| Drug-small molecule complex,including co-crystal and co-amorphous,is new solid form in which active pharmaceutical ingredient(API)and co-crystal former(CCF)or co-amorphous former(CAF)are connected by hydrogen bonding,which is new technology and new method to improve the physicochemical properties of API.Azelnidipine(AZE)is a new dihydropyridine calcium antagonist which is used to treat moderate and severe hypertension.Compared with analogous drugs,AZE has diuretic,cardiac protection,renal protection,anti-atherosclerosis and other advantages.AZE is a polymorphic drug which has ? and ? forms with lower water-solubility.They have different physicochemical properties because of the different arrangement of molecular and space docking.For example,? form has slightly higher solubility than ? form.However,? form is a metastable phase and easily transforms to form ? at suitable conditions.Previous studies have shown that the formation of drug-coformer complex is a powerful approach in enhancing the solubility,dissolution rate,stability and even bioavailability of poor water-soluble drugs.Objective: One of the purposes of this paper is to screen drug-CCF(or CAF)complexes used the two crystal forms of the AZE as the model API.The complexes were prepared and characterized and the physochemical properties of the complexes were evaluated.The interaction forces between the two components in the supramolecular complex were analyzed.The effects of different crystal forms on the formation and physicochemical properties of the complex were investigated.Another objective of this paper is to study the interaction between the complexes and human serum albumin(HSA)at simulating the physiological environment,so as to explore the change in the interaction mode and rule of human serum albumin with API-small moleculesupramolecular complex at the molecular level.Methods: Used the two crystal forms of the AZE as the model API and two different biocompatible small molecules of oxalic acid(OXA)and piperazine(PEZ)as co-former molecules,the complexes were screened by solvent(ethanol)assisted solvent grinding(SG)and neat powder grinding(NG)mothods.In addition,the interaction between the complex and human serum albumin were studied by fluorescence spectroscopy and UV-Vis spectroscopy.1 Complex characterization: AZE-OXA complex were prepared at AZE/OXA molar ratio of 2:1,1:1 and 1:2.The effect of the different polymorphs of AZE on the micro-structure of the complexes were investigated by powder X-ray diffraction(PXRD),differential scanning calorimetry(DSC),thermogravimetric analysis(TG),temperature modulated differential scanning calorimetry(TMDSC),cryo-field emission scanning electron microscope system(SEM),fourier transform infrared spectroscopy(FT-IR),and solid-state nuclear magnetic resonance spectroscopy(ss NMR).2 Evaluation of Physiochemical Properties of Complex: The shake-flask test method was used to determin the equilibrium solubility of ?-AZE,?-AZE and AZE-OXA complex and AZE-PEZ complex in 0.1 and 0.01 mol/L HCl solution.While,the dissolution rate of ?-AZE,?-AZE and AZE-OXA complex and AZE-PEZ complex in 0.1 mol/L HCl solution were determined by paddle method.The hygroscopicity of ?-AZE,?-AZE and AZE-OXA complex and AZE-PEZ complex were determined at 25±1°C and 75%±1%RH using the hygroscopicity determination method recorded in pharmacopeia.The effects of different crystal forms on the formation and physicochemical properties of the complex were investigated by measuring the solubility,dissolution rate and hygroscopicity of the complexes.3 The difference on the interaction of the ?-AZE,the ?-AZE-PEZ 1:2(NG)complex,the ?-AZE-PEZ 1:1(NG)complex and the ?-AZE-OXA 1:1(NG)complex with human serum albumin under the simulated physiological condition were investigated by fluorescence spectrometry and UV-visspectrophotometry.The mechanism of fluorescence quenching,the binding constants and binding sites were analyzed by Stern-Volmer equation and modified Stern-Volmer equation.Moreover,the Van't Hoff equation was used to obtain the thermodynamic parameters which was used to analysis the nature of binding forces of drug-HSA system.The effects of different CCF(CAF)and the different molar ratios of same CCF(CAF)on the action of API and human serum albumin were investigated.Results:1 Based on the two polymorphs(? and ? form)of AZE,12 complexes of AZE and OXA were prepared by solvent-assisted grinding(SG)and neat powder grinding(NG)methods at the AZE/OXA molar ratios of 2:1,1:1,and1:2,which were characterizated by PXRD and proved formation two co-crystal samples and ten co-amorphous samples.The two co-crystal samples were ?-AZE-OXA 2:1(SG)and ?-AZE-OXA 2:1(SG);the other ten combinations were in co-amorphous forms,including the NG samples with ?(or ?)-AZE/OXA molar ratios of 2:1,1:1(SG and NG)and 1:2(SG and NG).The melting points of the two co-crystal samples were 130.4 oC form DSC and TG analysis.The Tgs were found to be 112.6°C,134.1°C,134.4°C,76.3°C,110.4°C and 112.5°C,134.7°C,134.8°C,77.3°C,110.8°C for ?-AZE-OXA2:1(NG),1:1(SG),1:1(NG),1:2(SG),1:2(NG)and ?-AZE-OXA 2:1(NG),1:1(SG),1:1(NG),1:2(SG),1:2(NG),respectively.SEM morphology showed that the co-crystal sample were piece or slice shape;while the pictures of ?-AZE-OXA co-amorphous and ?-AZE-OXA co-amorphous combinations showed sphere-shape particles without special shape.Based on the results of FT-IR and ssNMR,we suggested that the interaction of the two components in AZE-OXA complex involved the hydrogen bonds of N-H···O and O-H···O.The equilibrium solubility of samples in 0.1 mol/L HCl solution were all higher than in 0.01 mol/L HCl solution.The equilibrium solubility of intact?-AZE and intact ?-AZE in 0.1 mol/L HCl solution were 268.40±3.2 ?g/mL and 222.46±2.8 ?g/mL,and the highest equilibrium solubility of samples were541.35±5.2 ?g/mL for ?-AZE-OXA 1:2(NG)and 495.05±6.0 ?g/mL for?-AZE-OXA 1:2(NG),respectively.The t50 values of intact ?-AZE and?-AZE in 0.1 mol/L HCl solution were 292.61 min and 803.44 min,respectively.However,the minimum t50 values of ?-AZE-OXA 1:2(NG)and?-AZE-OXA 1:2(NG)co-amorphous were 55.59 min and 53.89 min,respectively.The statistics analysis showed significant differences(P<0.05)in t50 value of ?-AZE-OXA 1:2(NG)and ?-AZE-OXA 1:2(NG)compared with respective intact AZE.2 Although both ? and ? forms could form co-amorphous complexes with OXA,the ?-AZE was clearly easier to form complex than that of ?-AZE.The PXRD showed that ?-AZE-OXA complex was producced after 20 min grinding,while ?-AZE-OXA complex was producced after prolonged 60 min grinding which was proved by typical diffraction halo in PXRD pattern.In the FT-IR spectra,When OXA was introduced at ?-AZE-OXA molar ratio of 2:1and 1:1,the ester C=O stretching displayed a 16 cm-1 shift to a lower wave-number,which could be attributed to the transformation of one hydrogen bond in the homogeneous dimer of ?-AZE to another hydrogen bond in the heterodimer co-amorphous complex.Moreover,when the ?-AZE/OXA molar ratio was increased to 1:2,unbound ester C=O stretching appeared at1750 cm-1,suggesting that the introduction of more OXA into the?-AZE-OXA mixture destroyed the intermolecular interaction in the crystalline structure of AZE and released the unbound ester C=O.However,for ? co-amorphous system,an obvious new peak at near 1750 cm-1,which can be attributed to the unbound ester C=O,appeared even when just a small amount of OXA was introduced,and the absorption peaks at 3414 and 3338cm-1 for N-H in AZE were too broad to be recognized in the complex.The appearance of unbounded ester C=O with the introduction of small amounts of OXA in the complex indicated that the crystalline structure of ?-AZE was destroyed easily than the ? form.3 Based on the two polymorphs(? and ? form)of AZE,12 complexes of AZE and PEZ were prepared by solvent-assisted grinding(SG)and neat powder grinding(NG)methods at the AZE-PEZ molar ratios of 2:1,1:1,and1:2,which were characterizated by PXRD.It was proved that the two co-crystal samples(?-AZE-PEZ 1:2(SG),?-AZE-PEZ 1:1(SG))and three co-amorphous samples(?-AZE-PEZ 1:2(NG),?-AZE-PEZ 1:1(NG),?-AZE-PEZ 2:1(NG))were produed.However,the new complex were not formed when ?-AZE was used as API.The melting point of the two co-crystal samples were 163.2oC from DSC and TG analysis;the Tgs were found to be74.6°C,66.8°C and 77.9°C for ?-AZE-PEZ 1:2(NG),1:1(NG)and 2:1(NG)respectively.SEM morphology showed that the co-crystal sample was piece shape;while the pictures of ?-AZE-PEZ co-amorphous combinations showed sphere-shape particles without special shape.Based on the results of FT-IR and ssNMR,we suggested that the molecule interaction of AZE and PEZ in the complex involved the hydrogen bonds of N-H···O and O-H···O.The equilibrium solubility of intact ?-AZE in 0.1 mol/L HCl and 0.01 mol/L HCl solution were 268.40±3.2 ?g/mL and 104.85±1.0 ?g/mL.The highest equilibrium solubility in 0.1 mol/L HCl and 0.01 mol/L HCl solution were552.81±2.2 ?g/mL for ?-AZE-PEZ 1:2(SG)and 108.37±5.6 ?g/mL for?-AZE-PEZ 2:1(NG),respectively.The t50 values of intact ?-AZE in 0.1mol/L HCl solution were 292.61 min.However,the minimum t50 values of?-AZE-PEZ 1:2(NG)co-amorphous was 38.70 min.The statistics analysis showed significant differences(P<0.05)in t50 value of ?-AZE-PEZ 1:2(NG)compared with respective intact ?-AZE.4 The fluorescence spectra showed that binding of ?-AZE,?-AZE-PEZ1:2(NG),?-AZE-PEZ 1:1(NG)and ?-AZE-OXA 1:1(NG)to HSA caused strong fluorescence quenching of HSA at 291 K,298 K,310 K.Furthermore,the result of UV-vis spectrophotometry indicated that the quenching mechanism of four samples to HSA were all static quenching.The result of synchronous spectrophotometry indicated that when ??-value between excitation and emission wavelength was set at 15 nm,the fluorescence intensity of HSA with four samples were steadily declined without any peak shift.However,when ??-value between excitation and emission wavelength was set at 60 nm,the fluorescence intensity of HSA with four samples weredeclined and the red shift(from 283 to 287 nm)of the maximum emission wavelength occured at the investigated concentrations range.At the temperature of 291 K,298 K,310 K,the quenching constants for ?-AZE were1.767,1.729,1.584×104 L/M;the quenching constants for ?-AZE-PEZ 1:2(NG)were 1.858,1.757,1.528×104 L/M;the quenching constants for?-AZE-PEZ 1:1(NG)were 1.753,1.681,1.606×104 L/M;the quenching constants for ?-AZE-OXA 1:1(NG)were 4.590,4.557,4.148×104 L/M.The binding constants for ?-AZE were 0.839,0.977,1.862×103 M-1;the binding constants for ?-AZE-PEZ 1:2(NG)were0.754,0.786,1.099×103 M-1;the binding constants for ?-AZE-PEZ 1:1(NG)were 0.566,2.110,3.134×103 M-1;the binding constants for ?-AZE-OXA 1:1(NG)were 9.168,8.568,5.800×103M-1.Conclusions:1 The new peaks of PXRD,the new melting point in DSC and TG curve and the piece or slice particles in SEM pictures indicated the formation of AZE-OXA co-crystal and AZE-PEZ co-crystal.The typical halo patterns of XRD,the single glass transition temperature(Tg)in TMDSC curve and the sphere-shape particles in SEM photos were indicated the formation of AZE-OXA co-amorphous and AZE-PEZ co-amorphous.The FT-IR spectra suggested that the hydrogen bonding of AZE-OXA were produced by the C=O in OXA with the N-H group in AZE and the C=O in AZE with the O-H group in OXA,while the hydrogen bonding of AZE-PEZ were produced by the C=O in AZE with the N-H group in PEZ.The difference of various co-amorphous were confirmed by different peak position of FT-IR and ssNMR spectroscopy.2 Compared with intact AZE,the significant improvment on equilibrium solubility and dissolution rate occurred for AZE complex.It indicated that AZE complex could potentially improve the dissolution rate and had the feasibility of enhancing in vivo absorption of AZE.3 PXRD patterns and IR spectra ndicated that it was easier for form ? to form a complex with OXA than the thermodynamically stable form ?.FT-IR spectra suggested that the intermolecular interactions were different between ?and ? co-amorphous systems.4 The result of the interaction for ?-AZE,?-AZE-PEZ 1:2(NG),?-AZE-PEZ 1:1(NG)and ?-AZE-OXA 1:1(NG)with HSA indicated that the formation of AZE complex lead to the change of quenching constants and binding constants.The binding constants were different when different CAF were used;that is,the quenching constants and binding constants of different molar ratio for AZE-PEZ samples and the same molar ratio for AZE-PEZ and AZE-OXA were also different.The high binding constants at different temperature indicated that the drug could be storaged and transported by HSA.The binding constants Kb decreasing with the increasing of temperature for the sample of AZE-OXA 1:1(NG)suggested that the AZE-OXA 1:1(NG)-HSA compound was not stable and could decompose,and the drug-HSA complex could release the drug to when it reach the position of treatment. |
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