ZHANG Yn-Jie LOU Ben-Yong HUANG Y-Li ZHANG Mei LIN Qi, b

?

Crystal Structure, Stability and Dissolution of a Drug-drug Molecular Salt Hydrate of Berberine with Protocatechuic Acid①

ZHANG Yan-JieaLOU Ben-Yonga②HUANG Ya-LiaZHANG MeiaLIN Qia, b

a(350108)b(350108)

berberine, protocatechuic acid, molecular salt, hydrate, non-hygroscopic;

1 INTRODUCTION

Salt formation represents an effective approach to improve physicochemical properties of active phar- maceutical ingredients (APIs)[1-3]. Although cocrys- tallization represents an emerging crystal engineering strategy[4-6], salt formation has remained a primary choice for ionized acids or bases. Especially, drug- drug molecular salts based on two kinds of different APIs have been exploited as new salt forms during recent years[7-9].

Berberine is a kind of natural alkaloid extracted from Huanglian (Rhizoma Coptidis), a herb that has been used for thousands of years in traditional Chinese medicine due to its antimicrobial activity[10, 11]. Much research work showed that berberine is also a potential drug in the treatment of diabetes, hyper- lipidemia and cancer[12-14]. Berberine is commonly marketed as hydrochloride salt[15]. The salt exhibited solid state instability since it could undergo solid state transformations among anhydrate, dehydrate and tetrahydrate depending on relative humidity[16]. Recently, Fang and Sun developed two organic salts of berberine with the sweeteners acesulfame and saccharine through anion exchange reactions[17]. The two sweet salts exhibited good stability against high humidity and acceptable tableting performance.

Protocatechuic acid is a kind of natural organic acid with antimicrobial activity which could be extracted from many vegetables[18]. Like berberine, it has also a potential in the treatment of cardiovascular disease and cancer[19]. Hence, drug-drug molelcular salt based on berberine and protocatechuic acid may have enhanced pharmacological effects. In this work, we prepared a drug-drug molecular salt hydrate, [C20H18NO4]+[C7H5O4]-?H2O (1), through anion exchange reaction between berberine chloride and protocatechuic acid. The structural characterization and preliminary physicochemical properties were studied.

2 EXPERIMENTAL

2. 1 Synthesis of [C20H18NO4]+[C7H5O4]-?H2O (1)

A mixture of berberine chloride (370 mg, 1 mmol), NaOH (40 mg, 1 mmol) and protocatechuic acid (154 mg, 1 mmol) with 0.1 mL ethanol added, was ground with a LAB WIZZ 320 ball mill in a 25 mL steel vessel for 15 min with a 15 mm steel ball at 30 Hz. The resulting powder was washed by water to remove the by-product NaCl and then re-crystallized in EtOH to give rise to yellow needle crystals.

2. 2 Structure determination

A yellow single crystal (0.25mm × 0.10mm × 0.05mm) was mounted on a glass fiber for data collection which was performed on a SuperNova CCD diffractometer at 293 K, using Cu-radiation (= 1.54178 ?). A total of 7774 reflections together with 4480 independent ones (int= 0.0226) were collected in the range of 4.36<<73.67° by using an-2scan mode, of which 3993 were observed with> 2() and used in the succeeding refinement. The empirical absorption corrections were applied by using the CrysAlisPro program[20]. The structure was solved by direct methods and refined on2by full-matrix least-squares methods with anisotropic displacement parameters for non-hydrogen atoms using the SHELXTL-97 program package[21]. Two C atoms, C(15) and C(16), of berberine cation are disordered over two positions (C(15)/C(15A) and C(16)/C(16A)) and were refined with a fixed occupancy ratio of 0.75:0.25. H atoms on C atoms were located geometrically (C–H = 0.95～0.99 ?) withiso(H) = 1.2eq(C) or 1.5eq(C). H atoms on O atoms were located by difference maps and the displacement factors were freely refined. The final= 0.0488,= 0.1378 (= [2(F2) + (0.0746)2+ 0.5748], where= (F2+ 2F2)/3), ()max= 0.596, (?)min= ?0.478 e/?3, (/)max= 0.000 and= 1.043.The selected bond lengths and bond angles for 1 are listed in Table 1. Hydrogen bond lengths and bond angles are given in Table 2.

Table 1. Selected Bond Lengths (?) and Bond Angles (°)

Table 2. Hydrogen Bond Lengths (?) and Bond Angles (°)

Symmetry codes: (a) 1+,,; (b) 1–, –, 2–z

2. 3 Powder X-ray diffractions (PXRD) and thermal analysis

PXRD data were collected on a Rigaku MiniFlex 600 diffractometer, equipped with Scintillation Counter detector, with Curadiation (40 kV and 15 mA). Each pattern wascollected with a step size of 0.02° in the 2range of 5～50°. PXRD data after DVS experiments were collected on a PANalytical Empyrean PXRD diffractometer. The thermal analysis was determined on a NETZSCH STA 449C analyzer with a heating rate of 10 °C/min under N2gas atmosphere.

2. 4 Dynamic Vapor Sorption (DVS)

DVS was measured via a SMS (Surface Measure- ment Systems) DVS Intrinsic at 25 oC. The relative humidity at 25 oC was calibrated against delique- scence point of LiCl, Mg(NO3)2and KCl. The nitrogen flow rate was 200 mL/min. The sample equilibrated at each step with the equilibration criteria of either dm/dt≤0.002% or maximum equilibration time of 3 h. Once one of the criteria was met, the relative humidity (RH) was changed to the next target value, following the 40-95-0-95% sorption and desorption cycle with a step size of 10% RH.

2. 5 Solubility and dissolution studies

The solubility of 1 was determined at 37 °C in pure water. Excess amounts of 1 were suspended in 10 mL of water in screw-capped glass vials, respec- tively. These vials were kept at 37 °C and stirred at 100 rpm using a magnetic stirrer. After 48 h, the suspensions were filtered through 0.2 μm syringe filter. The filtered aliquots were sufficiently diluted, and the absorbance was measured at 261 nm in triplicate. Finally, the concentration of 1 after 48 h in each sample was determined from the standard graph. A standard graph for compound 1 was made by measuring the absorbance of varied concentrations of 1 (2～14 mg/L) in water solution using a Shimadzu UV-2500 spectrophotometer at 261 nm. The calibrated plot showed a good correlation coefficient (= 0.067– 0.003,2= 0.999).

The samples of 1 were micronized and sieved using standard-mesh sieves (meshsize 150m). Three 1000 mL round-bottomed flasks containing 900 mL water were equilibrated at 37.0 °C. The 150 mg samples of 1 containing 100 mg berberine were added to the flasks, and the resulting slurry was stirred at 100 rpm. At specific time intervals, 2 mL of the slurry was withdrawn from the three flasks and filtered through a 0.2m syringe filter. Additional 2 mL fresh solvent was added into the flasks after sampling. The concentration in each sample was determined from the standard graph.

3 RESULTS AND DISCUSSION

3. 1 Crystal structure of 1

Fig. 1 ORTEP drawing of 1 with 30% probability displacement ellipsoids

Fig. 2. 1D double-chainstructure of protocatechuic anion and water in 1 along theaxis

Fig. 3. Packingstructure in 1 viewed along theaxis

3. 2 Thermal analysis and powder X-ray powder diffraction

The thermogravimetric analysis (TGA) of 1 lost water molecules (4.2%) during 50～130 oC (Calcd: 3.5%) and it begins to decompose rapidly after 200 oC. The differential scanning calorimetry (DSC) of 1 shows one wide endothermic peak at 130 oC and one sharp peak at 210 oC, which corresponds dehydration and decomposition process, respectively.The patterns of powder X-ray powder diffraction (PXRD) of 1 are in accordance with simulated patterns from single crystal diffractions, which verified the purity of the hydrate (Fig. 4).

Fig. 4. PXRD patterns of 1a) Simulated patterns of 1; b) Experimental patterns of 1; c) Patterns of 1 after DVS experiments

3. 3 Dynamic Vapor Sorption (DVS)

Berberine chloride is reported to undergo solid state transformations among anhydrate, dehydrate and tetrahydrate depending on relative humidity[16, 17].contrary, compound 1 is non-hygroscopic and absorbs less than 0.4% of moisture even at 95% RH (Fig. 5). The hydrate also exhibits good stability even at 0% RH. PXRD of samples of 1 after DVS indicated that the crystal structure kept unchanged after complete absorption at 95% RH. The good stability of 1 during 0～95% RH could be attributed to the strong hydrogen-bonding interactions between water molecules and carboxylate anions of proto- catechuic acid.

Fig. 5. DVS result of 1

3. 4 Solubility and dissolution studies

The solubility of 1 measured in water at 37 °C was 740 mg/L. This corresponds to 490 mg berberine and 250 mg protocatechuic acid dissolved in 1 L water. The solubility is much lower than that of berberine chloride (4.9 g/L)[17]. However, for the typical dose of 100 mg of berberine, the molecular salt hydrate is soluble according to the Biopharmaceutics Classi- fication System (BCS) definition[17]. For dissolution experiment, above 60% of samples of 1 could be dissolved in water at the first 10 minutes and 100% dissolution could be achieved within 1 h (Fig. 6).

Fig. 6. Dissolution profiles of 1

4 CONCLUSION

In conclusion, a 1:1:1 drug-drug salt hydrate of berberine and protocatechuic acid has been prepared. Protocatechuic acid lost its carboxylic proton and turned to be a protocatechuic anion. The carboxy- late of protocatechuic acid is simultaneously hydro- gen-bonded to two hydroxyl groups of protoca- techuic anion to give a 1D hydrogen-bonded chain of anions. Water molecule is hydrogen-bonded to carboxylate anions to form a synthon R24(8) which linked the 1D chains into a centrosymmetric double chain of protocatechuic anions. The hydrate exhibited good solid state stability during 0～95% RH, which may result from strong hydrogen- bonding interactions between water molecules and carboxylate anions. The hydrate also exhibited acceptable solubility and dissolution rate.

(1) Stahl, P.H.; Wermuth, C.G.. Wiley-VCH, Chichester2002.

(2) Sanphui, P.; Tothadi, S.; Ganguly, S.; Desiraju, G. R. Salt and cocrystals of sildenafil with dicarboxylic acids: solubility and pharmacokinetic advantage of the glutarate salt.2013, 10, 4687–4697.

(3) Black, S. N.; Collier, E.; Davey, R. J.; Roberts, R. J. Structure,solubility, screeningandsynthesis of molecularsalts.. 2007, 96, 1053–1068.

(4) Almarsson, ?.; Zaworotko, M. J. Crystal engineering of the composition of pharmaceutical phases. Do pharmaceutical co-crystals represent a new path to improved medicines?. 2004, 40, 1889–1896.

(5) Sun, C. C. Cocrystallization for successful drug delivery.. 2013, 10, 201–213.

(6) Desiraju, G.R.; Vittal, J.J.; Ramanan, A.. World Scientific, Singapore2011.

(7) Thorat, S.H.; Sahu, S.K.; Patwadkar, M.V.; Badiger, M.V.; Gonnade, R.G. Drug-drug molecular salt hydrate of an anticancer drug gefitinib and a loop diuretic drug furosemide: an alternative for multidrug treatment.. 2015, 104, 4207–4216.

(8) Bag, P.P.; Ghosh, S.; Khan, H.; Devarapalli, R.; Reddy, C.M. Drug-drug salt forms of ciprofloxacin with diflunisal and indoprofen.2014, 16, 7393–7396.

(9) Gopi, S. P.; Ganguly, S.; Desiraju, G. R. A drug-drug salt hydrate of norfloxacin and sulfathiazole: enhancement ofbiological properties via improved physicochemical properties.2016, 13, 3590–3594.

(10) Baird, A.W.; Taylor, C. T.; Brayden, D. J.Non-antibiotic anti-diarrhoeal drugs: factors affecting oral bioavailability of berberine and loperamide in intestinal tissue..1997, 23, 111–120.

(11) Habtemariam, S. Berberine and inflammatory bowel disease: a concise review.. 2016, 113, 592–599.

(12) Jin, Y.; Khadka, D. B.; Cho, W. J. Pharmacological effects of berberine and its derivatives: a patent update.. 2016, 26, 229–243.

(13) Sun, Y. Y.; Xu, K. L.; Wang, Y. T.; Chen, X. P. A systematic review of the anticancer properties of berberine, a natural product from Chinese herbs.. 2009, 20, 757–769.

(14) Liu, C. S.; Zheng, Y. R.; Zhang, Y. F.; Long, X. Y. Research progress on berberine with a special focus on its oral bioavailability.2016, 109, 274–282.

(15) Chen, C. Q.; Tao, C. H.; Liu, Z. C.; Lu, M. L.; Pan, Q. H.; Zheng, L. J.; Li, Q.; Song, Z. S.; Fichna, J.A randomized clinical trial of berberine hydrochloride in patients with diarrhea-predominant irritable bowel syndrome.. 2015, 29, 1822–1827.

(16) Tong, H. H.; Chow, A. S.; Chan, H.; Chow, A. H.; Wan, Y. K.; Williams, I. D.; Shek, F. L.; Chan, C. K. Process-induced phase transformation of berberine chloride hydrates.. 2010, 99, 1942–1954.

(17) Wang, C.; Perumalla, S. R.; Lu, R.; Fang, J.; Sun, C. C. Sweet berberine.2016, 16, 93–939.

(18) Yamabe, N.; Park, J. Y.; Lee, S.; Cho, E. J.; Lee, S.; Kang, K. S.; Hwang, G. S.; Kim, S. N.; Kim, H. Y.; Shibamoto, T. Protective effects of protocatechuic acid against cisplatin-induced renal damage in rats.2015, 19, 20–27.

(19) Masella, R.; Santangelo, C.; D’Archivio, M.; LiVolti, G.; Giovannini, C.; Galvano, F. Protocatechuic acid and human disease prevention: biological activities and molecular mechanisms.. 2012, 19, 2901–2917.

(20) Rigaku Oxford Diffraction. CrysAlisPro. Empirical absorption correction using spherical harmonics, implemented in SCALE3 ABSPACK scaling algorithm. Agilent Technologies Ltd, Yarnton 2015.

(21) Sheldrick, M. A short history of SHELX.2008, 64, 112–122.

19 July 2017;

2 November 2017 (CCDC 1526052)

①The work was supported by the National Natural Science Foundation of China (21503105), the Natural Science

of Fujian Province (2015J01599, 2017J01584) and Research Project for Fujian Provincial Universities (JK2015038)

. Lou Ben-Yong. Research interest: Pharmaceutical material chemistry. E-mail: lby@mju.edu.cn

10.14102/j.cnki.0254-5861.2011-1789