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The native CUR amorphous phase showed high Tg of 355.7K (355.7K showed in Fig.S2), but CUR-piperazine CAP with ratio of CUR/piperazine of 1:2 showed lower Tg of 306.2K, which seemingly followed the observation reported by Dengale et al., stating that the Tg of CAP has tendency to be closer to the Tg of the component existing in excess within CAP. So, Although Tg value of the native piperazine amorphous phase was not obtained in our study, the low Tg value of CUR-piperazine CAP depended largely on piperazine. However, the CUR-piperazine CAP has a very low Tg of 306.2K, which would make it difficult to develop as an actual pharmaceutical composition.

3.1.3 FT-IR spectroscopy As mentioned above, the introduction of piperazine molecule induced the orderliness of crystalline structure of CUR to transform to disorderliness, which especially resulted in the conformational and structural variations in hydrogen bonds donor and acceptor functional groups. FTIR spectroscopy was used to detect these variations and the FTIR spectra of CUR, piperazine and CAP were shown in Fig. 4. CUR showed characteristic absorption peaks at 1624 cm−1 and 3505 cm−1, which were assigned to the carbonyl C=O and O-H stretching vibration, respectively (Fig. 4b). Piperazine showed characteristic absorption peaks at 3256 cm−1, which was assigned to the N-H stretching vibration (Fig. 4a). Peak at 1624 cm−1 assigned to the carbonyl C=O in CUR shifted to 1619 cm−1 in the CAP (Fig. 4c), indicating that the C=O was involved in the hydrogen bond formation in CAP. Moreover, owing to changing of molecular arrangement and structural disorderliness in CAP, the IR absorption peak of CAP became broadening [32]. In the spectrum of the CAP, the peak assigned to the N-H was too aboard

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to allow for detection of any change. Nevertheless, it was speculated that the hydrogen bonding between C=O in CUR and the N-H group in piperazine was produced in CAP. CUR molecule exists as keto-enol tautomers and the predominant form is related with acid-base character of solution. In acidic and neutral solution, CUR presented keto form predominantly, but the enol formed in alkaline solution [33]. Based on the CUR/piperazine ratio of 1:2, it was reasoned that the hydrogen bonding between CUR and piperazine could modify the predominant form of keto-enol tautomers. In the EtOH-assisted grinding procedure, the predominant di-keto form was favorable for formation of the CAP at the molar ratio of CUR/piperazine 1:2; the double C=O were involved in the formation of hydrogen bonding with two piperazine molecules. The introduction of piperazine stabilized the di-keto structure of CUR in CAP. Piperazine is idea hydrogen bond donor and a good co-former for the formulation of CUR-based CAP drug systems.

Fig.4 FTIR spectra of the CUR /piperazine system: (a) piperazine;(b) CUR; (c) CUR/piperazine CAP.

3.1.4 Solid-state NMR spectroscopy (ssNMR) Besides IR spectra, the differences of molecular mobilityand conformations before 16


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and after formation of CAP would present in the ssNMR spectroscopy

[34] 13

.

C ssNMR

spectra were conducted to further investigate the inter-molecular interaction between CUR and piperazine in CAP. We reported that significant changes in chemical shifts for azelnidipine-maleic acid co-crystal and CAP; the inter-molecular interaction between azelnidipine with and maleic acid in binary complex could induce the corresponding changes in NMR chemical shifts

[35,36]

. Differences of a similar magnitude for chemical

shift were observed for CUR-piperazine CAP (Fig.5). The spectra of the parent CUR and CAP showed significant differences at almost all positions for the

13

C chemical shift,

indicating molecular re-arrangement in CAP hetero-dimer. The double peaks at the positions of C9 and C11 showed combination in one broadened peak, indicating the variation of conjugation effects with the keto and enol groups. The parent solid CUR exists as keto-enol tautomers, in which C9 and C11 showed different chemical shift. The introduction of piperazine stabilized the di-keto structure and the two C=O groups interacted with two piperazine molecules in 1:2 CAP binary complex, then, only one broad peak appeared in the spectrum for binary CAP. Besides, owing to the conjugation effects with the double C=O groups, the chemical shifts for C8 and C12, C7 and C13, C4 and C14, C2 and C18 showed significant changes and the peaks broadened. However, small change occurred for C20 and C21 because of weak conjugation effect from C=O groups. For piperazine molecule, the only peak at 46.5 ppm downshifted to 44.0 ppm in the CAP. The significant shift was due to participating of N-H in formation of hydrogen bonding with CUR and destroying of molecular arrangement in the lattice in intact piperazine after formation of binary amorphous phase. Just because of the losing the

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order of molecular arrangement in crystalline parent API and co-former, broadening and shift of NMR peaks occurred for CAP, which was consistent with the finding of IR spectra, that is, the abroad peaks in the spectra of CUR-piperazine CAP indicated its amorphous nature.

Fig. 5

13

C ssNMR spectra of the CUR /piperazine system: (a) CUR; (b) piperazine;

(c) CUR-piperazine CAP. The peaks in the CAP are broaden and shifted relative to the intact CUR.

3.2 Physicochemical properties of the CAP 3.2.1 Apparent solubility The apparent solubilities of crystalline CUR and CUR-piperazine CAP after 48h in 0.1 mol·L-1 HCl and 30% EtOH-water at different temperature were presented in Table 1. The solubility of crystalline CUR is high under basic conditions, EtOH and acetone medium and low at aqueous, acidic and neutral pH

[15]

. As can be seen in Table 1, the

solubility of crystalline CUR was lower in 0.1 mol·L-1 HCl and showed slight improvement in 30% EtOH-water media at different tempertatures; slight increasing of 18


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solubility occurred as temperature rose. In the case of CUR-piperazine CAP, the solubility showed no obvious improvement in above two medium (P>0.05) at 304, 310 and 315K, which were near or above Tg (values at 306.2K from TMDSC) of amorphous phase. However, the solubility of CAP was about 4 times higher than that of crystalline CUR at temperature of 293K which was below Tg. Babu et al. Reported that the solubility of co-crystal was almost equivalent to that of the amorphous drug [37], and amorphous drug solubility was 4~14-times higher than that of the crystalline phase

[38]

. Obviously,

the solubility of CAP of CUR-piperazine also matched this improvement fold. However, compared with intact CUR, CUR-piperazine CAP showed higher solubility below Tg, such as at 293K, but, showed almost unchanged solubility exceed Tg, such as at 304K, 310K, 315K. The CUR-piperazine CAP maintained its amorphous phase nature with higher solubility below Tg, but transferred to the rubbery phase above temperature of Tg, which retarded dissolution of CUR from the solid phase. The reverse relationship between solubility and temperature is related with the glass and rubbery phase below and above Tg of CAP. Compared with intact CUR, CUR-piperazine CAP exceeded saturated solubility of intact CUR to form a supersaturated solution in 0.1 mol·L-1 HCl and 30% EtOH-water media below Tg. For procedure of nucleation and growth in solution, supersaturation was the thermodynamic driving force, which may cause the dissolved drug to re-precipitate in soluble media. The PXRD of excess solid sampled from solubility experiments were scanned (Fig. S4); all samples maintained the initial amorphous phase after solubility determination, implying that it was difficult for CUR to re-crystallize after formation of

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CAP with piperazine. Table 1 Apparent solubility of free CUR and CUR-piperazine CAP (n=3, x±s) Sample

CUR

CUR /piperazine CAP

Temperature

Solubility (µg·mL-1) 0.1 mol·L-1 HCl

30% EtOH-water

293K

7.2 ± 3.2

12.7 ± 2.0

304K

8.9 ± 2.3

16.8 ± 1.6

310K

10.1 ± 3.3

17.9 ± 2.8

315K

11.2 ± 2.1

18.8 ± 2.4

293K

31.4 ± 2.6

43.6 ± 1.5

304K

10.6 ± 2.1

22.2 ± 2.3

310K

10.9 ± 3.1

22.5 ± 2.2

315K

12.1 ± 2.1

23.4 ± 2.2

3.2.2 Powder dissolution rate As CUR has an extremely low aqueous solubility, in vitro standard powder dissolution test in 0.1 mol·L-1 HCl and 30% EtOH-water at 310K and 293K, respectively, were used to compare the dissolution profiles of CUR with and CAP (Fig.6). As mentioned above, owing to low Tg, CAP showed higher solubility at 293K than that of temperature above Tg. In the case of powder dissolution rate, it showed the same pattern as the solubility in 30% EtOH-water medium (Fig.6B). Intact CUR dissolved faster than CAP at 310K, while, the powder dissolution rate of CAP was quicker than that of CUR at 293K in 30% EtOH-wate medium; the t50 were 243.1 min for CUR and 4.378 min for CAP. Obviously, extremely low dissolution rate occurred for intact CUR and the CAP in 0.1mol·L-1 HCl (Fig.6A). Furthermore, it seems that the dissolution rate of CUR from CAP is not only critical to the solubility in 0.1 mol·L-1 HCl medium. It was speculated that the variation of the conditions for determining solubility and powder dissolution rate made this seeming conflicting result. In solubility measurement procedure, the solid 20


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powder experienced a long equilibrium procedure to produce a supersaturated condition. On the contrary, it was unsaturated system in dissolution profile measurement procedure in the large-volume 0.1 mol·L-1 HCl apparatus, in which rubbery phase above Tg didn’t delay the dissolution rate of CUR. The mechanism of dissolution improvement of CUR-piperazine CAP might be related with the retaining CUR as amorphous form with the introduction of piperazine [39]. Owing to high free energy, amorphous drug showed intermediate nature and elemental randomness in the atomic positions, Therefore, the releasing from the solid bulk becomes more easily

[40]

. Based on the thermodynamic theory, crystalline phase showed lower

dissolution rate compared to its counterpart amorphous form. However, in our case, intact CUR dissolved faster compared to CAP in some determination condition. Besides solubility nature, physicochemical properties of powder, such as rubbery phase resulted from lower Tg, poor dispersibility and higher hygroscopic ability, would make impact on the dissolution profile. 3.3 Spectroscopic studies on the interaction of CUR-based CAP with BSA 3.3.1 Fluorescence Measurements Fluorescence quenching of BSA or HSA can provide adequate information including binding mechanism, binding-specific parameters, and structural changes of the protein, which are applied to characterize the interaction of API molecules with proteins. Fluorescence quenching assumes decline of the fluorescence intensity of a certain fluorophore through diversity of molecular interactions. Fluorescence spectra of BSA were measured ranging from 220 nm to 450 nm at a excitation wavelength of 280 nm in

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Fig. 6. Powder dissolution profile of free CUR and CUR-piperazine CAP in 0.1 mol·L-1 HCl (A) and 30% EtOH-water (B) ( ∆:310K; • 293K)(a) free CUR (b) CUR-piperazine CAP. (n=3, x±s)

the presence of various amounts of CUR (Fig.S5). It was important to note that free CUR and CUR-piperazine CAP showed no fluorescence intensity near the maximum emission wavelength of BSA. Both CUR and CAP were capable of linear quenching the BSA fluorescence, and CUR-BSA binding did not induce any changes either in the emission wavelength or peak shape of the BSA. The fluorescence intensity of BSA declined with adding CUR or CAP without any shift in the maximum emission wavelength, suggesting the interaction of BSA with CUR

[41]

. Various kinds of intermolecular interactions such

22


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as energy transfer, ground-state complex formation, rearrangement of molecules, excited-state reactions, and collisional quenching could induce the quenching in fluorescence intensity of BSA[42]. 3.3.2

Fluorescence quenching mechanism of BSA

Molecular interactions between BSA and quencher molecule (here CUR or CAP) could induce declining of BSA fluorescence intensity through decreasing of quantum yield of fluorescence from a fluorophore. Generally, above mentioned molecular interactions were considered to produce complex between CUR and BSA. Stern–Volmer quenching equation was used to analyze fluorescence intensity data: F0 / F = 1 + K sv [Q ]

(1)

where F and F0 are the fluorescence intensity in the presence and the absence of quencher (here CUR or CAP), respectively. [Q] is the concentration of CUR. Ksv is quenching constant of Stern-Volmer. The temperature-dependent behavior of Ksv can be used to judge quenching mechanisms

[43]

. With increasing temperature, collisional quenching

involved in the dynamic quenching mechanisms was increased because of the faster diffusion of molecules. However, the decomposition of weakly bound complexes occurred with increasing temperature, which induced the decreasing of static quenching. Consequently, an increase in the dynamic quenching constant while decreasing in the static quenching constant occurred with increasing temperature [44]. Used Stern-Volmer Eq.(1), the fluorescence quenching data at different temperatures (i.e., 298, 310 and 315 K) were used to reveal the quenching mechanism in CUR-BSA interaction. The KSV values obtained from Stern-Volmer plots of F0 / F against

23



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Page 24 of 41

CUR concentration (provided in Fig.S6) were summarized in Table 2. As can be seen from Table 2, with increasing temperature, value of KSV decreased. Furthermore, at all applied temperatures, the values of the bimolecular quenching constant (Kq) were found to be 1011 order of magnitude, which exceeded 2×1010·s-1·mol-1, reported for the value of the maximum dynamic quenching constant [45]. Table 2 Static and dynamic quenching constants for CUR-BSA interaction for free CUR and CUR-piperazine CAP at different temperatures. T/K

298 310 315

Ksv/(103L·mol-1)

Kq /(1011L·mol-1s-1)

Free CUR

CUR-piperazine CAP

Free CUR

CUR-piperazine CAP

15.54 11.30 9.85

14.64 9.72 8.80

15.54 10.93 9.85

14.64 9.72 8.80

Obviously, there was inverse correlation between KSV values and temperature and the values of Kq were appeared to be large than maximum dynamic quenching constant. Based on above quenching theory, it clearly indicated the CUR-BSA complex formation, which was confirmed by inverse correlation between Kq values and temperature, induced the static fluorescence quenching of BSA. When piperazine molecules were introduced into the structure of CUR, CAP was formed, in which rearrangement of molecules occurred. In order to find the quenching mechanism for CUR-piperazine CAP quencher molecule, the fluorescence quenching data at 298, 310 and 315 K were also calculated using the Stern-Volmer Eq. (1) and the KSV values for CAP system were listed in Table 2. Same as free CUR, value of KSV from CAP decreased with increasing temperature, and the values of Kq were also more than the maximum dynamic quenching constant. Just like free CUR, CUR-piperazine CAP seemed to follow static quenching mechanism. However, KSV values for free CUR were 24


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higher than those of CUR-piperazine CAP, indicating that the hydrogen bonding between CUR and piperazine affected the interaction between CUR and BSA to a certain degree. 3.3.3 Binding constant Used the fluorescence quenching data at different temperatures and modified Stern-Volmer equation Eq. (2), the binding constants (Kb) for CUR-BSA interaction can be calculated: log[( F0 − F ) / F ] = log K b + n log[Q]

(2)

where F and F0 are the fluorescence intensities in the presence and absence of the quencher, respectively, n is the average number of biding site per protein molecule, and Kb is the binding constant. The y-axis intercept of Stern-Volmer plots yielded the values of Kb, which were shown in Fig.7 and listed in Table 3 for free CUR and CUR-piperazine CAP. As evident from Table 3, the Kb value for free CUR had fallen in the range of 3.3~10×103 L·mol-1, suggesting that the binding affinity between free CUR and BSA was moderately strong. For a number of therapeutic drugs, these moderately strong binding affinities were reported

[46,47]

. Such binding affinity seemed to be favorable for CUR to

be transported in the blood circulation and next released at the target sites in vivo. Moreover, the destabilization of CUR-BSA complex with increasing of temperature resulted in decreasing of Kb values, so, we also found an inverse correlation between the temperatures and the Kb values. However, it seemed for CUR-piperazine CAP that the Kb values decreased compared with free CUR (Table 3). Generally, there are four types of interactions of electrostatic interactions, hydrogen bonding, van der Waals and hydrophobic involved in the

25



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Page 26 of 41

interaction between biological macro-molecules and small drug molecules

[48,49]

. It is

presumed that interaction between CUR and BSA is hydrogen bonding, which may be affected by the hydrogen bonding between CUR and piperazine in CAP. As illustration from IR and NMR above, two hydrogen bondings between two C=O groups in one CUR molecule and the two N-H groups from two piperazine molecules was produced in CAP trimer. Thus, the occupied hydrogen bonding binding sites in CAP weakened the ability of hydrogen bonding formation with BSA, which resulted in the decline of Kb values. Table 3 Binding and thermodynamic parameters CUR-piperazine-BSA (B) systems at different temperatures

System

A

B

T/ K

log［(F0 −F)/F］ -log [Q]

Kb/ 3 (10 L·mol-1)

298

Y=0.9455x+4.0007

10.0

310

Y=0.8396x+3.5352

3.4

315 298

Y=0.8561x+3.5214 Y=0.9383x+3.9591

3.3 9.1

310

Y=0.8054x+3.3623

2.3

315

Y=0.8085x+3.3215

2.1

of

free

∆H θ / kJ·mol1

CUR–BSA

∆S θ / -1

J·mol ·K

(A)

and

∆G θ / -1

kJ·mol-1 -22.7

-54.2

-105.7

-21.5 -20.9 -22.5

-71.5

-164.4

-20.5 -19.7

Fig.7. The modified Stern-Volmer plots of (A) CUR and (B) CUR-piperazine CAP interaction with BSA at (a) 298 K, (b) 310K, (c) 315K. CBSA = 5×10-6 mol·L-1. The insert in figure was Van’t Hoff plots for the quenching of BSA. 26


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3.3.4 Thermodynamic parameters and binding forces For the binding reaction between CUR and BSA, enthalpy changes ( ∆H θ ), entropy changes ( ∆S θ ) and free energy changes( ∆G θ ) are important to evaluate the binding forces. If ∆H θ almost maintains constant in the temperature range studied, then its value as well as that of ∆S θ can be calculated using van’t Hoff equation: ln K b = − ∆H θ / RT + ∆S θ / R

(3)

Kb is the binding constant at certain temperature, T is the absolute temperature and R is

the gas constant (8.31 J·mol-1·K-1). The slope and the intercept values of the linear van’t Hoff plot between ln Kb and 1/T yielded the ∆H θ and ∆S θ values, respectively (inserted in Fig.7). The values of ∆G θ of the binding reaction at studied temperatures were calculated by substituting the values of ∆H θ and ∆S θ in the following equation: ∆G θ = ∆H θ − T ∆ S θ

(4)

The magnitude and sign of ∆H θ and ∆S θ can be used to predict the types of force(s) in CUR-BSA interaction

[50]

. Positive ∆H θ and negative ∆S θ values indicates the

presence of hydrophobic interactions, while negative ∆H θ and ∆S θ values suggested both hydrogen bonds and/or van der Waals forces in API-protein complex. On the other hand, a positive ∆S θ value as well as a very small value of ∆H θ indicated the existance of electrostatic forces. Table 3 listed the values of ∆H θ , ∆S θ , and ∆G θ at different temperatures obtained using Eqs. (3~4) for free CUR-BSA and CUR-pipearzine-BSA system. The negative ∆G θ values suggested the feasibility of CUR-BSA complex formation. The negative ∆H θ indicated an exothermic binding reaction and the negative values of

27



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∆S θ were for the enthalpy-driven binding reaction between CUR and BSA. The large

negative values of ∆H θ and ∆S θ obtained in this study indicated that hydrogen bonds and/or van der Waals forces were main interactions force(s) in CUR-BSA complexation. This was further supported by Kb values for free CUR and CUR-piperazine CAP. As above mentioned, binding constant Kb values for CUR-piperazine CAP was lower than that of free CUR, which was owing to decreasing of hydrogen bond binding focre from CUR after formation of CUR-piperazine CAP. 3.3.5 Conformational investigations Owing to the polarity of the environment around tryptophan (Trp) and tyrosine (Tyr) in BSA relating with the maximum emission wavelength of residues, the alteration of protein conformation was investigated by that of emission wavelength using synchronous fluorescence spectra. As the wavelength interval (∆λ=λem -λex) was 60 nm, synchronous fluorescence provided information of the Trp residues, while it offered the characteristics of Tyr residues when ∆λ is 15 nm [51]. In the presence of CUR or CAP, any change in the microenvironment around residues could be used to predict its binding sites on BSA. Under various concentrations of CUR or CAP, the fluorescence spectra of Trp and Tyr residues of BAS were recorded (provided in Fig.S7). Trp residues contributed significantly to the quenching of the intrinsic fluorescence by virtue of its’ stronger fluorescence intensity quenching than that of Tyr residues. In the free CUR-BSA system, following the CUR addition to BSA, the maximum emission wavelength with ∆λ=15 nm showed no shift. However, the maximum emission wavelength with ∆λ=60 nm presented slightly blue shift (from 290 to 288.1nm), suggesting that the interaction of CUR with BSA induced the changing of the conformation of the region around Trp residues but did 28


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not affect that of Tyr residues. Meanwhile, in the CAP-BSA system, maximum emission wavelength of Trp residues showed a slightly blue shift (from 305 to 304.4nm). For Tyr residues, the fluorescence intensity also had the obvious decrease, but the emission peak position did not change, suggesting that the hydrophobicity around both the residues increased and the conformation of BSA changed. 3.3.6 The interactions of tryptophan with CUR and CUR-piperazine CAP simulated by DFT calculation Besides that interactions of CUR-BSA were investigated utilized quench the fluorescence of BSA, DFT simulation was used to investigate the CUR-BSA interaction. Synchronous fluorescence spectra indicated that tryptophan residues contributed greatly to the quenching of the intrinsic fluorescence. So, in DFT simulation calculation, we selected the tryptophan as representative amino acid model from the BSA. Fig.8 showed the contour maps of the molecular electrostatic potentials for CUR(a), tryptophan(b), CUR-piperazine complex (1:2) (c), with the color ranges: red, more positive than 20 kcal·mol-1; yellow, -3 – 20 kcal·mol-1; green, -20 – -3 kcal·mol-1; blue, more negative than -3 kcal·mol-1. The most positive and negative electrostatic potentials were denoted as VS, max and VS, min, respectively. For CUR, a negative region existed outside the two C=O bonds, with the VS, min value -49.1 kcal·mol-1. For tryptophan, there was a positive region outside the C-H bond, with the VS, max value 46.6 kcal·mol-1. For CUR-piperazine 1:2 complex, the negative region of electrostatic potentials still existed outside the two C=O bonds. However, the VS,min value became less negative to -35.1 kcal·mol-1. Based on the above analyses of molecular electrostatic potentials, the molecular graphs of

29



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CUR-piperazine 1:2 complex, tryptophan-CUR, and tryptophan-CUR-piperazine were displayed in Fig.8 (d), (e) and (f), respectively. In the CUR-piperazine 1:2 complex, there were two hydrogen bonds: N(a)-H(a)···O(a) and N(b)-H(b)···O(b) hydrogen bond, which were consistent with the result of IR and ssNMR analysis. In the tryptophan-CUR complex, the N(c)-H(c)···O(a) hydrogen-bonded interaction formed, with the interaction energy -26.1 kJ·mol-1. In the tryptophan-CUR-piperazine complex, the N(c)-H(c)···O(a) bond became somewhat weaker, with the interaction energy -17.9 kJ·mol-1. QTAIM

[26]

is a very useful theory to analyze the chemical bonds [52,53]. Based on

the QTAM, the electron density (ρb) at the bond critical points (BCP) could reflect the strength of the interactions. The larger ρb value, the interaction is stronger. As can be seen in the molecular graphs for CUR-piperazine complex (1:2), tryptophan-CUR, and tryptophan-CUR-piperazine displayed in Fig.8 (d) to (f), the ρb values at the BCPs of N(a)-H(a)···O(a) and N(b)-H(b)···O(b) hydrogen bond are 0.0114 in the CUR-piperazine CAP. In the tryptophan-CUR complex, the ρb value at the BCP of e N(c)-H(c)···O(a) hydrogen bond is 0.0152. While, in the tryptophan-CUR-piperazine complex, the N(c)-H(c)···O(a) bond became 0.0148, and the ρb values at the BCPs of N(a)-H(a)···O(a) and N(b)-H(b)···O(b) hydrogen bond became smaller 0.0110. That is to say, compared with CUR-tryptophan, the N(c)-H(c)···O(a) bond became weaker, and at the same time, the N(a)-H(a)···O(a) and N(b)-H(b)···O(b) hydrogen bonds also became weaker in the tryptophan-CUR-piperazine complex. Just like investigation of the above fluorescence spectra, it seemed for CUR-piperazine CAP that the binding constant Kb values decreased compared with free CUR. Furthermore, the negative value of ∆H and ∆S for the binding

30


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reaction indicated that van der Waals forces as well as hydrogen bonds were involved in CUR-BSA complexation. Thus, the occupied hydrogen bonding binding sites in CAP weakened hydrogen bonding with BSA, which resulted in the decreasing of Kb values and theoretical interaction energy.

Fig.8. The electrostatic potentials on the 0.001 a.u. contour of the molecular electron density for CUR (a), tryptophan (b), CUR-piperazine complex (1:2) (c), and the molecular graphs for CUR-piperazine complex (1:2) (d), tryptophan-CUR (e), and tryptophan-CUR-piperazine (f)

Conclusion CUR-piperazine CAP were obtained using ethanol-assisted grinding methods in this paper. Molecular interactions in the CAP blends were systematically investigated and identified using FTIR, TMDSC, ssNMR characterization. The introduction of piperazine stabilized the di-keto structure of CUR and two hydrogen bonding of C=O…H-N were produced in CAP with CUR/piperazine mole ratio of 1:2. CUR-piperazine CAP showed lower dissolution rate than that of intact CUR above Tg temperature, but higher dissolution rate below Tg. Most importantly, the present binding study of CUR and BSA 31



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Page 32 of 41

utilized the ability of CUR to quench the fluorescence of BSA along with the DFT simulation to characterize the CUR-BSA interaction. The binding mechanism was identified as static quenching due to CUR-BSA complex formation both for free CUR and CUR-piperazine CAP with the different binding parameters calculated; thermodynamic parameters suggested a spontaneous binding involves bothvan der Waals forces

and

hydrogen

bonds.

The

decreasing

of

binding

constant

Kb

for

CUR-piperazine-BSA complex indicated decreasing of interaction force between CUR and BSA. Meanwhile, DFT simulation proposed that hydrogen bonds became weaker in the tryptophan-CUR-piperazine complex compare with CUR-tryptophan. With the various roles played by serum albumins, chiefly as drug carriers, it may be suggested that BSA may facilitate the delivery of CUR to its site of action. Moreover, since only unbound fraction had the pharmacological effect, BSA binding would has effect on CUR’s biological half-life in vivo. However, with introduction of piperazine into the structure of CUR, the binding constant of BSA and CUR in CAP decreased, indicating that unbound fraction increased. Compared with intact CUR, the increasing of unbound fraction of CUR in CAP would change the absorption, distribution, metabolism, and excretion properties of CUR. Hence, the work described in this study can be principally employed to investigate the pharmacological behavior of CUR-based CAP for application when administered to patients. Acknowledgments The financial supports for this work by the National Natural Science Funds of China (grants Nos: 81202504 and 21371045), and the Natural Science Foundation of Hebei

32


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Province (grant No: H2017206214) are greatly acknowledged. Thanks are also due to Education Department of Hebei Province of China through innovative hundred talents support program (SLRC2017047). References [1] Aaltonen J.; Rades T. Towards Physico-Relevant Dissolution Testing: The Importance of Solid-State Analysis in Dissolution. Dissolution Technol. 2009, 16, 47-54. [2] Hauss D.J. Oral lipid-based formulations. Advanced Drug Delivery Reviews. 2007, 59, 667-676. [3] Hancock B.C.; Zografi G. Characteristics and significance of the amorphous state in pharmaceutical systems. J. Pharm. Sci. 1997, 86(1), 1-12. [4] Jena S.K.; Singh C.; Dora C.P.; Suresh S. Development of tamoxifen-phospholipid complex: novel approach for improving solubility and bioavailability. Int. J. Pharm. 2014, 473 (1-2), 1-9. [5] Aditya N.P.; Yang H.; Kim, S.; Ko S. Fabrication of amorphous curcumin nanosuspensions using lactoglobulin to enhance solubility, stability, and bioavailability. Colloids Surf. B. 2015, 127, 114-121.

[6] Kola I.; Landis J. Can the pharmaceutical industry reduce attrition rates? Nat. Rev. Drug Discovery. 2004, 3, 711-715. [7] Zhang F.; Koh G. K.; Jeansonne D. P.; Hollingworth J.; Russo P. S.; Vicente G.; Stout, R. W.; Liu Z. A Novel solubility enhanced curcumin formulation showing stability and maintenance of anticancer activity. J. Pharm. Sci. 2011, 100, 2778-2789. [8] Gupta N. K.; Dixit V. K. Bioavailability enhancement of curcumin by complexation

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Comparative Studies on Group III

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σ-Hole and π-Hole Interactions. J. Comput. Chem. 2016, 37, 1321-1327.

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Preparation of curcumin-piperazine co-amorphous phase and

fluorescence spectroscopic and DFT simulation studies on the interaction with bovine serum albumin Wenzhe Pang, Jie Lv, Shuang Du, Jiaojiao Wang, Jing Wang*, Yanli Zeng*

Compared with CUR-tryptophan, the N-H∙∙∙O bond became weaker in the tryptophan-CUR-piperazine complex; the occupied hydrogen bonding binding sites in CAP weakened hydrogen bonding with BSA.


Preparation of curcumin-piperazine co-amorphous phase and

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