About 40% of marketed drugs and over 75% of drugs under development are poorly water-soluble,and this causes problems in drug development and their clinical use,representing one of the major challenges in the pharmaceutical science [1, 2]. Current strategies to address this low solubility problem include cocrystals, solid dispersions,and nanosizing [3, 4, 5]. Most of these approaches can be explained with the “spring-and-parachute” concept that drugs are dissolved first and make a supersaturated solution in the gastrointestinal tract,and then the supersaturated state is maintained for an extended period of time to maximize the oral absorption . Along the line with this concept,it is highly desirable to develop additives which can enhance the drug solubility in the aqueous solution,as they can be used to reduce the actual supersaturation level and hence prolong the supersaturation in vivo.
Among the studied solubilizing additives,arginine is an interesting example. Arginine is a natural amino acid with a zwitterionic head (NH3+-CHR-COO-) and a guanidinium tail group,which are connected by a short aliphatic segment (i.e.,3 methylene groups) in its molecule structure. The guanidinium group is a planar group with a high pKa (12.5 for arginine in water ),thus it is positively charged in aqueous solutions at all physiologically relevant pH. Due to this special structure,the guanidinium group is capable of forming ionic bonds and hydrogen bonds with anionic and hydrogen-bond-active groups (including water),respectively,in the guanidinium plane [7, 8]. However,the guanidinium group is poorly hydrated above and below the plane,and can form strong cation-π interaction with hydrophobic aromatic groups along the direction perpendicular to the guanidinium plane [9, 10, 11, 12, 13]. With this special structure, arginine has been used to suppress the aggregation of proteins, probably due to the interactions between the guanidinium groups and the protein surface residues [11, 14, 15, 16, 17, 18, 19]. Recently,it was reported that arginine can also increase the solubility of small aromatic compounds (e.g.,coumarin,benzyl alcohol,and alkyl gallates),but not of non-aromatic caffeine,suggesting that the solubilization effect is due to the cation-p interactions between the guanidinium group of arginine and the aromatic compounds [9, 10, 13, 20].
However,one problem with arginine is that high concentration is often needed to be effective on drug solubilization. For example, 1 mol/L (174 mg/mL) arginine is needed to double the coumarin solubility . We hypothesized that a polymer of argininederived monomers should have a high local concentration of guanidinium groups and may have stronger interaction with aromatic compounds (Fig. 1),i.e.,the aromatic plane may bind simultaneously with two guanidinium groups or a guanidinium group may interact with two aromatic compounds . Here,we designed a guanidinium-containing polymer based on an argininederived monomer (Scheme 1),and compared with arginine for their effect on the solubility of three model compounds (i.e., coumarin,pyrene and doxorubicin).
|Fig. 1.Hypothesized interactions between aromatic compounds with the guanidinium side groups along the polymer chains.|
2. Experimental 2.1. Materials
L-Arginine,methanol and coumarin were purchased from J&K Chemicals. Methacrylic anhydride,triethylamine (TEA),ammonium persulfate (APS) and N,N,N',N'-tetramethyl ethylene diamine (TMEDA) were purchased from Alfa Aesar. 3-(Trimethylsilyl)-1- propanesulfonic acid sodium salt (DSS) and pyrene were purchased from Sigma-Aldrich. Doxorubicin hydrochloride (Dox-HCl) was purchased from Melone Pharma (Liaoning,China). Thionyl chloride (SOCl2) was purchased from Aladdin. Dioxane and acetone were purchased from Beijing Chemical Company. Phosphate buffer saline (PBS) tablets were purchased from Amresco. All reagents were of analytical grade and used as received.2.2. Synthesis of N-methacryl arginine (M-Arg,I)
M-Arg was prepared following the method in literature  with modifications. L-Arginine (2 g,11.5 mmol) was dissolved in a mixed solvent of deionized water (20 mL) and dioxane (8.5 mL). Then TEA (4.5 mL,32.3 mmol) was added and the solution was cooled with an ice/water bath. Methacrylic anhydride (3 mL, 18.9 mmol) was added dropwise over a period of 10 min under stirring. Then the ice/water bath was removed and the mixture was stirred overnight at room temperature. The product was precipitated in acetone (400 mL). The precipitates were then redissolved in water and precipitated again in acetone. The precipitation step was repeated two times. White powder was obtained and dried under vacuum at room temperature (yield 75%).
1H NMR (D2O,400 MHz,Fig. S1a in Supporting Information): δ 1.60 (m,2H,-CH2-CH2-CH2-),1.74 and 1.87 (m,2H,-CH(COOH)- CH2-),1.92 (s,3H,-CH3),3.18 (t,2H,-NH-CH2-),4.22 (q,1H, -NH-CH(COOH)-),5.44 and 5.70 (s,2H,=CH2). 13C NMR (D2O, 400 MHz,Fig. S1b): δ 17.74 (-CH3),24.56 (-CH2-CH2-CH2-),28.81 (-CH(COOH)-CH2-),40.67 (-NH-CH2-),54.86 (-NH-CH(COOH)- ),121.17 (=CH2),139.04 (=C(CH3)-),156.75 (-NH-C(NH)-NH2), 171.25 (-NH-CO-),178.61 (-COOH). MS (ESI,Fig. S1c): m/z 243 (M+H+,theoretical value M = 242),485 (2M+H+,theoretical value 2M = 484).2.3. Synthesis of poly(M-Arg) (II)
The monomer M-Arg (I) was polymerized via the redoxinitiated radical polymerization,using APS and TMEDA as the redox initiator pair. M-Arg (2 g,8.3 mmol) and APS (80 mg, 0.4 mmol) were dissolved in deionized water (40 mL) and nitrogen was passed through the solution for 30 min to remove oxygen. TMEDA (80 μL,0.5 mmol) was added under stirring. The reaction proceeded at room temperature for 24 h,and then the precipitate in the solution were obtained by filtration and washed with water for three times. The resulted white powder was dried under vacuum at room temperature (yield 88%).
1H NMR (D2O,400 MHz,Fig. S2a in Supporting Information): δ 0.75-1.25 (m,3H,-CH3),1.44-2.24 (broad,6H,-CH2-CH2-CH2-,- CH(COOH)-CH2-,-(CH3)C(COR)-CH2-),3.22 (m,2H,-NH-CH2--), 4.23 (m,1H,-NH-CH(COOH)-). 13C NMR (D2O,400 MHz,Fig. S2b): δ 17.09 (-CH3),25.15 (-CH2-CH2-CH2-),27.83 (-CH(COOH)- CH2-),40.68 (-NH-CH2-),45.05 (-(CH3) C (COR)-),52.31-55.01 (-NH-CH(COOH)-,-(CH3)C(COR)-CH2-),156.74 (-NH-C(NH)- NH2),175.48 (-NH-CO-),178.94 (-COOH).2.4. Synthesis of poly (M-Arg methyl ester hydrochloride) (poly (M-Arg-OMe-HCl),III)
The suspension of poly(M-Arg) (1 g,corresponding to 4.1 mmol M-Arg unit) in methanol (20 mL,0.5 mol) was cooled with an ice/ water bath. SOCl2 (0.5 mL,6.9 mmol) was added dropwise under stirring and the suspension gradually turned into a clear solution. The solution was then refluxed at 70 ℃ for 24 h. The solvent was evaporated with a rotary evaporator,and the residue was then redissolved in methanol and precipitated in acetone. The precipitation step was repeated twice. White powders were obtained after filtration and dried under vacuum at room temperature (yield 89%).
1HNMR (D2O,400MHz,Fig. 3a): d 0.75-1.24 (m,3H,-CH3),1.47- 2.16 (broad,6H,-CH2-CH2-CH2-,-CH(COOCH3)-CH2-,-(CH3) C(COR)-CH2-),3.25 (m,2H,-NH-CH2-),3.76 (m,3H,-COOCH3), 4.28 (m,1H,-NH-CH(COOCH3)-). 13CNMR(D2O,400MHz,Fig. 3b): δ 16.97 (-CH3),25.25 (-CH2-CH2-CH2-),27.75 (-CH(COOH)-CH2-), 40.77 (-NH-CH2-),45.23 (-(CH3)C(COR)-),52.97 (-COOCH3),53.29- 54.46 (-NH-CH(COOH)-,-(CH3)C(COR)-CH2-),156.82 (-NH-C(NH)- NH2),174.17 (-NH-CO-),179.28 (-COOCH3).
|Fig. 3.The (a) 1H NMR spectrum (400 MHz, D2O), (b) 13C NMR spectrum (400 MHz, D2O), and (c) the circular dichroism spectrum of poly(M-Arg-OMe).|
The solubility of coumarin,pyrene and Dox in PBS with additives at different concentrations (0-100 mmol/L; for polymers, the concentration refers to the concentration of repeating unit) were measured as follows. Stock solutions of additives at different concentrations were prepared in PBS (10 mmol/L phosphate, 137 mmol/L NaCl and 2 mmol/L KCl) and were adjusted to pH 7.4 using concentrated NaOH or HCl solutions. Excess amounts of coumarin or pyrene (～10 mg/mL) powder were added into the additive solutions. For the group of Dox,Dox-HCl suspension was prepared at 20 mg/mL in PBS and pH was adjusted to 7.4. Then the suspension was added into additive stock solutions and each of the final solutions contained 10 mg/mL Dox-HCl. The samples of coumarin,pyrene or Dox were then placed in a shaker incubator (TS-100C,Tensuc) with a shaking speed of 300 r/min at 30 ℃. After 24 h,the samples were centrifuged (16,000 r/min,20 min,TG16- 11,Pingfan) to remove the undissolved solid.
The supernatantswerediluted (400-foldfor coumarin,10-fold for pyrene and 8-fold for Dox) with PBS. The concentrations of soluble coumarin or Dox in the supernatants were measured by UV absorbance (at 277 nm for coumarin and 485 nm for Dox) with a UV-Vis spectrophotometer (Agilents-8435,Agilent) and calculated with the pre-determined calibration curves. The concentrations of solublepyrene inthe supernatantsweremeasuredbyfluorescence at 383 nm(I3) on excitation at 334 nm(excitation slit andemission slit: 2.5 nm,PMTVolta: 700 V) with afluorescence spectrometer (F-7000, Hitachi),and also calculated with a pre-determined calibration curve. All the measurements were conducted in triplicate.2.6. Precipitation of Dox with and without poly(M-Arg-OMe)
Dox-HCl stock solution was prepared at 2 mg/mL in D2O. Poly(M-Arg-OMe) stock solutions at different concentrations were prepared in PBS (20 mmol/L,274 mmol/L NaCl and 4 mmol/L KCl, prepared in D2O with 1 mg/mL DSS added as the internal standard) and pH was adjusted to 7.4 (pD 7.8). Then,0.25 mL of the Dox stock solution was mixed with 0.25 mL of the polymer stock solutions in glass bottles respectively,and then the solutions were transferred from the bottles into NMR tubes. The final solutions contained 1 mg/mL Dox·HCl (0.94 mg/mL Dox),0.5 mg/mL DSS and 0- 20 mmol/L polymer in terms of the guanidinium groups (0 mmol/L,5 mmol/L,10 mmol/L and 20 mmol/L respectively), pH ～ 7.2 (pD ～ 7.6). Then 1H NMR spectra of the samples were measured every 5 min,and the concentrations of unprecipitated Dox in the samples were calculated by relative peak area of the phenyl ring protons of Dox (7.3 ppm (2H) and 7.6 ppm (1H)) and the H peak area of -CH3 of DSS (0 ppm,9H).3. Results and discussion
Poly(M-Arg-OMe-HCl) was synthesized through 3 steps (Scheme 1). We first followed the exact method in literature  to make M-Arg,but in our hand,the final products contained significant amount of impurities,which were also difficult to remove. Therefore,we modified the synthesis protocol as follows: organic base TEA was used as acid scavenger instead of NaHCO3, and dioxane was added as a co-solvent to dissolve TEA. After precipitation in acetone,M-Arg products of high purity (Fig. S1) were obtained.
Then,M-Arg was polymerized into poly(M-Arg). Poly(M-Arg) was initially designed as the final polymer to be used in our solubilization studies. Unfortunately,the zwitterionic polymer was found to be poorly water soluble itself unless the pH was adjusted to be below 3 or above 13. The poor solubility of poly(MArg) at pH 3-13 may be due to the strong inter-chain association between the carboxyl groups and the guanidinium groups. To improve its aqueous solubility,the zwitterionic polymer was esterified to obtain a polycation,i.e.,poly(M-Arg-OMe),which showed good water solubility. The 1H NMR spectrum and 13C NMR spectrum of poly(M-Arg-OMe) are shown in Fig. 3a and b respectively,where the characteristic peaks of each H and C were clearly assigned and the relative peak area ratios in the 1H NMR spectrum were consistent with the target structure. The circular dichroism spectrum demonstrated that the conformation of poly(M-Arg-OMe) was mostly random coil in water (Fig. 3c). The molecular weight of the polymer could not be measured by GPC because the polymer was found to bind the column too strongly to be eluted. Instead,we measured the size of single polymer chains in water (Zetasizer Nano ZS90,Malvern) to be 3-10 nm in diameter,and using the calibration curve made of poly(ethylene glycol) ,the molecular weight of poly(M-Arg- OMe) was estimated to be in order of magnitude of 103-104 Da.
After the guanidinium-containing polymer poly(M-Arg-OMe) was successfully synthesized,the effects of the polymer and arginine on aromatic organic compounds were studied. Coumarin was used as the first compound because it was already proved to be solubilized by arginine . Coumarin solubility in the pH 7.4 PBS at 30 8C was found to be 2.69 mg/mL (18.5 mmol/L) in our study, which is consistent with literature . As shown in Fig. 4a,both arginine and poly(M-Arg-OMe) additives significantly increased the solubility of coumarin,and arginine seemed to be more effective than poly(M-Arg-OMe). For example,the solubility of coumarin was increased by 50% in the presence of 100 mmol/L arginine and by 26% with 100 mmol/L poly(M-Arg-OMe) (weight concentration of the additives are about 1.7 wt% and 2.5 wt%, respectively).
|Fig. 4.Solubility of (a) coumarin and (b) pyrene in the presence of arginine and poly(M-Arg-OMe) at different concentrations at pH 7.4 in PBS. The concentration of the polymer refers to the concentration of guanidinium groups.|
In contrast,poly(M-Arg-OMe) was found to be more effective than arginine in enhancing the solubility of pyrene (Fig. 4b). Pyrene is a molecule with a larger aromatic ring (Fig. 2),and it is often used as a fluorescent probe with very low aqueous solubility. Its solubility in pH 7.4 PBS at 30 8C was determined as 0.06 μg/mL (3 × 10-7 mol/L) in our study,also consistent with the literature value . As shown in Fig. 4b,the solubility of pyrene was increased by one- and sixfold in the presence of 100 mmol/L arginine and poly(M-Arg-OMe),respectively.
According to Li et al. ,a possible mechanism for the solubilization effect of arginine is that the guanidinium group of arginine binds to aromatic compounds via the cation-π interaction to prevent their hydrophobic association,while the hydrophilic polar head extends out to make the complex water soluble. The different performance of arginine and poly(M-Arg-Ome) in the pyrene and coumarin cases may be due to the fact that pyrene has an aromatic plane large enough to take the advantage of the multivalent guanidinium structure of poly(M-Arg-OMe),as hypothesized in Fig. 1,while the smaller coumarin compounds can not.
To further test the hypothesis that compounds with larger aromatic planes benefit more from the solubilizing effect of the polymer,the solubility of doxorubicin was studied. Dox is an anticancer drug,and similar to pyrene,it contains a large aromatic molecule plane. Dox is soluble in acidic water but poorly soluble at neutral pH. In our study,the solubility of Dox was found to be 0.22 mg/mL (0.4 mmol/L) at pH 7.4 in 10 mmol/L PBS at 30 ℃. As shown in Fig. 5,the solubility of Dox was enhanced more significantly in the presence of poly(M-Arg-OMe) than arginine at the same guanidinium concentrations. For example,it was increased by 2-fold by 100 mmol/L arginine and 11-fold by 100 mmol/L poly(M-Arg-OMe). The result is consistent with the hypothesis that poly(M-Arg-OMe) would be much more effective to solubilize organic compounds with larger aromatic molecule plane.
|Fig. 5.Solubility of Dox in the presence of arginine and poly(M-Arg-OMe) at different concentrations at pH 7.4 in PBS. The concentration of the polymer refers to the concentration of guanidinium groups.|
Finally,we tested whether or not that poly(M-Arg-Ome) is effective to prolong the supersaturation of doxorubicin. Fig. 6 shows the precipitation process of 1 mg/mL supersaturated Dox-HCl solution (corresponding to 0.94 mg/mL Dox) in PBS with different concentrations of the additive in 1 h. The concentration of Dox without the additve was about 0.6 mg/mL in 1 h after the the sample was prepared,still higher than the equilibrium solubility of Dox. The plateau concentration of Dox increased as the concentration of the added polymer was increased from 0 to 20 mmol/L. It is 12%,25% and 57% higher than that without the additive, coresponding to the addition of 5 mmol/L,10 mmol/L and 20 mmol/L poly(M-Arg-OMe),respectively. The picture inset in Fig. 6 shows the amount of precipitates in the NMR tubes 24 h after the measurement (from left to right,0,5,10,and 20 mmol/L of polymer additives),and the sample with 20 mmol/L polymer remained a clear solution without any visible pricipitates, indicating that the polymer did increase the solubility of Dox and inhibit its precipitation.
|Fig. 6.The concentration–time profiles of Dox supersaturated solution with different amount of polymer additives. The photo was taken 24 h after the NMR measurement. From left to right, the concentration of the polymer was 0 mmol/L, 5 mmol/L, 10 mmol/L and 20 mmol/L, respectively.|
In this study,we designed and synthesized a guanidiniumcontaining polymer and evaluated its potential as a solubility enhancing additive. The polymer significantly increased the solubility of pyrene and Dox in aqueous solutions,much more effective than arginine at the same guanidinium group concentration. In contrast,the polymer was less effective on the solubility of the smaller coumarin,suggesting that compounds with larger aromatic groups may benefit more from the polymer structure. However,the exact mechanism of the solubilizing effects of the polymer and arginine still need to be further studied.Acknowledgment
This study is supported by the Natural Science Foundation of China (No. 21434008).Appendix A. Supplementary data
Supplementary data associated with this article can be found,in the online version,at http://dx.doi.org/10.1016/j.cclet.2015.04.009.
|||L. Di, E.H. Kerns, G.T. Carter, Drug-like property concepts in pharmaceutical design, Curr. Pharm. Des. 15 (2009) 2184-2194.|
|||T. Takagi, C. Ramachandran, M. Bermejo, et al., A provisional biopharmaceutical classification of the top 200 oral drug products in the United States, Great Britain, Spain, and Japan, Mol. Pharm. 3 (2006) 631-643.|
|||Y.B. Huang, W.G. Dai, Fundamental aspects of solid dispersion technology for poorly soluble drugs, Acta Pharm. Sin. B 4 (2014) 18-25.|
|||Y. Zhang, M.Y. Xu, T.K. Jiang, W.Z. Huang, J.Y. Wu, Low generational polyamidoamine dendrimers to enhance the solubility of folic acid: a "dendritic effect" investigation, Chin. Chem. Lett. 25 (2014) 815-818.|
|||L. Wang, L.L. Li, H.L. Ma, H. Wang, Recent advances in biocompatible supramolecular assemblies for biomolecular detection and delivery, Chin. Chem. Lett. 24 (2013) 351-358.|
|||H.R. Guzman, M. Tawa, Z. Zhang, et al., Combined use of crystalline salt forms and precipitation inhibitors to improve oral absorption of celecoxib from solid oral formulations, J. Pharm. Sci. 96 (2007) 2686-2702.|
|||K.A. Schug, W. Lindner, Noncovalent binding between guanidinium and anionic groups: focus on biological-and synthetic-based arginine/guanidinium interactions with phosph[on]ate and sulf[on]ate residues, Chem. Rev. 105 (2005) 67-114.|
|||N. Sakai, S. Matile, Anion-mediated transfer of polyarginine across liquid and bilayer membranes, J. Am. Chem. Soc. 125 (2003) 14348-14356.|
|||T. Arakawa, Y. Kita, A.H. Koyama, Solubility enhancement of gluten and organic compounds by arginine, Int. J. Pharm. 355 (2008) 220-223.|
|||A. Hirano, T. Kameda, T. Arakawa, K. Shiraki, Arginine-assisted solubilization system for drug substances: solubility experiment and simulation, J. Phys. Chem. B 114 (2010) 13455-13462.|
|||D. Shukla, B.L. Trout, Interaction of arginine with proteins and the mechanism by which it inhibits aggregation, J. Phys. Chem. B 114 (2010) 13426-13438.|
|||P.E. Mason, G.W. Neilson, J.E. Enderby, et al., The structure of aqueous guanidinium chloride solutions, J. Am. Chem. Soc. 126 (2004) 11462-11470.|
|||J.G. Li, M. Garg, D. Shah, R. Rajagopalan, Solubilization of aromatic and hydrophobic moieties by arginine in aqueous solutions, J. Chem. Phys. 133 (2010) 054902.|
|||K. Tsumoto, M. Umetsu, I. Kumagai, et al., Role of arginine in protein refolding, solubilization, and purification, Biotechnol. Prog. 20 (2004) 1301-1308.|
|||B.M. Baynes, D.I.C. Wang, B.L. Trout, Role of arginine in the stabilization of proteins against aggregation, Biochemistry 44 (2005) 4919-4925.|
|||U. Das, G. Hariprasad, A.S. Ethayathulla, et al., Inhibition of protein aggregation: supramolecular assemblies of arginine hold the key, PLoS One 11 (2007) e1176.|
|||J. Arakawa, M. Uegaki, T. Ishimizu, Effects of L-arginine on solubilization and purification of plant membrane proteins, Protein Expr. Purif. 80 (2011) 91-96.|
|||M.M. Varughese, J. Newman, Inhibitory effects of arginine on the aggregation of bovine insulin, J. Biophys. 2012 (2012) 434289.|
|||D.X. Zhao, Z.X. Huang, Effect of arginine on stability of GST-ZNF191 (243-368), Chin. Chem. Lett. 18 (2007) 355-356.|
|||A. Hirano, T. Arakawa, K. Shiraki, Arginine increases the solubility of coumarin: comparison with salting-in and salting-out additives, J. Biochem. 144 (2008) 363-369.|
|||J.G. Cheng, X.M. Luo, X.H. Yan, et al., Research progress in cation-p interactions, Sci. China Ser. B: Chem. 51 (2008) 709-717.|
|||Y. Kim, S. Binauld, M.H. Stenzel, Zwitterionic guanidine-based oligomers mimicking cell-penetrating peptides as a nontoxic alternative to cationic polymers to enhance the cellular uptake of micelles, Biomacromolecule 13 (2012) 3418-3426.|
|||Z.L. Luo, G.Z. Zhang, Scaling for sedimentation and diffusion of poly(ethylene glycol) in water, J. Phys. Chem. B 113 (2009) 12462-12465.|
|||L.C. Cartwright, Vanilla-like synthetics, solubility and volatility of propenyl guaethyl, bourbonal, vanillin, and coumarin, J. Agric. Food Chem. 1 (1953) 312-314.|
|||D. Mackay, W.Y. Shiu, Aqueous solubility of polynuclear aromatic hydrocarbons, J. Chem. Eng. Data 22 (1977) 399-402.|