Development of a Targeted Polymorph Screening Approach for a Complex Polymorphic and Highly Solvating API
ANTHONY M.CAMPETA,BRIAN P.CHEKAL,YURIY A.ABRAMOV,PAUL A.MEENAN,MARK J.HENSON,BING SHI, ROBERT A.SINGER,KEITH R.HORSPOOL
Pharmaceutical Sciences,P?zer Global Research&Development,Groton,Connecticut06340
Received19February2010;revised21April2010;accepted22April2010
Published online22June2010in Wiley InterScience(https://www.doczj.com/doc/7114461975.html,).DOI10.1002/jps.22230
ABSTRACT:Elucidation of the most stable form of an active pharmaceutical ingredient(API)is
a critical step in the development process.Polymorph screening for an API with a complex
polymorphic pro?le can present a signi?cant challenge.The presented case illustrates an
extensively polymorphic compound with an additional propensity for forming stable solvates.
In all,5anhydrous forms and66solvated forms have been discovered.After early polymorph
screening using common techniques yielded mostly solvates and failed to uncover several key
anhydrous forms,it became necessary to devise new approaches based on an advanced under-
standing of crystal structure and conformational relationships between forms.With the aid of
this analysis,two screening approaches were devised which targeted high-temperature deso-
lvation as a means to increase conformational populations and enhance overall probability of
anhydrous form production.Application of these targeted approaches,comprising over100
experiments,produced only the known anhydrous forms,without appearance of any new forms.
The development of these screens was a critical and alternative approach to circumvent
solvation issues associated with more conventional screening methods.The results provided
con?dence that the current development form was the most stable polymorph,with a low
likelihood for the existence of a more-stable anhydrous form.?2010Wiley-Liss,Inc.and the
American Pharmacists Association J Pharm Sci99:3874–3886,2010
Keywords:axitinib;polymorphism;polymorph screen;crystallization;desolvation;compu-
tational chemistry;crystal structure
INTRODUCTION
Polymorphism can be thought of as the state in which a solid chemical compound exists in more than one crystalline form1with only one polymorph being the thermodynamically most stable form at a speci?c temperature and pressure.This phenomenon is very common to most pharmaceutical APIs.It is well known that polymorphs of the same substance can have dramatic differences in pertinent pharmaceu-tical properties,such as solubility and stability that can often have a signi?cant impact on bioavailability and overall drug product performance.A number of excellent texts on polymorphism and their in?uence on pharmaceutical development are available.2Thus,identifying the most appropriate solid form,typically the thermodynamically stable form,is a key element in the early developmental process for a new drug candidate.In regard to polymorph screening approaches,there are many common techniques employed that are designed to typically uncover all metastable and low-energy polymorphic forms.These include,for example,crystallizations through solvent evaporations,antisolvent crystallizations,slow and fast cooling of saturated API solutions to induce precipitation,and slurrying of solid API for extended periods of time.3A signi?cant number of solvents and cosolvents of varying polarity and chemical composi-tion are usually employed,while variable tempera-tures are also incorporated in the design to assess enantiotropic behavior.These approaches have been incorporated into our practices for polymorph screen-ing and are typical throughout the pharmaceutical industry.
In most cases,a thoroughly designed API form screen employing the approaches previously dis-cussed should typically identify the thermodynami-cally stable polymorph.
Additional Supporting Information may be found in the online version of this article.
Mark J.Henson’s present address is Molecular Biometrics,Inc., New Haven,CT06511.
Correspondence to:Anthony M.Campeta(Telephone:860-441-5844;fax:860-715-2454;E-mail:anthony.m.campeta@p?https://www.doczj.com/doc/7114461975.html,) Journal of Pharmaceutical Sciences,Vol.99,3874–3886(2010)
?2010Wiley-Liss,Inc.and the American Pharmacists Association
exceptions in the literature4,5that describe the appearance of a lower energy form at late stages in development.In these cases,common polymorph screening approaches were clearly unsuccessful,as unique physical and structural properties of the molecule hindered the anticipated cascade to the most stable form based on Ostwald’s rule.6In this article, we describe another such example.
Axitinib(Fig.1)is an oncology candidate under development at P?zer.This API targets the vascular endothelial growth factor(VEGF)to prevent the growth and proliferation of cancer cells via interrup-tion of tumor angiogenesis(formation of vascular supply tissue).7This compound has shown consider-able promise in the treatment of carcinomas in a number of target tissues and organs and is currently in late stage clinical development.8
Understanding the polymorphism of axitinib has been a subject of considerable focus and effort,which we have initially reported.9In this work,polymorph investigations using the traditional approaches pre-viously mentioned,which incorporated well over 300experiments,identi?ed a surprisingly high total of23unique solid forms.Distinction of these forms was assigned on the basis of powder X-ray diffraction (PXRD)patterns and thermal characteristics such as melting onset,melting enthalpy,and desolvation temperatures.This group of solid forms included three anhydrous forms with the remainder solvates (refer to Tab.1).The anhydrous form IV was characterized as a robust developmental form with acceptable solid-state properties and was advanced for early clinical studies.
It was apparent that axitinib had a high tendency to form solvates.There was some question whether certain polymorph screening approaches may be challenged by this phenomenon,and the risk of not observing critical anhydrous forms(of which three had been already discovered)could exist.In parti-cular,axitinib had a propensity to form relatively stable solvated structures,as a majority of these solvates were characterized as possessing relatively high temperatures of desolvation(desolvation tem-
peratures signi?cantly higher than the
normal
Figure1.Structure of axitinib.Table1.Summary of Axitinib Solid Forms
Name Form Solvent
Form I(1)Anhydrate—
Form II(2)Hydrate Water
Form III(3)Solvate Ethyl acetate(EtOAc) Form IV(4)Anhydrate—
Form V(5)No solid form designation
Form VI(6)Anhydrate—
Form VII(7)Solvate Isopropyl alcohol(IPA),
IPA/water
Form VIII(8)Solvate Dioxane,tetrahydrofuran(THF) Form IX(9)Hydrate Water
Form X(10)Solvate Dimethylformamide(DMF),
DMF/water
Form XI(11)Solvate THF/water,THF
Form XII(12)Solvate Dichloromethane(DCM),
ethanol(EtOH)
Form XIII(13)Solvate Acetonitrile(ACN)
Form XIV(14)Solvate Acetic acid
Form XV(15)Solvate EtOH
Form XVI(16)Solvate IPA
Form XVII(17)Solvate Acetone
Form XVIII(18)Solvate Methylisobutyl ketone(MIBK) Form XIX(19)Solvate Methylethyl ketone(MEK) Form XX(20)Solvate Methyl benzoate
Form XXI(21)Solvate2,2,2-CF3CH2OH/ether/hexane Form XXII(22)Solvate1-Pentanol
Form XXIII(23)Solvate Pyridine
Form XXIV(24)Solvate Chloroform
Form XXV(25)Anhydrate—
Form XXVI(26)Solvate THF/water,THF
Form XXVII(27)Solvate Dimethylsulfoxide(DMSO) Form XXVIII(28)Solvate Benzyl alcohol
Form XXIX(29)Solvate Trichloroethylene
Form XXX(30)Solvate Dimethylformamide
(DMF)/octanol(1:1)
Form XXXI(31)Solvate Octanol
Form XXXII(32)Solvate Methanol
Form XXXIII(33)Solvate1-Butanol
Form XXXIV(34)Solvate3-Methyl-1-butanol
Form XXXV(35)Solvate MEK
Form XXXVI(36)Solvate Pyrrole/1-pentanol
pyrrole/p-cymene
Form XXXVII(37)Solvate Allyl alcohol
Form XXXVIII(38)Solvate Pyrrole allyl alcohol
Form XXXIX(39)Solvate Acetic acid
Form XL(40)Solvate EtOH
Form XLI(41)Anhydrate—
Form XLII(42)Solvate2-Butanol
Form XLIII(43)Solvate2-Methyl THF
Form XLIV(44)Solvate2-Methyl THF
Form XLV(45)Solvate Toluene
Form XLVI(46)Solvate N-Methylpyrrolidone Form XLVII(47)Solvate Isoamyl acetate
Form XLVIII(48)Solvate Methylcyclohexane
Form XLIX(49)Solvate Cyclohexanone
Form L(50)Solvate Cyclohexanone
Form LI(51)Solvate1,2-Dichloroethane
Form LII(52)Solvate Propionic acid
Form LIII(53)Solvate Tert-butanol
Form LIV(54)Solvate Dimethoxymethane
Form LV(55)Solvate2-Pentanone
Form LVI(56)Solvate Dimethyl acetate(DMA) Form LVII(57)Solvate Nitromethane
Form LVIII(58)Solvate1,2,3,4-tetrahydronaphthalene Form LIX(59)Solvate Tetramethylene sulfone Form LX(60)Solvate Methyl acetate
Form LXI(61)Solvate p-Xylene
Form LXII(62)Solvate Trichloroethylene
Form LXIII(63)Solvate n-Butyl acetate
Form LXIV(64)Solvate Isobutyl alcohol
Form LXV(65)Solvate Cyclohexanol
Form LXVI(66)Solvate Isopropyl acetate
Form LXVIII(67)Solvate p-Cymene/pyrrole(1:1) Form LXIX(68)Solvate t-Amyl alcohol
Form LXX(69)Solvate4-Methyl-2-pentanone Form LXXI(70)Solvate Cyclohexane
Form LXXII(71)Solvate1,2-Dichlorobenzene Form LXXIII(72)Solvate p-Cymene/acetone(1:1)
TARGETED POLYMORPH SCREENING APPROACH DEVELOPMENT FOR AN API3875
boiling point of the corresponding solvent),suggest-ing strong bonding within the crystal.These solvates may be anticipated to be thermodynamically stable in their corresponding mother liquor and may resist further solvent-mediated transformation to an anhy-drous form.Solvent content was often found to be nonstoichiometric,suggesting nonsite-speci?c sol-vent incorporation.Also,many solvates formed from different solvent systems demonstrated isomorphic10 structures,as evidenced by very similar PXRD patterns,which made differentiation of solid forms complex.In addition,multiple polymorphs of solvates were observed from the same solvent,such as ethanolate forms XII and XV.
During process development and re?nement stu-dies with form IV,a new crystalline anhydrous form was discovered.9This new form,form XXV,was characterized as a thermodynamically more stable form than form IV,though it was not observed in any of the aforementioned polymorph screening studies. Crystal structure analysis would eventually show that the molecular conformation of most of the solvate structures was very similar to that of the anhydrous form IV.This coincides with the observation that the solvates would desolvate to form IV during DSC and variable temperature PXRD heating experiments. This suggested that the molecular conformation is mostly retained upon desolvation,resulting in the favored formation of form IV.Equally important was the?nding that the crystal packing and molecular conformation of the new form XXV were found to be different from those of form IV.In particular,the molecular arrangement of form XXV generally would be dif?cult to achieve through desolvation.Thus,it is believed the propensity to initially form stable solvates as well as structural properties that would favor desolvation to form IV contributed to the lack of appearance of form XXV in our polymorph screens.
Though there was con?dence that form XXV was the thermodynamically stable form of axitinib, another more-stable anhydrous form,form XLI, was later discovered during development.It became obvious that the polymorph environment of axitinib could not be con?dently assessed through the use of conventional polymorph screening experiments.In this article,the discovery and characterization of this new form XLI are described,including a thorough evaluation and understanding of the crystal struc-ture,and its structural relationship relative to known polymorphs.Finally,with this detailed understand-ing of the structural properties,the development of two speci?c polymorph screening approaches were designed around desolvation to more completely assess the polymorphic environment of axitinib and improve con?dence that form XLI was indeed the thermodynamically stable form.EXPERIMENTAL
Materials
A sample of form XVI(isopropanol solvate)of axitinib (purity99.8%)was obtained from P?zer Ireland Pharmaceuticals,Inc.,Little Island,CO.Cork,Ire-land.Samples of anhydrous forms IV,VI,XXV,and XLI were obtained from P?zer Global Research& Development,Groton,CT.Full details on methods to prepare forms I,IV,and VI are presented by Ye et al.,11whereas methods to prepare forms XXV and XLI are described by Campeta et al.12Solvents used in this study were obtained from Sigma–Aldrich,St Louis,MO,Pharmco-AAPER,Brook?eld,CT,or Mallinckrodt Baker,Phillipsburg,NJ.
Solubility Measurements of Various Forms in Ethanol Solubility measurements of various forms in ethanol were completed using a100-mL jacketed glass vessel. When operated at temperatures above the normal boiling temperature of ethanol(788C),the vessel was pressurized to suppress boiling.During the solubility measurement,a measured amount of a particular form of axitinib was added to a measured amount (typically30–40g)of ethanol.The mixture was heated quickly to5–108C below the expected dissolu-tion temperature,and then heated slowly(0.25–0.58C/min)until all the solids were dissolved.The dissolution was monitored using a Mettler-Toledo Lasentec S400FBRM probe.
Powder X-Ray Diffraction
The PXRD pattern measurement was carried out on a Bruker D5000diffractometer using copper radiation (CuK a,wavelength:1.54056A?).The tube voltage and amperage were set to40kV and40mA,respectively. The divergence and scattering slits were set at1mm, and the receiving slit was set at0.6mm.Diffracted radiation was detected by a Kevex PSI detector.A theta-two theta continuous scan at2.48/min(1s/0.048 step)from 3.0to4082u was used.An alumina standard was analyzed to check the instrument alignment.Samples were prepared by placing them in a quartz holder.
Single Crystal Diffractometry
The single crystal X-ray diffraction measurements were carried out at room temperature on either a Bruker SMART APEX CCD area detector system equipped with a graphite monochromator and sealed tube Cu radiation(1.54178A?)source or a Bruker FR591rotating anode with Motel Multilayer Optics, Cu radiation(1.54178A?),equipped with an APEX II detector.Single crystal X-ray diffraction data were collected at room temperature in order to provide a calculated powder pattern that best matches
3876CAMPETA ET AL.
experimental powder patterns for API samples, which are measured at room temperature.Tempera-ture changes may result in the contraction or elongation of unit cell dimensions to such an extent that the calculated powder patterns at100K and room temperature may be signi?cantly different.All crystallographic calculations were facilitated by the SHELXTL software suite system.In general,hydro-gens bonded to heteroatoms were located by differ-ence Fourier techniques.The remaining hydrogen atoms were placed in idealized positions.The hydro-gen parameters were added to the structure factor calculations but were not re?ned.Crystals suitable for this analysis were grown through crystallization techniques including evaporations of saturated solu-tions,vapor and liquid diffusion,and antisolvent crystallization.
Thermal Analysis
Differential scanning calorimetry(DSC)was carried out on a Mettler Instruments DSC821e,Columbus, OH.The instrument was calibrated for cell constant and heat capacity using indium and zinc.Samples were prepared by weighing about3mg of into a40m L aluminum pan which was then covered with an aluminum lid.The lid is pierced prior to measure-ment.Data were analyzed using STAR e SW8.10.The experiments started at ambient temperature and heated the sample at58C/min to3008C under a nitrogen gas purge(?ow rate was60mL/min). Thermogravimetric analysis(TGA)was carried out on a TA Instruments,New Castle,DE,DSC Q500 V6.7Build203.The instrument was weight cali-brated with200and1000mg weights.Samples were prepared by weighing about6mg of sample into a 50m L aluminum pan.Data were analyzed using Universal Analysis2000for Windows2000/XP version4.2E,Build4.2.0.38.The experiments started at ambient temperature and heated the sample at 108C/min to3008C under a nitrogen gas purge(?ow rate was50mL/min).
Conventional Polymorph Screen With Form XXV
The polymorph screen consisted of generating solid samples using a variety of techniques and solvents.A total of12solvents and8aqueous cosolvent mixtures were used.Techniques for generating samples included fast evaporations,slow cools of saturated solutions,and slurries consisting of excess solid in saturated solutions at different temperatures.Resul-tant solids were examined under a microscope for birefringence and morphology,and analyzed at a minimum by PXRD.Selected samples were also assessed by thermal methods.Approximately70total experiments were performed.Fast evaporation experiments:Solutions were prepared in various solvents and sonicated to assist in dissolution.The ?nal solutions were typically?ltered through a 0.2-m m nylon?lter.The solutions were allowed to evaporate at ambient(20–258C)in a vial.The solids that formed were isolated and analyzed.Slow cooling experiments:Saturated solutions were prepared in various solvents at elevated temperatures(approxi-mately608C)and rapidly?ltered through a0.2-m m nylon?lter into open vials.The vials were covered and allowed to cool slowly to room temperature.The presence or absence of solids was noted.If there were no solids present,or if the amount of solids was judged too small for PXRD analysis,the vial was placed in a refrigerator overnight.Again,the presence or absence of solids was noted and if there were none, the vial was placed in a freezer overnight.Solids that formed were isolated by?ltration and allowed to dry prior to analysis.Slurry experiments:Solutions were prepared by adding enough solids to a given solvent so that excess solids were present.The mixtures were then agitated in sealed vials at either ambient or an elevated temperature,typically608C.After a given amount of time(typically7days for ambient and1day for608C),the solids were isolated by vacuum ?ltration.
Preparation of Solvates and Three-Component Desolvation Screen
The solvate–desolvation polymorph screen was run by?rst preparing a variety of axitinib solvates,and then desolvating using multiple methods.The solvate samples were prepared on a1g scale starting from form XVI(isopropanol solvate)and were formed by reslurrying in a single solvent or mixed solvent system for3days at508C.The isolated solids were initially characterized by PXRD to determine if a unique solid form had been generated.Additional characterization utilized DSC and TGA to con?rm the presence of desolvation events.
Once the solvated forms were characterized, parallel desolvation experiments were run on sam-ples of the solvated forms.The desolvation techniques utilized were a)vacuum drying at1008C,b)reslurry in ethanol at908C in a sealed vial,and c)reslurry in heptane at1058C in a sealed vial.Desolvation studies were carried-out on a100mg scale.Samples exhibit-ing the initial solvate PXRD powder pattern were exposed again to the same desolvation conditions.If a unique powder pattern was observed,additional thermal analysis(DSC and TGA)was conducted to characterize the material.
Raman Spectroscopy
The solid form of larger scale reslurry studies was monitored via Raman spectroscopy with a Kaiser Optical Systems HoloLab5000equipped with a 785nm laser.A?ber optic cable linked the instru-ment to an MkII probe head?tted with a Hastelloy1
TARGETED POLYMORPH SCREENING APPROACH DEVELOPMENT FOR AN API3877
1.3cm diameter immersion probe.The probe incor-porated a short focal length sapphire optic.Data collection was controlled via Kaiser’s HoloReact software(v.4.0.0233).The software resampled experi-mental data to a spectral resolution of0.6cmà1. Wavelength calibration was performed using cyclo-hexane.Ten second spectral accumulation times were found to be suf?cient to achieve slurry signal levels within the recommended segment of the detector dynamic range.Dark background spectra were acquired prior to each spectrum,and automatic cosmic ray rejection was employed.Utilization of these two options extended the total acquisition time for each spectrum to40s.During the experiments, spectra were acquired at5-min intervals.As Raman spectra were being acquired,the univariate intensity trending of key spectral bands in real time was performed using the HoloReact package based on MatLab.Subsequent of?ine spectral data analysis was performed using Delight v.3.
2.1(DSquared Development,Inc.,LaGrande,OR).
High Boiling Solvent Polymorph Screen Approximately40axitinib solvates were?rst pre-pared on a1g scale starting from form XVI (isopropanol solvate)and were formed by reslurrying in a single solvent or mixed solvent system for3days at508C.These samples were then reslurried at1508C in p-cymene for at least7days before isolating.The isolated solids were initially characterized by PXRD to determine if a unique solid form had been generated.If needed,thermal analysis was completed by DSC and TGA.
Additional high-temperature reslurry experiments in p-cymene at1508C were completed on a50mL scale using the Biotage AS2350reactor system starting from the anhydrous forms XLI and XVI with the solid form monitored in situ by Raman spectroscopy.An additional set of1308C reslurry experiments were completed in3:1p-cymene/benzyl alcohol(v/v) mixture and3:1p-cymene/o-cresol(v/v)mixture starting with form XVI.Isolated material was characterized by PXRD.
Computational Chemistry Methods
Crystallographic and generated conformations of axitinib were optimized using aqueous media at the PBE/DNP/COSMO level of theory(de?ned by theo-retical method and a basis set)as implemented in DMol3.13This utilizes density functional theory (DFT)PBE approximation14with all-electron dou-ble-numeric-polarized basis set.The effect of bulk water is estimated by conductor-like screening model (COSMO)as implemented in DMol3.15The generated cosmo?les by the PBE/DNP/COSMO calculations for each conformer are further used for the prediction of solubility and conformational distribution in different solvents adopting COSMO-RS theory16as implemen-ted in COSMOTherm software.17
RESULTS AND DISCUSSION
Conventional Polymorph Screen of Form XXV
The properties and characteristics of the newly discovered anhydrous form XXV have been recently described,which demonstrated that it was a more stable polymorph at room temperature than the early development form IV.9After discovery,an additional polymorph screen with this new form was performed to reassess the polymorph environment of axitinib. The screening approaches were similar to those used for polymorph screening of form IV,which comprised well-precedented techniques previously mentioned. Solubility assessment showed that form XXV had solubilities roughly2?lower than form IV in most solvent and aqueous cosolvent mixtures.It was acknowledged that the lower solubilities may hinder solvent-mediated transformations in slurry-type experiments.At this point in time,extensive crystal structure information of axitinib solid forms had not been discerned,including those of forms XXV and IV. Thus,though it was unclear as to de?nitive reasons for the lack of appearance of form XXV in previous form screens,conducting another traditional screen in the same manner as with form IV was considered the best?rst approach for the screening of form XXV. These experiments discovered no new anhydrous forms.Form XXV was the predominant anhydrous form,while a number of new solvates were found through the use of some solvents not evaluated in previous studies.As a result,a total of39forms of axitinib had been found,see Table1.
Discovery of Form XLI
Once form XXV was identi?ed as the lead develop-ment form,chemical process development studies identi?ed facile conditions to isolate the desired form. The process utilized a two-step reslurry process to convert the crude API to the?nal form.An initial reslurry in isopropanol converted the crude API form to an isopropanol solvate,which was subsequently converted to the desired?nal form via a reslurry in ethanol with the addition of form XXV seed crystals to control the polymorph conversion.In the initial manufacturing campaign for producing form XXV, the conversion of the isopropanol solvate to form XXV required extended processing time possibly due to poor dispersion of the form XXV seed crystals in the crystallization vessel attributed to mixing.Subse-quent lab-scale development studies demonstrated that the seeded ethanol reslurry process conducted at re?ux with a high jacket temperature resulted in a
3878CAMPETA ET AL.
faster and more robust conversion to form XXV.The rationale for the faster form conversion was not determined but may have been due to the higher solubility difference between the solvate form and form XXV at higher temperatures,and/or it may be due to the improved solid –liquid mixing in the crystallization vessel due to the re ?uxing solvent.The lab-scale process was modi ?ed to utilize the higher jacket temperature for the subsequent man-ufacturing campaign.During the ?nal isolation,an in process control sample was pulled and analyzed by PXRD to determine if the conversion to the desired form XXV was complete.PXRD analysis indicated a previously unknown powder pattern.The slurry was seeded with form XXV a second time,and the process was allowed to reslurry at re ?ux for 12h.Analysis of a subsequent sample indicated the same unknown powder pattern with no indication of form XXV,which suggested that the unknown form (form XLI)was thermodynamically favored versus form XXV.Characterization of Form XLI and Comparison to Known Anhydrous Forms
With the discovery of form XLI,an intensive evaluation of its physical properties was conducted to characterize this new form and understand its relationship to the other known anhydrous forms.DSC experiments showed form XLI possessing a single endotherm,with a melting temperature and heat of fusion value higher than other known anhydrous forms,see Table 2.Together,a total of ?ve anhydrous polymorphs of axitinib were now known,which presents an unusual case.On the basis of the DSC data and Burger ’s rule,18form XLI is shown to be monotropically related to all other
anhydrous forms and is thus expected to be the most stable solid form at all temperatures.Form XLI also has the lowest solubility of all the anhydrous forms,providing further support for its designation as the lowest energy form.Figure 2illustrates the signi ?-cantly lower solubility of form XLI in ethanol.
Table 2provides the rank order of the relative stabilities of the anhydrous forms.The calculated density values for all forms do not correlate with relative stability,suggesting this parameter is not a reliable measure of stability,as is often the case.18As will be described later,the various forms have different conformations,which makes comparisons based on the density rule invalid.In regard to relationships between the other forms,forms XXV and VI are enantiotropically related and very close in energies.The average heat of fusion of form VI is slightly higher,though the range of values from multiple determinations of each form is overlapping and thus may be within the experimental sensitivity of the instrument.
Table 2.Thermal Properties and Calculated Density of Axitinib Anhydrous Forms
Melt Onset (8C)
D H f (J/g)Density (g/mL)a
Form XLI 225.9153 1.369Form XXV 217.2129–132b 1.382Form VI 211.6132–136b
1.330Form IV 218.7122 1.293Form
I 210.5
115
1.333
a Calculated from single-crystal structure information.
b
Range of values from multiple DSC (5–10)determinations at 58C/min heating
rate.
Figure 2.Solubility in ethanol of ethanol solvate,forms IV,VI,XXV,and XLI.
TARGETED POLYMORPH SCREENING APPROACH DEVELOPMENT FOR AN API 3879
Form IV is enantiotropically related to forms XXV and VI.The transition temperature between forms IV and XXV,determined from solubility,showed form XXV to be the stable form below758C.9Form I is unstable in the solid state under high humidity and converts to the monohydrate(form IX),which can be converted back to form I upon drying.
Further insights into the structural relationships between these forms were obtained through a subsequent computational analysis of their crystal structures.Ultimately,parallel efforts were under-taken to understand the structural intricacies of axitinib forms and utilize this knowledge into the design of new experimental approaches to re-assess the polymorphic environment.Results from each approach helped con?rm form XLI as the low energy form.
Crystal Structure Information
The crystal structures of forms I,IV,VI,XXV,and XLI have been determined and crystallographic information is presented in Table 3.The crystal packing diagrams for these forms are shown in Figure3.Crystallographic data for forms IV and XXV have been reported previously.9The axitinib molecule contains two hydrogen bond donor groups(the pyrazole and benzamide NH),and three acceptor groups(the pyridine and pyrazole aromatic nitrogens
Table3.Crystallographic Data for Anhydrous Axitinib Forms
API Form Space Group Z0Unit Cell Dimensions and Volume Re?nement R-Factor(%) I P-11a?7.74A?,b?11.88A?,c?12.15A?;a?65.678,b?72.648,and g?76.198;V?963.2A?38.43
IV P-12a?11.86A?,b?12.40A?,c?15.00A?;a?81.78,b?81.18,and g?65.98;V?1984.7A?3 6.57
VI P-11a?8.15A?,b?10.73A?,c?12.68A?;a?68.18,b?88.38,and g?70.68;V?965.1A?3 4.68
XXV P21/c1a?4.54A?,b?11.75A?,c?34.83A?;b?92.138;V?1858.1A?3 6.25
XLI P21/c1a?16.08A?,b?8.10A?,c?15.58A?;b?112.28;V?1875.3A?3
5.42
Figure3.Crystal packing patterns of axitinib anhydrous forms:(a)form I,(b)form
IV,(c)form VI,(d)form XXV,and(e)form XLI.
3880CAMPETA ET AL.
and benzamide oxygen).All anhydrous forms display hydrogen bonding between the pyrazole secondary amine and benzamide oxygen.In addition,hydrogen bonding between the benzamide NH and either the pyridine nitrogen(in forms IV,XXV,and XLI),or the pyrazole nitrogen(in forms I and VI)is observed. Structure Evaluation:Hydrogen-Bonding Analysis
An assessment was performed for all the anhydrous forms to(a)evaluate the stability of form XLI relative to the other anhydrous forms and(b)evaluate the likelihood that a more stable form of axitinib could exist.This was based on an analysis of agreement between the observed hydrogen-bonding network and predicted hydrogen-bonding propensities.Though there are other intermolecular interactions taking place in organic crystals,hydrogen bonding is the most important and typical interaction for active pharmaceutical ingredients.Hydrogen bonding is a very speci?c(both selective and directional)inter-molecular interaction,which makes a crucial con-tribution to both thermodynamic and kinetic behavior of a crystalline phase.For the hydrogen bonding propensity analysis,computational(s HB charges analysis19,20)tools were adopted.
It has been demonstrated19,20that the hydrogen-bonding energy displays a high correlation with the corresponding atomic s HB surface charges16as esti-mated within COSMOTherm software.17For axitinib, the analysis of the relative strength of the acceptors and donors was performed based on the calculated atomic s HB charges(PBE/DNP/COSMO level of theory)and visualized by COSMOTherm software. The largest positive and negative s HB values corre-spond to the strongest acceptor and donor features, respectively.According to these calculations,the strongest donor–acceptor pair for this molecule in all conformations(all anhydrous solid forms)should be the pyrazole amine–amide oxygen hydrogen bond.The rest of the donors and acceptors should display a noticeably weaker hydrogen-bonding propensity according to these calculations.
Computational Assessment of the Relative Stability of Form XLI
The stability of the anhydrous form XLI which is monotropically related to other forms can be estimated based on relative conformational(internal)and inter-molecular energies.
Intermolecular Contribution
The hydrogen bond between the pyrazole amine and amide oxygen(calculated to be the strongest)was observed to occur in all known axitinib crystal structures.For all anhydrous forms except form XLI,this bond leads to the formation of molecular dimers,which are connected to each other by additional weaker hydrogen bonds(Fig.3a–d).Only in form XLI do the strongest hydrogen bonds (between the pyrazole amine and amide oxygen)form an extended network throughout the crystal(see Figure3e,which depicts in the forefront a network of three axitinib molecules connected by the single strongest hydrogen bond).The presence of this strong hydrogen bond network is favorable for the thermo-dynamic and mechanical stability of the crystal.For example,a thermal destruction of the long order packing(through melting)of all forms except form XLI can be achieved by imposing an amount of energy suf?cient to break only the weak hydrogen bonds connecting molecular dimers.For form XLI,greater energy is required(higher melting point)to break the stronger hydrogen-bonded extended chains.More-over,the strong hydrogen bond distance is the shortest in form XLI among all the anhydrous crystal forms(Tab.4),demonstrating stronger intermolecu-lar interactions.
Intramolecular Contribution
DFT calculations at the BP-TZVP level of theory21 were shown in the previous publication9to correctly describe the conformations of forms IV and XXV. However,this level of theory failed to reproduce the correct geometry of the observed molecular conforma-tion of form XLI.Therefore,in the current study all the molecular conformational and energetic analyses were performed adopting an improved DFT func-tional,PBE.14The molecular conformations observed in axitinib crystal structures can be characterized by a conformation of the propenylpyridine fragment combined with the orientation of the methylbenza-mide terminal group(Fig.4).The crystallographic conformations were closely reproduced by the PBE/ DNP/COSMO optimizations resulting in highest RMSD of0.30A?,which demonstrates that the selected level of theory is appropriate for describing the geometry of axitinib.The relative calculated conformational energies are presented in Table5, indicating that at the adopted level of theory,form XLI displays the least stable molecular conformation compared to the other anhydrous forms.This internal Table4.Intermolecular Hydrogen Bond Distances Between Pyrazole N-Donor and Amide O-Acceptor Observed for Anhydrous Forms
Form NáááO HB Distance(A?) I 2.83
IV 2.80
VI 2.82
XXV 2.83
XLI 2.78
TARGETED POLYMORPH SCREENING APPROACH DEVELOPMENT FOR AN API3881
energy difference is thought to be counterbalanced by stronger intermolecular interactions to explain the empirical relative stability of form XLI.
In summary,with the appearance of such a diverse set of solid forms,which included ?ve anhydrous forms,the application of these computational tools provided valuable information to assist in the under-standing of axitinib polymorphism.Though confor-mationally less stable and unique relative to the other anhydrous forms,form XLI can be expected to be the most stable form based on the strong,extended hydrogen bonding network within the crystal lattice which is absent in the other forms.The signi ?cantly higher melting point and melting enthalpy observed in the DSC data (see Tab.2)corroborate this conclusion.The hydrogen-bonding patterns in form XLI were different relative to the other forms,and did not form molecular dimers as in the other forms.Based on hydrogen-bonding patterns within the dimers,weaker hydrogen bond centers are expected to be assessable for solvate formation.9
Based on our knowledge of form XLI,it was recognized a different approach was needed to assess the polymorphic environment.Conformations like that found in form XLI which can have an extended hydrogen bonding would be expected to be signi ?-cantly more stable than dimer-based structures.It was recognized that experimental methods that may promote more conformational adaptability and restructuring may lead to the appearance of
additional form XLI-like forms.Due to the high propensity of solvate formation observed in common screening methods,approaches modeled around desolvation at high temperatures were devised.Three-Component Solvate Desolvation Screen
Based on the dif ?culty to resolve the polymorphism of axitinib through common screening techniques (such as evaporation,cooling,slurry,and precipitation methods),different approaches were devised based on the understanding of the structural characteristics and experience with prior screens.This API prefer-entially forms stable solvates,and as such,attention was concentrated to better understanding this phenomenon.The study of solvates,their correspond-ing desolvation products,and proposed desolvation mechanisms have been the subject of several articles.For example,it has been found that depending on the desolvation conditions,desolvated forms which are essentially isostructural with the solvate as well those having a completely different structure can be obtained.22These authors,as well as others,23,24have suggested two different desolvation mechanisms are typical.In the ?rst case,solvent removal leads only to relaxation of the crystal to an energy minimum,thus resulting in little change in the structure.In this case,the appearance of an isomorphic desolvate may occur.The second mechanism involves destruction of the initial crystal structure,resulting in the formation of a metastable intermediate phase,followed by reor-dering and growth of the new crystal structure.The mechanism involved for a particular solvate will certainly be dependent on bonding strength and molecular strain within the crystal.A common theme noted is that different desolvation conditions (e.g.,temperature,pressure,solvent)can affect the result-ing desolvated product.In addition,the resulting desolvated form may not necessarily correlate with the class of solvate,as different solvate types (isolated site vs.channel solvate)have been shown to desolvate to the same form.25
For axitinib,where a large population of stable solvates has been observed,evaluation of their desolvation endpoints under different conditions may potentially reveal previously unobserved non-solvated forms.Indeed,the study of desolvation,in conjunction with molecular modeling tools,has been proposed as a complementary approach to conven-tional polymorph screening as a means to study polymorphism when solvates are present.26It was postulated that desolvation was a key pathway to the ultimate formation of axitinib anhydrous forms.Therefore,a desolvation-based form screen approach was developed.
In this screen,approximately 60different solvates were ?rst prepared,then desolvated through three different methods.Solvates were prepared using a
Table 5.Relative Energies of the Crystallographically Observed Conformers Optimized at the PBE/DNP/COSMO Level of Theory
Form D E (PBE/DNP/COSMO)(kCal/mol)
IV,VI
0.00XXV,IV,I 0.59XLI
3.34
Figure 4.Aligned crystallographic molecular conforma-tions of axitinib anhydrous forms.The conformation of form XLI is presented in orange.
3882CAMPETA ET AL.
wide variety of solvents of different polarities and chemical composition,many of which had not been used in previous screens.New solvates were char-acterized as unique based on PXRD and thermal properties.As such,a number of new solvated forms were prepared.In all,71solid forms of axitinib have been discovered,which are summarized in Table 1.The prepared solvates were exposed to three different desolvation conditions:vacuum drying at 1008C,heptane reslurry at 1058C,and ethanol reslurry at 908C.The duration at each condition was up to 7days.The vacuum drying conditions were selected as this elevated temperature and reduced pressure would be expected to desolvate most solvates.Heptane was one of the few solvents that did not solvate with axitinib and would be expected to be partially miscible with most solvents at high temperature and would be expected to facilitate desolvation.Ethanol was used in the ?nal step during manufacturing of earlier anhydrous forms and was signi ?cantly different in polarity and chemical composition than heptane and thus it was of interest to evaluate the outcome using this solvent for the reslurry of a variety of solvates.
The two solvent-based desolvations were performed at high temperatures,which should facilitate deso-lvation of the stable solvated structures as well as potentially overcome conformational energy barriers to promote structural rearrangements to any anhy-drous form.In all,approximately 120desolvation experiments were carried out.
The results from the studies are summarized statistically in Figure 5.For the vacuum drying method,approximately half of all solvates desolvated to form IV.This result can be rationalized based on the structural similarities between form IV and most solvates.Under these conditions,desolvation to form IV might not be expected to cascade further to more-stable anhydrous forms in the solid state since form IV has demonstrated itself as a physically stable form.Desolvation was not achieved for about 15%of solvates with this method,which shows the relative stability of many of the solvate structures formed with axitinib.
Solvent-mediated desolvation experiments in hep-tane and ethanol produced signi ?cantly higher instances of form XLI,the thermodynamically
stable
Figure 5.Summary of axitinib forms obtained from desolvation experiments using heptane reslurry at 1058C,vacuum drying at 1008C,ethanol reslurry at 908C,and p -cymene reslurry at 1508C.
TARGETED POLYMORPH SCREENING APPROACH DEVELOPMENT FOR AN API 3883
form.In general,solvent-mediated conditions along with the high temperatures are expected to provide greater opportunity to overcome conformational barriers and allow the cascade to the most stable form.The difference in the relative form population obtained between the heptane and ethanol systems may be theorized to be attributed to solubility differences.Both solvents were expected to desolvate the axitinib solvated forms.However,adequate solubility in these solvents must exist for the desolvated form,typically the metastable form IV, to undergo a further solvent-mediated transforma-tion and conversion to form XLI or any other more-stable form.27The absolute solubility of axitinib in heptane is very low((1mg/mL at208C),and also in a relative sense when compared to that in ethanol(see Fig.2).Though the heptane desolvation produced form XLI,the low energy form,in one-third of the cases,form IV still presented as the endpoint form in half the cases,suggesting solubility was insuf?cient to facilitate a further solvent-mediated transforma-tion within the time frame(7days)of the experiment. Desolvation also did not occur for11%of the solvates, suggesting a potential solvent nonattraction in these cases.In the ethanol system,all of the solvates demonstrated conversion to form XLI.This medium likely provided adequate solubility at the elevated temperatures to facilitate transformation to the low energy form.Of primary importance was that no new anhydrous forms evolved from this screen,providing additional con?dence that form XLI was the lowest energy form.
Polymorph Screen in High Boiling Point Solvents
As mentioned,early polymorph screens resulted in extensive solvate formation,not the(as expected) formation of lower energy anhydrous forms.For axitinib,a structural assessment of the lowest energy molecular conformations of the previously known anhydrous forms XXV,IV,VI,and I,as well as the solvated forms,showed direct assembly of pyrazole amine–amide oxygen dimers in solution.The simi-larity of the structural arrangement of these anhy-drous forms and commonality of the dimer formation, particularly of form IV,may explain the propensity of solvate formation with this compound.Form IV also has two molecules in the asymmetric unit,one with a conformation associated with most solvates,which also can explain why solvated forms tended to desolvate to form IV.Calculations performed to determine the basis for the low energy of form XLI relative to the other forms shows that form XLI does not show the dimer pair formation but instead has a molecular conformation which allows an extended two-dimensional intermolecular hydrogen bonding network to form,which preferentially stabilizes form XLI relative to the other anhydrous forms.While strong hydrogen bonds also exist in the other forms, these molecules hydrogen bond in dimer pairs rather than in an extended network.
According to the results of a full conformational search,there are other molecular conformations (‘‘form XLI-sister’’conformers),which were not observed but could potentially also produce anhy-drous forms with an extended network of the strongest hydrogen bonds.These‘‘form XLI-sister’’conformers differ from form XLI only by the conformation of the propenylpyridine fragment and are energetically less favorable based on their conformational energies(Tab.6).These form XLI-sister forms were not observed in either the early conventional polymorph screening or the previously discussed desolvation https://www.doczj.com/doc/7114461975.html,putational approaches identi?ed a greater likelihood of the appearance of the form XLI-sister conformers at elevated temperatures.It is also reasonable to assume that at room temperature,conformers of forms I,IV,VI,and XXV are stabilized relative to the conformer of form XLI by the formation of molecular dimers in saturated solutions.Therefore,a poly-morph screening approach that incorporates a sig-ni?cant increase of the experimental temperature may result in breaking up those dimers,reducing the formation of solvates,and increasing the populations of unobserved or lower populated conformations, including the form XLI-sister conformers.Although the high-temperature screening approach was applied initially to the desolvation screen discussed in the previous section,with now more sophisticated knowledge of the conformational and hydrogen bonding traits of form XLI relative to the other Table6.Relative Energies of the Form XLI and XLI-Sister Conformers
Propenylpyridine D E(PBE/DNP/COSMO)
(kCal/mol)
0(Form
XLI)
0.85
0.93
1.53
3884CAMPETA ET AL.
anhydrous forms,it was sought to build upon and apply this approach to a more advanced level.
For validation of this rational,the temperature dependence of the conformational populations of axitinib in a number of high boiling solvents (Tab.7)was calculated using the COSMOTherm program.The calculations demonstrated an increase of the absolute and relative populations of the form XLI-sister forms with temperature in all solvents. However,the population of the form XLI conformer also correspondingly increases with temperature and stays noticeably higher than the population of any of its sister-forms,theoretically favoring formation of the form XLI polymorph.
To apply this principle,several desolvation experi-ments were performed at high temperatures(130–1508C)using several of the solvents listed in Table7. It was advantageous that the chemical stability of axitinib is quite robust and,as such,high-tempera-ture experiments could be conducted without chemi-cal decomposition occurring.Initial high-temperature slurry-based desolvation experiments were performed in p-cymene at1508C.It has been previously demonstrated that p-cymene does not form a solvate.This study was run in parallel with the three-component screen discussed in the previous section,and thus employed using the same solvates that were prepared.The results of this high boiling solvent screen are shown in Figure5and indicated a preponderance of conversion to form XLI(83%).The remaining samples were either known metastable anhydrous forms or mixtures of known anhydrous forms.No new forms of axitinib were observed from this desolvation study.
Additional targeted high-temperature desolvation studies were conducted to verify these results.The polymorph conversions of form XVI(an isopropanol solvate which had previously shown to readily converts to other forms)via slurry experiments in p-cymene and in p-cymene/benzyl alcohol mixture were performed at elevated temperature.An online Raman spectroscopic immersion probe was inserted directly into the slurry,allowing collection of spectral data in real time at all temperatures of the slurry. This was very useful to identify any potential intermediate phase transformations that would occur during the desolvation process.Online Raman spectroscopy successfully tracked the axitinib form while reslurrying in these solvents and showed the conversion from the initial solvate form XVI to the metastable anhydrous form IV,which then trans-formed to the most stable anhydrous form XLI.Based on the Raman spectral trends,full conversion of the API in the slurry to form XLI appeared to be complete within a total elapsed time of5h.Of?ine PXRD analysis con?rmed the identity of the?nal product to be form XLI.No evidence for additional polymorphic forms was seen in the analysis of the Raman data. These conditions did not produce new forms(parti-cularly form XLI-sister forms),thus supporting the robustness of form XLI as the most stable polymorph.
A further experiment involved heating a slurry of form XLI in p-cymene at1508C for3h prior to cooling to1008C,for an additional6h of reslurrying.In situ analysis of the solid form by Raman indicated no conversion of form XLI to any new form.Analysis of the isolated material from the end of the reslurry experiment indicated the presence of form XLI alone. An additional1308C reslurry experiment starting with form XVI using3:1p-cymene/benzyl alcohol resulted in the formation of form XLI as observed by PXRD.
CONCLUSIONS
Axitinib has shown to be a polymorphically challen-ging https://www.doczj.com/doc/7114461975.html,mon screening approaches did not lead to the discovery of the low energy form,form XLI, although early clues were yielded as to the solid-state complexity of this API as evidenced by the large numbers of solvates that were discovered.It was recognized that overcoming solvation tendencies and focusing on desolvation pathways were critical to formation of anhydrous forms.After the discovery and characterization of this new anhydrous form,a thorough evaluation of the crystal structures of all the known anhydrous forms was undertaken.This lead to a detailed understanding of the molecular conforma-tions and hydrogen bonding patterns,which enabled the design of speci?c polymorph screening methodol-ogies that targeted desolvation mechanisms.The results from the desolvation screens con?rmed that form XLI was the low-energy form.This work provides an example of applying computationally derived structural knowledge to the design and implementation of targeted polymorph screening methodologies.This allowed the high propensity of forming solvates to be circumvented;this tendency
Table7.High Boiling Point Solvents Used in the
COSMOTherm Calculations of Conformational Populations
Solvent BP(8C)
Anisole155
o-Cymene175
p-Cymene175
m-Cymene175
o-Cresol200
p-Cresol200
m-Cresol200
Benzyl alcohol205
2-Methoxyethanol124
Tert-amyl alcohol102
n-Butanol118
TARGETED POLYMORPH SCREENING APPROACH DEVELOPMENT FOR AN API3885
had prevented the successful use of conventional screening approaches to identify the low-energy anhydrous form.
Experimental and calculated powder X-ray diffrac-tograms of axitinib forms I,IV,VI,XXV,and XLI are available as Supporting Information.The crystal-lographic data of these forms are also available (reference numbers773991-773995)with the Cam-bridge Crystallographic Data Center,CCDC. ACKNOWLEDGMENTS
A number of individuals contributed to this project. The authors thank the following group for their assistance in the manufacture and characterization of axitinib:Shane Horgan,John O’Connell,John Barry,Philomena Enright,Jerome McCormick,Aoife Nagle,Conor McSweeney,and Pat McGauley.The authors also thank Ivan Samardjiev,Jon Bordner, and Ilie Saracovan for solid form characterization studies.The structural informatics support and manuscript review of Neil Feeder and George Qual-lich,respectively,are also appreciated. REFERENCES
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3886CAMPETA ET AL.
药物晶型 物质在结晶时由于受各种因素影响，使分子内或分子间键合方式发生改变，致使分子或原子在晶格空间排列不同，形成不同的晶体结构。同一物质具有两种或两种以上的空间排列和晶胞参数，形成多种晶型的现象称为多晶现象(polymorphism)。虽然在一定的温度和压力下，只有一种晶型在热力学上是稳定的，但由于从亚稳态转变为稳态的过程通常非常缓慢，因此许多结晶药物都存在多晶现象。固体多晶型包括构象型多晶型、构型型多晶型、色多晶型和假多晶型。 同一药物的不同晶型在外观、溶解度、熔点、溶出度、生物有效性等方面可能会有显著不同，从而影响了药物的稳定性、生物利用度及疗效,该种现象在口服固体制剂方面表现得尤为明显。药物多晶型现象是影响药品质量与临床疗效的重要因素之一，因此对存在多晶型的药物进行研发以及审评时，应对其晶型分析予以特别的关注。目前鉴别晶型主要是针对不同的晶型具有不同的理化特性及光谱学特征来进行的，现将几种常用且特征性强、区分度高的方法介绍如下，以供参考。 1 X-射线衍射法（X-ray diffraction） X-射线衍射是研究药物晶型的主要手段，该方法可用于区别晶态和非晶态，鉴别晶体的品种，区别混合物和化合物，测定药物晶型结构，测定晶胞参数（如原子间的距离、环平面的距离、双面夹角等），还可用于不同晶型的比较。X-射线衍射法又分为粉末衍射和单晶衍射两种，前者主要用于结晶物质的鉴别及纯度检查，后者主要用于分子量和晶体结构的测定。 1.1 粉末衍射粉末衍射是研究药物多晶型的最常用的方法。粉末法研究的对象不是单晶体，而是众多取向随机的小晶体的总和。每一种晶体的粉末X-射线衍射图谱就如同人的指纹，利用该方法所测得的每一种晶体的衍射线强度和分布都有着特殊的规律，以此利用所测得的图谱，可获得出晶型变化、结晶度、晶构状态、是否有混晶等信息。该方法不必制备单晶，使得实验过程更为简便，但在应用该方法时，应注意粉末的细度，而且在制备样品时需特别注意研磨过筛时不可发生晶型的转变。 1.2 单晶衍射单晶衍射是国际上公认的确证多晶型的最可靠方法，利用该方法可获得对晶体的各晶胞参数，进而确定结晶构型和分子排列，达到对晶型的深度认知。而且该方法还可用于结晶水/溶剂的测定，以及对成
晶型药物常用的检测分析方法 (2012-02-08 13:54:05) 物质在结晶时由于受各种因素影响，使分子内或分子间键合方式发生改变，致使分子 或原子在晶格空间排列不同，形成不同的晶体结构。同一物质具有两种或两种以上的空间排列和晶胞参数，形成多种晶型的现象称为多晶现象(polymorphism)。虽然在一定的温度和压力下，只有一种晶型在热力学上是稳定的，但由于从亚稳态转变为稳态的过程通常非常缓慢，因此许多结晶药物都存在多晶现象。固体多晶型包括构象型多晶型、构型型多晶型、色多晶型和假多晶型。 药物分子通常有不同的固体形态，包括盐类，多晶，共晶，无定形，水合物和溶剂合物；同一药物分子的不同晶型，在晶体结构，稳定性，可生产性和生物利用度等性质方面可能会有显著差异，从而直接影响药物的疗效以及可开发性。如果没有很好的评估并选择最佳的药物晶型进行研发，可能会在临床后期发生晶型的变化，从而导致药物延期上市而蒙受巨大的经济损失，如果上市后因为晶型变化而导致药物被迫撤市，损失就更为惨重。因此，药物晶型研究和药物固态研发在制药业具有举足轻重的意义。 由于药物晶型的重要性，美国药监局(FDA)和中国药监局(SFDA)在药物申报中对此提 出了明确规定，要求对药物多晶型现象进行研究并提供相应数据。正因如此，任何一个新药的研发，都要进行全面系统的多晶型筛选，找到尽可能多的晶型，然后使用各种固态方法对这些晶型进行深入研究，从而找到最适合开发的晶型；选定最佳晶型后，下一步就是开发能始终如一生产该晶型的化学工艺；最后一步是根据制剂对原料药固态性质的要求，对结晶工艺进行优化和控制，确定生产具有这些固态性质的最佳工艺参数，从而保证生产得到的晶型具有理想的物理性质，比如晶体表象，粒径分布，比表面积等。这种通过实验设计来保证质量的方法必须对药物晶型具有非常全面深刻的理解才能实现。 原研药公司对药物分子的晶型申请专利，可以延长药物的专利保护，从而使自己的产 品具有更长时间的市场独享权。而对于仿制药公司来说，为了确保仿制药和原研药在生物利用度上的等同性，也需要对原料药的晶型进行研究，以确保原料药和制剂的质量，正因为如此美国药监局在ANDA申报中也对仿制药多晶型控制有明确的指南；另外，开发出药物的 新晶型从而能够打破原研药公司对晶型的专利保护，提早将仿制药推向市场，也是近年来仿制药公司一个至关重要的策略，而且如果能找到在稳定性，生物利用度，以及生产工艺方面具有优越性的新晶型，还可以申请晶型专利保护，从而大大提升自己的市场竞争力。总之，不管是新药开发，还是仿制药生产，药物晶型研究都是必不可少的中心环节。 目前鉴别晶型主要是针对不同的晶型具有不同的理化特性及光谱学特征来进行的，现 将几种常用且特征性强、区分度高的方法介绍如下，以供参考。 1 X-射线衍射法（X-ray diffraction） X-射线衍射是研究药物晶型的主要手段，该方法可用于区别晶态和非晶态，鉴别晶体的品种，区别混合物和化合物，测定药物晶型结构，测定晶胞参数（如原子间的距离、环平面的距离、双面夹角等），还可用于不同晶型的比较。X-射线衍射法又分为粉末衍射和单晶 衍射两种，前者主要用于结晶物质的鉴别及纯度检查，后者主要用于分子量和晶体结构的测定。
1.药物分析的任务是什么？ ①.药物成品的化学检验工作 ②.药物生产过程的质量控制 ③.药物贮存过程的质量考察 ④.临床药物分析工作 2.高效液相色谱法检查药物的杂质方法有几种? ①.内标法加校正因子测定供试品中某个杂质含量 ②.外标法测定供试品中某个杂质含量 ③.加校正因子的主成分自身对照法 ④.不加校正因子的主成分自身对照法 ⑤.面积归一法 3.杂质有哪些来源和途径? 来源； ①.从药物生长过程中引入 ②.由药物储藏过程中引入 途径：在合成药的生产过程中，未反应完全的原料、反应的中间体和副产物，在精致时未能完全出去，就会成为产品中的杂质。药品在储藏过程中，在外界条件的影响下，或因微生物的作用，可能发生水解、氧化、分解、异构化、晶型转变、聚合、潮解和发霉等变化，产生有光的杂质。具有酚羟基、巯基、亚硝基、醛基以及长链共轭多烯等结构的药物，在空气中易被氧化，引起药物变色、失效甚至产生毒性的氧化产物等。 4.铁盐检查法中加入硫酸铵的目的是什么？ 加入氧化剂过硫酸铵，一方面可以氧化供试品中Fe2＋成Fe3＋，同时可防止光线导致的硫氰酸铁还原货分解褪色。 5.为什么标准铅液、标准铁液、标准砷液都要事先配制成储备液存放，用时稀释？铅离子、铁离子和亚砷酸根离子在低浓度、近中性溶液中易水解，故先配成高浓度的酸性储备液，使其稳定。用时稀释即可。 6.薄层色谱法检查杂质的类型有哪几种？ ①.选用实际存在的待检杂质对照品法 ②.选用可能存在的某种物质作为杂质对照品 ③.高低浓度对比法 ④.在检查条件下，不允许有杂质斑点。 7.用对照法检查杂质，应注意哪些方面的平行？ 供试液的处理和对照液的处理在所用试剂、反应条件、反应时间、实验顺序登方面要相同，以保证结果的可比性。 8.重金属检查的常用四种方法反别在什么情况下应用？ 第一法（硫代乙酰胺法）：适用于无需有机破坏，在酸性条件下可溶解的，无色的药物的重金属检查。 第二法（炽灼破坏后检查重金属）：适用于含芳香环、杂环以及不溶于水稀酸及乙醇的有机药物的重金属检查。 第三法（硫化钠法）：适用于溶于碱而不溶于稀酸或在稀酸中生成沉淀的药物。第四法（微孔滤膜法）：适用于含2～5μg重金属杂质及有色供试液的检查。 9.制定杂质检查项目和限量的原则是什么？ 凡是影响疗效和对人体健康有害的杂质均应制定相应的检查项目和限量。
药物分析学 药物分析学是药学学科下设的二级学科，是我国高等教育药学专业教学计划中规定设置的一门主要专业课程，也是我国执业药师考试中指定考试的专业课程之一。本课程主要介绍药品质量标准及其制订和药品质量检验的基本知识，通过本课程的学习为从事药品质量检验和新药的研究开发工作奠定基础。 第一节药物分析的性质和任务 药物分析学是药品全面质量控制的一个重要学科，它主要运用物理学、化学、生物化学的方法与技术研究、解决化学结构已经明确的合成药物或天然药物及其制剂的质量控制问题，也研究有代表性的中药制剂和生化制剂的质量控制方法。因此，药物分析学是一门研究与发展药品质量控制的“方法学科”，是药学学科的重要组成部分。 药物分析学科和药物分析工作者的任务，除了药品的常规理化检验以及药品质量标准的研究和制订外，尚需深入到生物体内过程并进行综合评价的动态分析研究中；所采用的分析方法应该更加准确、灵敏、专属和快速，并力求向自动化、最优化和智能化方向发展。 第二节药物分析与药品质量标准 一、我国药品质量标准体系 我国药品质量标准体系包括：法定标准和非法定标准；临时性标准和正式标准；内部标准和公开标准等。其中，法定标准又可分为中国药典、局颁标准和地方标准等三级药品标准。 （一）法定药品质量标准 我国现行的法定的药品质量标准体系包括：中华人民共和国药典、国家药品监督管理局药品标准、省（自治区、直辖市）药品标准，人们习称为“三级标准”体系。 中华人民共和国药典，简称为中国药典。是由国家药典委员会主持编纂、国家食品药品监督管理局批准并颁布实施。中国药典是我国记载药品标准的法典，是国家监督管理药品质量的技术标准。凡生产、销售和使用质量不符合药典标准规定的药品均为违法行为。 国家食品药品监督管理局标准（简称局颁标准），系由国家食品药品监督管理局批准并颁布实施的药品标准 省（自治区、直辖市）药品标准，系由各省、自治区、直辖市的卫生行政部门〔卫生厅（局）〕批准并颁布实施的药品标准，属于地方性药品标准（简称地方标准），主要收载具有地方性特色的药品标准。 （二）临床研究用药品质量标准 该标准仅在临床试验期间有效，并且仅供研制单位与临床试验单位使用，属于非公开的药品标准。
第四节药物晶型研究 内容： 1.药物的晶型物质存在状态。 2.不同晶型物质间的形式差异。 3.晶型对药物理化性质的影响。 4.晶型对药物稳定性的影响。 5.晶型对药物临床有效性的影响。 6.晶型对药物安全性的影响。 第五节优势药物晶型 药用优势药物晶型是指对于具有多种形式物质状态的晶型药物而言，应具备晶型物质相对稳定、能够最好的发挥防治疾病作用、毒副作用较低的晶型特质状态。研究药用优势药物晶型，就是在多晶型药物研究中选择优势药物晶型的过程，药用的优势药物晶型研究主要内容包括三个方面： 一、晶型的稳定性 晶型药物物质状态不同，其晶型稳定性间亦可存在较大差异，作为药物晶型物质必须具备一定的稳定性质，这是保证药品质量的最基本的要求。药物晶型稳定性一方面是指晶型自身的稳定性，即在不同环境条件下能够保持晶型物质状态的稳定；此外，由于药品都是以剂型形式存在，也应保证药物制剂中的优势药物晶型和各种药用辅料物质在临床应用过程中的稳定。所以，只有符合药物稳定性要求的晶型物质才有可能成为一个理想的优势药物晶型。 二、不同晶型物质对药物生物利用度的影响 不同晶型物质会影响药物在机体内的吸收，吸收差异性可是数倍乃至数十倍。药物晶型引起的吸收变化会直接影响到药物在临床中发挥作用。因此，吸收性质是药用优势药物晶型选择的关键条件。但是，生物利用度的提高并不能作为药用晶型优劣筛选的单纯条件依据。对于不同药物而言，生物利用度提高可能会产生更好的药理作用，也可能会产生更多的不良反应。而导致这种差异的原因是来自于每种药物的自身性质和在生物体内分布的特点，这是在药物晶型选择中必须要考虑的重要因素。 三、优势药物晶型的选择需要观察药物的有效性和毒性反应。 药物晶型不仅影响着药物的吸收，同时不会影响到药物在体内的作用和在体内产生的不良反应。对于体内分布不均一的药物，在生物利用度提高的情况下，会导致个别靶器官浓度过高而引起毒性反应。同样，对于作用的靶器官药物浓度的提高会产生更好的疾病治疗作用。因此，在对优势药物晶型进行评价的过程中必须对药物的有效性、安全性进行全面的考察和评价研究。 第六节晶型药物的临床疗效 尽管药物的不同晶型并不影响药物的化学结构和组成，其主要的化学性质也可以没有明显影响，但是，这并不意味着不同晶型药物就完全相同。事实上，同一种药物由于晶型不同，其不仅物理性质会有所不同，而且其生物活性也有明显的差异。有些药物不同的晶型的生物活性不仅差异非常显著，而且干扰了药物的临床应用。 一、同一药物不同产品的差异 为控索和揭示引起国产药品与进口药品间、国内制药企业的药品间、同一企业的不同生产批号药品间的临床疗效与质量差异问题，我们进行了深入的调查研究，就最为常用的各种固体药物剂型而言，国产仿制药与进口药相比较，可以有如下情况发生： 1.没有差异的药品两者临床疗效完全相同。这类药品突出的表现是药品的质量相同，固体剂型类型完全一致，虽然进口与国产药品两者的价格差别较大，但事实上在药品临床疗效中不存在差异性。 2.符合标准的药品差异国产药品使用的质量控制标准与国外的进口药品标准非常接近，但是，在临床应用中却被屡屡发现国产药品与进口药品间疗效存在有很大的差异性，即：国产药品的临床疗效明显低于进口的药品。 3.药品临床疗效不稳定的现象国产同一药品的不同生产企业或同一生产企业的不同生产批次药品
化学结构相同的药物,可因结晶条件不同而得到不同的晶型, 积溶出97.以上0%,一旦成为溶液状态,就不存在两种晶型之别, 这种现象称为多晶型。药物的晶型不同,其物理性质如密度、熔点、因此它们的生物利用度无显著差别是合理的。药物的多晶型现象 [1] 溶解度和溶出速度均有不同。在一定温度与压力下,多晶型中只极为普遍,例如38种巴比妥药物中63%有多晶型, 48种甾体化合 有一种是稳定型,溶解度最小,化学稳定性好,而其他晶型则为亚物中67%有多晶型,目前仍有部分多晶型药物的生物利用度差异 。盐酸金霉素有α,β两种晶型,β型比α型溶解度大,动物口子排列,然而由于较难得到足够大小和高纯度的单晶,因此多采用 [12 -]13 服后β型的血药浓度较高,人口服后尿排泄量较多,说明β型的生粉末衍射法。不同晶型的晶胞参数如晶面距、晶面夹角等不 [4] 物利用度较高。利福定用不同溶剂结晶可以得到4种晶型,其中同,所得到的衍射光谱也必然不一样。如奈非西坦两种晶型的X - 利福定Ⅳ型为有效剂型,其血药浓度高峰是利福定Ⅱ型血药浓度高射线粉末衍射光谱就 不同。近年来出现的小分子衍射区域检测器 [5] 峰的10倍。阿司匹林存在两种晶型,志愿者试验表明,服用Ⅱ型为分析较小晶体或纯度不够的晶体提供了可能。 [6] 的血药浓度比服用Ⅰ型的高70%。3)红外光谱(IR)法:红外吸收光谱是共价键运动能级跃迁的 药物多晶型对生物利用度的影响,最典型的例子是无味氯霉结果,同一物质的不同晶型由于分子内共价键的电环境不一样,共 [7] [14] 素(氯霉素棕榈酸酯)。无味氯霉素有A, B,种晶型及无定型C 3 , 价键强度也会有变化,因此必然导致多晶型IR光谱的改变。此 其中B晶型与无定型有效,而A、C两种晶型无效。1975年以前,我方法较为简便、快速,但对于不同晶型间红外光谱差异较小的药物 国生产的无味氯霉素原料、片剂及胶囊剂均为无效剂型,后来经过则难以区别,同时图谱的差异也可能来自其他方面的原因,如样品 进一步研究,改进生产工艺后生产出了有活性的B型,并在质量标纯度不够、同系物错标、晶体大小、溴化钾(KBr)压片过程的晶型转 准中增加了非活性晶型的含量限度,确保了药物的疗效。变等。比如在盐酸丁螺环酮的多晶型研究中,为避免KBr压片时压 [15]
药物分析方法进展 摘要: 药物分析的发展已从一种专门技术逐步发展成为一门日臻成熟的科学，所涉及的研究范围包括药品质量控制、临床药学、中药与天然药物分析、药物代谢分析、法医毒物分析、兴奋剂检测和药物制剂分析等。随着药物科学的迅猛发展，各相关学科对药物分析不断提出新的要求，它已不再仅仅局限于对药物进行静态的质量控制，而是发展到对制药过程、生物体内和代谢过程进行综合评价和动态分析研究。 关键词药物分析研究进展 药物是预防、治疗、诊断疾病和帮助机体恢复正常机能的物质。药品质量的优劣直接影响到药品的安全性和有效性，关系到用药者的健康与生命安危。虽然药品也属于商品，但由于其特殊性，对它的质量控制远较其他商品严格。因此，必须运用各种有效手段，包括物理、化学、物理化学、生物学以及微生物学的方法，通过各个环节全面保证、控制与提高药品的质量。传统的药物分析，大多是应用化学方法分析药物分子，控制药品质量。然而，现代药物分析无论是分析领域，还是分析技术都已经大大拓展。从静态发展到动态分析，从体外发展到体内分析，从品质分析发展到生物活性分析，从单一技术发展到联用技术，从小样本分析发展到高通量分析，从人工分析发展到计算机辅助分析。 具体一点的讲，药物分析是分析化学技术在药学领域中的具体应用。分析化学的进步，尤其是近年仪器分析和计算机技术的进展，为药物分析的发展提供了坚实的基础。药物分析的任务是在药学各个领域中，对出于不同的目的和要求, 不同来源和组成的样品中的某些成分进行检出、鉴别和测定。药物分析发展的主要趋向就是如何能够简便、快速地从复杂组成的样品中，灵敏、可靠地检测一些微量成分。 药物分析学的研究范围包括药物质量控制、临床药学、中药与天然药物分析、药物代谢分析、法医毒物分析、兴奋剂检测和药物制剂分析、创新药物研究，以及药品上市后的再评价等，哪里有药物，哪里就有药物分析。 1、药物分析技术的发展 光谱法如紫外分光光度法、核磁共振光谱法、质谱法、拉曼光谱法、红外光谱法、荧光、磷光及化学发光光谱法、原子吸收和原子发射光谱法以及X 2射线衍射谱法等，方法较多。近年来发展虽不如色谱那么迅速，在药典中所占的比重有下降的趋势，但是仍出现了很多新方法，如二维核磁共振谱法、近红外光谱法、激光拉曼光谱以及色谱光谱联用技术等。在新的世纪中,这些方法会有更快的发展，并广泛地应用于药学科学各领域中。电化学部分分别为化学传感器、离子选择性电极和动力电化学方法与应用。近几年生物传感器的发展，成为电分析化学中活跃的研究领域。微电极技术是一种新的电化学测试技术，在活体分析中，微电极用作电化学微探针，检测动物神经传递物质的扩散过程，成为微柱液相色谱和高效毛细管电泳的电化学检测器。在将来药物分析的发展中，将会显示出光辉的应用前景。 复杂样品中微量成分的检测是在药物分析工作中比较困难的问题。色谱法对复杂样品具有较高的分离能力，是药物分析中常用的分析技术。 薄层色谱法主要用于药物及制剂的鉴别、杂质检查以及中药成分分析，已成为当今药
药物分析的方法学验证所要做到的事项（一） 新药申报时，药品质量标准中分析方法必须验证；药物生产工艺变更、制剂的组分变更、原分析方法进行修订时，则质量标准分析方法必须进行验证；2005版药典中分析方法验证指导原则只规定了项目和基本方法而没有合格标准:附录XIX A；中国GMP(98)非常关注验证的过程,分析方法验证不完善是常见的问题 药物分析检验时药品生产的GMP的药物分析的方法学验证，是保证药物分析结果准确性的前提和基础，也是实现药物分析检测GMP的必然要素。 构成药物分析中的检测方法验证，这要涉及到以下些方面的内容： 1、分析方法验证成功的前提条件： （！）仪器已经确认、校正并在有效期内（2）经过培训的人员（3）可靠稳定的对照品 （4）可靠稳定的实验试剂（5）确认受试溶液的稳定性，在规定时间内无降解。 2、分析方法学验证所要求验证的内容：（1）含量的测定（2）杂质的含量测定（3）药物的定性鉴定（4）药物的含量均匀度测定（5）药物的微生物检测（6）药物的细菌内毒素的检测 验证内容： 准确度：准确度是指用该方法测定的结果与真实值或参考值接近的程度，用百分回收率表示。 测定回收率R（recovery）的具体方法可采用加样回收试验法来进行测定。加样回收试验已准确测定药物含量P的真实样品+已知量A的对照品（或标准品）测定，测定值为M。数据要求:规定的范围内，至少用9次测定结果评价，如制备高、中、低三个不同浓度样品各测三次 精密度：（重复性、中间精密度和重现性精密度是指在规定条件下，同一个均匀样品，经多次取样测定所得结果之间的接近程度。用偏差（d）、标准偏差(SD)、相对标准偏差(RSD)（变异系数，CV）表示。 （1）重复性(repeatability):在相同条件下，由同一个分析人员测定所得结果的精密度；在规定的范围内，至少用9次测定结果评价，如制备三个不同浓度样品各测三次或把被测物浓度当作100%，至少测6次进行评价 （2）中间精密度同一实验室，不同时间由不同分析人员用不同设备
1.药物分析的任务是什么 ①.药物成品的化学检验工作 ②.药物生产过程的质量控制 ③.药物贮存过程的质量考察 ④.临床药物分析工作 2.高效液相色谱法检查药物的杂质方法有几种 ①.内标法加校正因子测定供试品中某个杂质含量 ②.外标法测定供试品中某个杂质含量 ③.加校正因子的主成分自身对照法 ④.不加校正因子的主成分自身对照法 ⑤.面积归一法 3.杂质有哪些来源和途径 来源； ①.从药物生长过程中引入 ②.由药物储藏过程中引入 途径：在合成药的生产过程中，未反应完全的原料、反应的中间体和副产物，在精致时未能完全出去，就会成为产品中的杂质。药品在储藏过程中，在外界条件的影响下，或因微生物的作用，可能发生水解、氧化、分解、异构化、晶型转变、聚合、潮解和发霉等变化，产生有光的杂质。具有酚羟基、巯基、亚硝基、醛基以及长链共轭多烯等结构的药物，在空气中易被氧化，引起药物变色、失效甚至产生毒性的氧化产物等。 4.铁盐检查法中加入硫酸铵的目的是什么 加入氧化剂过硫酸铵，一方面可以氧化供试品中Fe2＋成Fe3＋，同时可防止光线导致的硫氰酸铁还原货分解褪色。 5.为什么标准铅液、标准铁液、标准砷液都要事先配制成储备液存放，用时稀释铅离子、铁离子和亚砷酸根离子在低浓度、近中性溶液中易水解，故先配成高浓度的酸性储备液，使其稳定。用时稀释即可。
6.薄层色谱法检查杂质的类型有哪几种 ①.选用实际存在的待检杂质对照品法 ②.选用可能存在的某种物质作为杂质对照品 ③.高低浓度对比法 ④.在检查条件下，不允许有杂质斑点。 7.用对照法检查杂质，应注意哪些方面的平行 供试液的处理和对照液的处理在所用试剂、反应条件、反应时间、实验顺序登方面要相同，以保证结果的可比性。 8.重金属检查的常用四种方法反别在什么情况下应用 第一法（硫代乙酰胺法）：适用于无需有机破坏，在酸性条件下可溶解的，无色的药物的重金属检查。 第二法（炽灼破坏后检查重金属）：适用于含芳香环、杂环以及不溶于水稀酸及乙醇的有机药物的重金属检查。 第三法（硫化钠法）：适用于溶于碱而不溶于稀酸或在稀酸中生成沉淀的药物。第四法（微孔滤膜法）：适用于含2～5μg重金属杂质及有色供试液的检查。 9.制定杂质检查项目和限量的原则是什么 凡是影响疗效和对人体健康有害的杂质均应制定相应的检查项目和限量。 ①.质量标准中规定的杂质限量是指正常生产和储藏过程中可能引入的杂质。 ②.制定杂质的检查项目和限量要不断完善和提高 ③.杂志项目和限量的制定要有针对性，能反映生产水平的高低以及生产工艺是否正常 ④.严重危害人体健康和影响药物稳定性的杂质必须制定相应的检查项目，并严格控制其限量 ⑤.药物的杂质检查项目和限量制定与化学制剂的杂质控制项目和限量不同，不能混淆。 10.苯甲酸钠含量测定的原理。 苯甲酸钠易溶于水，水溶液呈弱酸性，可用盐酸直接滴定。但生成的苯甲酸
药物晶型常用的检测分析方法 物质在结晶时由于受各种因素影响，使分子内或分子间键合方式发生改变，致使分子或原子在晶格空间排列不同，形成不同的晶体结构。同一物质具有两种或两种以上的空间排列和晶胞参数，形成多种晶型的现象称为多晶现象(polymorphism)。虽然在一定的温度和压力下，只有一种晶型在热力学上是稳定的，但由于从亚稳态转变为稳态的过程通常非常缓慢，因此许多结晶药物都存在多晶现象。固体多晶型包括构象型多晶型、构型型多晶型、色多晶型和假多晶型。物质在结晶时由于受各种因素影响，使分子内或分子间键合方式发生改变，致使分子或原子在晶格空间排列不同，形成不同的晶体结构。同一物质具有两种或两种以上的空间排列和晶胞参数，形成多种晶型的现象称为多晶现象(polymorphism)。虽然在一定的温度和压力下，只有一种晶型在热力学上是稳定的，但由于从亚稳态转变为稳态的过程通常非常缓慢，因此许多结晶药物都存在多晶现象。固体多晶型包括构象型多晶型、构型型多晶型、色多晶型和假多晶型。 药物分子通常有不同的固体形态，包括盐类，多晶，共晶，无定形，水合物和溶剂合物；同一药物分子的不同晶型，在晶体结构，稳定性，可生产性和生物利用度等性质方面可能会有显著差异，从而直接影响药物的疗效以及可开发性。如果没有很好的评估并选择最佳的药物晶型进行研发，可能会在临床后期发生晶型的变化，从而导致药物延期上市而蒙受巨大的经济损失，如果上市后因为晶型变化而导致药物被迫撤市，损失就更为惨重。因此，药物晶型研究和药物固态研发在制药业具有举足轻重的意义。 由于药物晶型的重要性，美国药监局(FDA)和中国药监局(SFDA)在药物申报中对此提出了明确规定，要求对药物多晶型现象进行研究并提供相应数据。正因如此，任何一个新药的研发，都要进行全面系统的多晶型筛选，找到尽可能多的晶型，然后使用各种固态方法对这些晶型进行深入研究，从而找到最适合开发的晶型；选定最佳晶型后，下一步就是开发能始终如一生产该晶型的化学工艺；最后一步是根据制剂对原料药固态性质的要求，对结晶工艺进行优化和控制，确定生产具有这些固态性质的最佳工艺参数，从而保证生产得到的晶型具有理想的物理性质，比如晶体表象，粒径分布，比表面积等。这种通过实验设计来保证质量的方法必须对药物晶型具有非常全面深刻的理解才能实现。 原研药公司对药物分子的晶型申请专利，可以延长药物的专利保护，从而使自己的产品具有更长时间的市场独享权。而对于仿制药公司来说，为了确保仿制药和原研药在生物利用度上的等同性，也需要对原料药的晶型进行研究，以确保原料药和制剂的质量，正因为如此美国药监局在ANDA申报中也对仿制药多晶型控制有明确的指南；另外，开发出药物的新晶型从而能够打破原研药公司对晶型的专利保护，提早将仿制药推向市场，也是近年来仿制药公司一个至关重要的策略，而且如果能找到在稳定性，生物利用度，以及生产工艺方面具有优越性的新晶型，还可以申请晶型专利保护，从而大大提升自己的市场竞争力。
药品研发中多晶型问题浅议 发布日期20070913 栏目化药药物评价>>化药质量控制 作者张玉琥 审评四部审评七室张玉琥 多晶型现象在固体化学药品研发过程中比较常见。由于多晶型问题可能影响药品的安全有效性和质量可控性，应当针对不同情况采取相应的措施。本文就药品研发中涉及的多晶型现象及其相关问题作简要讨论，供药品研发和评价工作参考。 一、晶型与多晶型现象 固体药物有结晶型和非结晶型（无定形）之分。构成药物结晶的基本单元为晶格，在晶格中药物分子以一定的规律排列。而无定形是分子以无序的方式排列，不具有明确的晶格。若药物结晶中包含结晶溶剂分子，就称为溶剂化物。当该溶剂为水时（即含有结晶水），通常称为水合物。药物的不同晶型是由分子在晶格中排列方式的不同所致。 需要注意晶型与结晶形态（晶癖）的区别。前者由晶格中分子的排列来决定，后者是指形成的结晶的外观形状，如针状结晶、片状结晶等。同一晶型的药物，可能具有不同的外观形状；反之，外观形状相同的结晶，其晶型也可能不同。 若固体药物可存在不同的晶型，或存在结晶型与无定形，或存在非溶剂化物与溶剂化物、不含或含有结晶水等现象，就称为该药物存在多晶型现象。 一些方法可用于研究和区分多晶型现象。单晶X－射线衍射可对晶体结构提供直接的证据，是研究多晶型现象的可靠方法。需要注意的是若获取单晶所采用的结晶条件与药物生产中实际采用的结晶条件不同，则单晶X－射线衍射得到的晶体特征并不代表药物实际的晶体特征。粉末X－射线衍射是常用的研究和区分不同晶型的有效方法。该方法不仅可用于不同晶型的定性区分，在建立特征衍射峰与不同晶型含量之间的定量关系后，粉末X－射线衍射还可用于不同晶型比例的定量控制。其他方法，包括显微镜、热分析（差示扫描量热、热重分析、热台显微镜等）、光谱法（红外光谱、拉曼光谱、固相核磁共振）等，亦有助于进一步研究多晶型现象。 二、多晶型对药物性质的影响 存在多晶型现象的药物，由于晶格能的不同，其不同晶型可具有不同的的化学和物理性质。如不同的熔点、化学反应性、表观溶解度、溶出速率、光学和机械性质、蒸汽压、密度等。 多晶型药物不同晶型之间理化性质的不同，可能对原料药及制剂的制
?综述? ［收稿日期］!""#$#!$!%；［修回日期］!""!$"&$!’［作者简介］王洪亮（#(’)*），男，天津宝坻人，河北医科大学药学院药剂学教研室在读硕士研究生，从事药剂学研究。 药物多晶型的鉴别方法 王洪亮#，董 燕!综述，陈桂兰#，王淑月#审校 （#+河北医科大学药学院药剂学教研室，河北石家庄",""#’；!+山东省菏泽地区第三医院，山东荷泽!’%"-!）【主题词】药物；结晶；溶解度；光谱分析；差热分析；显微镜检查，电子，扫描【中图分类号】.(#-【文献标识码】/ 【文章编号】#""’$-!",（!""!）",$"-"’$"- 同一种药物，由于结晶条件的不同，可以生成完全不同类型的晶体（01234566789），这种现象称为药物的多晶型现象（:;62<;1:=73<），亦称同质异晶现象。有机药物中多晶型现象是普遍存在的。药物的晶型不同，会对生物利用度、药效、毒副作用、制剂工艺及 稳定性等诸多方面产生影响［#，!］，故对多晶型进行 准确、快速的鉴别很有必要。目前鉴别晶型的方法主要是针对不同晶型具有不同的理化特性及光谱学 特征来进行的［-］，现将一些常用且特征性强、区分 度高的方法综述如下。!熔点测定法 一般而言，由于多晶型晶格能差会使同质异晶体间存在熔点差异，故可用熔点测定法做定性鉴别。例如尼莫地平!种晶型的熔点：>型#!-!#!,?，@型##!!##%?， 差异十分显著。一般熔点较高的是稳定型，常用的测定熔点方法主要有以下几种。!"!毛细管法：该法是中国药典的法定方法，它操作简便，使用样品量少，但测得的数值常略高于真实熔点，且主观性较强，需要操作者具有较熟练的实验技能。尽管如此，它的精确度已可满足一般要求，对于晶型间熔点差别较大者，已能完全区分开。!"#熔点测定仪法：该法是借助光学显微镜观察，加热台进行加热，采用不同升温速率或方式持续升温，当样品熔化时，立即观测加热台上温度计温度，即得该样品的熔点。采用这种方法，精确度高，主观性低，且可同时清楚地看到样品的晶体形态和熔化全过程的固相变化。 另外，A ;B 691热台偏光显微镜法和热分析法也是测定多晶型熔点的常用方法。#溶解度与热力学参数的关系 固体药物的溶解度和温度有关，多数随着温度 的升高，溶解度逐渐增大。晶体的溶解度大小和其自由能有关。一般说来，自由能越大，越不稳定，溶解度也越大；反之则小。在实践中，常测定各晶型在不同温度下的溶解度，并绘制出溶解度（C 3）$温度（D ）曲线。通过C 3$D 曲线， 可以区分出稳定晶型和不稳定晶型。若有相交曲线亦可得到其热力学转变 温度（D : ）。A 17346等［%］ 通过对无环鸟苷的不同晶型的研究，发现两者之间的溶解度和溶解速度存在很 大差异。甲氰米胍的,种晶型溶出速率亦有明显差 别［,］。 对于难溶性药物，若以68C 3对#／D （绝对温度的倒数）回归，多可获得良好的线性关系。其二者遵循亨利定律：68C 3E*!>／.D F 0;834 。!>为溶解焓，晶型不同，其值不同。一般来讲，!>小者，溶解度大，熔点低，为亚稳定型或不稳定型；!>大者， 溶解度小，熔点高，为稳定型［&，’］。 王静康等［)，(］采用激光技术和间歇动态法对苄 青霉素钠进行了结晶热力学和动力学分析，得到了 相应的三相平衡相图和间歇蒸发结晶模型［(，#"］。 $红外分光光度法 红外光谱系分子的振动$ 转动能级跃迁引起的吸收光谱，不同的晶型，由于其内部分子的分子间力作用方式和作用强度不同，使形成的晶格能不同，造成红外光谱的差异，如吸收峰位置的移动和缔合、吸收强度的变化、吸收峰数目的增减等。一般来说，同一药物若得到彼此完全不同的红外图谱，几乎可以 肯定存在不同的晶型。例如西咪替丁［#"］、吲哚拉 新、棉酚、盐酸丁卡因等均如此。测定时，多采用石蜡油糊法（G H I ;6法），以避免在研磨时发生晶型转变，或压片时压力破坏晶胞。对于某些不会因研磨而发生转晶的药物，也可采用A J 1压片法测定。 红外分光光度法还可用于测定混晶的相对含 量。王绪明等［##］，应用标准曲线法，测定了联苯双 酯同质异晶体的相对含量。 但也有某些药物不同晶型的K .光谱间无明显 ? ’"-?第!-卷第,期 !""!年(月河北医科大学学报L M N .G /@M O>P J P KQ P R K C /@N G K S P .T K D U S ;6+!-G ;+,T 9: 4+!""!万方数据
【摘要】目的强调固体药物早期研究与开发阶段进行多晶型研究的重要意义，并介绍药物多晶型研究的几种手段。方法查阅相关文献并结合研究经验，归纳总结了有关药物晶型研究进展，讨论了固体药物晶型的鉴别研究方法、晶型药物对生物利用度的影响、影响固体药物晶型的因素及研究药物多晶型的意义。结果与结论应根据新药晶型的具体情况，选择适当的研究方法，以确定合适的目标晶型。 【关键词】固体药物；多晶型；生物利用度 固体物质按其内部原子、离子或分子的排列方式可分为晶型（包括假晶型）和无定形。晶型形成的基础是物质微粒之间的相互作用，药物微粒间的作用方式可以是金属键、共价键、范德华力等，因此晶体可分为金属晶体、共价键晶体、分子晶体等［1］。有机药物晶体大多是分子晶体, 可因结晶条件不同而得到不同的晶型，这种现象称为多晶型。药物的多晶型现象极为普遍，晶型不同，它们的物理性质如密度、熔点、硬度、外观、溶解度和溶出速度等方面差异均有显著性［2～3］。在一定温度与压力下，多晶型中只有一种是稳定型，溶解度最小，化学稳定性好，其他晶型为亚稳定型，它们最终可转变为稳定型。一般讲，亚稳定型的生物利用度高，为有效晶型，而稳定晶型药物往往低效甚至无效。因此，药物多晶型的研究已经成为新药开发和审批、药物的生产和质量控制以及新药剂型确定前设计所不可缺少的重要组成部分。 1 药物多晶型的鉴别研究方法 对多晶型药物，要确证其结构，除了要确定其分子中各原子的组成、数量及相互间的连接方式外，还要确定各分子在不同晶格中的填充、排列方式。由于分析方法的灵敏度及仪器分辨率的限制, 不同晶型间的差异常常出现在分析范围边缘, 因此同时采用多种方法进行研究。过去几十年中，常用的晶型研究方法有：热分析法、红外分光光度法、热载台显微镜法、溶解度测定法及X-射线衍射法等，近年来又发展了一些新的技术如拉曼分光光度法、固态核磁共振法、近红外分光光度法、热气压测量法以及一些传统方法的联用。 1.1 热分析法热分析法包括差热分析法（DTA）、差示扫描量热法（DSC）及热重分析法（TGA）。同一药物由于晶型不同，在加热（或放热）过程中，吸（或放）热峰会出现差异，因此可以根据吸（或放）热峰的不同来确定不同的晶型。在甲苯磺丁脲多晶型的研究中用DSC 对样品检测，晶型Ⅰ～Ⅳ在80～127 ℃范围内有不同的吸、放热峰［4］。对葛根素［5］采用四种不同溶剂进行结晶，根据DSC和TGA图显示具有四种晶型，熔点分别为206、185、182、211℃。在头孢呋辛酯［6］多晶型的差热分析中，低熔点晶型α则在175℃处出现一个小的吸热峰，而高熔点晶型β在205℃处出现一个尖锐的吸热峰。采用热分析法所需样品量少，方法简便灵敏，重现性好，是药物多晶型研究中常见的一种方法［7］。 1.2 红外光谱（IR）法同一物质的不同晶型，由于分子内共价键的电环境不一样，共价键强度也会有变化，红外吸收光谱是共价键运动能级跃迁的结果，因此必然导致多晶型IR 光谱的改变［8］。红外光谱法较为简便、快速，但同时图谱的差异也可能来自其他方面的原因，如样品纯度不够、同系物的错标、晶体的大小、KBr压片过程的晶型转变等。比如在盐酸丁螺环酮的多晶型研究中，为避免KBr压片时压力可能引起的晶型转变，采用石蜡糊法［9］，很好地测出了A、B两种晶型在红外光谱上的细微差别。当然多晶型也有晶型不同而IR相同的情况，如苯乙阿托品的晶型Ⅰ和Ⅱ的IR 就一样［10］。 1.3 X-射线粉末衍射法因为有机药物不容易得到足够大小和高纯度的单晶，因此多采用粉末衍射法［11,12］，即采用单波长多角度对样品粉末照射,仪器记录衍射强度I/ I0 对2θ（θ为入射角）的变化曲线，不同晶型的晶胞参数如晶面距、晶面夹角等不同，所得到的衍射光谱也必然不一样。近年来发展的小分子衍射区域检测器为分析较小晶体或纯度不够的晶体提供了可能。许多样品在使用热分析法、显微镜检查法或红外光谱法无法分辨出差别的情况下,用X－射线衍射方法却能得到满意的结果［13 ,14］。
1.简述采用紫外分光光度法鉴别药物时常用的方法，以及薄层色谱法检查药物中特殊杂质的方法。 答: 1)测定最大吸收波长，或同时测定最小吸收波长 2)规定一定浓度的供试液在最大吸收波长处的吸收度 3）规定吸收波长和吸收系数法 4）规定吸收波长和吸收度比值法 5）经化学处理后，测定其反应产物的吸收光谱特性 1）杂质对照品法 2）供试品溶液自身稀释对照法 3）杂质对照品与供试品溶液自身稀释对照并用法 4）对照药物法 2.试述古蔡法测砷原理。操作中为何要加碘化钾试液和酸性氯化亚锡试液？醋酸铅棉花起什么作用？ 答:1)原理：金属锌与酸作用产生新生态的氢与药物中微量的砷盐反应生成具挥发性的砷化氢，遇溴化汞试纸，产生黄色至棕色的砷斑，与一定量标准溶液所生成的砷斑比较，判断供试品中重金属是否符合限量规定。 2)五价砷在酸性溶液中也能被金属锌还原为砷化氢，但生成的砷化氢的速度较三价砷慢，故反应中加入碘化钾及氯化亚锡将五价砷还原为三价砷，碘化钾被氢化生成的碘又可被氯化亚锡还原为碘离子，后者与反应中产生的锌离子能形成稳定的配位离子，有利于生成砷化氢的反应进行，还可抑制锑化氢的生成，因锑化氢也能与溴化汞试纸作用生成锑斑。 3)锌粒及供试品种可能含少量硫化物，在酸性液中能产生硫化氢气体，与溴化汞作用生成硫化汞的色斑，干扰试验结果，故用醋酸铅棉花吸收硫化氢 3.简述薄层色谱法检查药物中的杂质，可采用高低浓度对比法检查，何为高低浓度对比法？答: 先配制一定浓度的供试品溶液，然后将供试品溶液按限量要求稀释至一定浓度作为对照溶液，将供试品溶液和对照溶液分别点样于同一薄层板上，展开、斑点定位。供试品溶液所显示杂质斑点与自身稀释对照品溶液或系列浓度自身稀释对照溶液的相应主斑点比较，不得更深。 4.药物分析在药品的质量控制中担任着主要的任务是什么？ 答: 保证人们用药安全、合理、有效，完成药品质量监督工作。 5.常见的药品标准主要有哪些，各有何特点？ 答: 国家药品标准(药典)；临床研究用药质量标准；暂行或试行药品标准；企业标准。 6.药品检验工作的基本程序是什么？ 答: 取样、检验(鉴别、检查、含量测定)、记录和报告。 7.简述RPHPLC法测定有机含氮类药物时色谱峰拖尾的原因，以及克服的措施。 一、造成色谱峰( 不对称)拖尾的原因 1.色谱柱本身填装问题,筛板堵塞或填料塌陷; 2.柱头有污染;
药物晶型的分析方法介绍 审评五部审评十室李志万 物质在结晶时由于受各种因素影响，使分子内或分子间键合方式发生改变，致使分子或原子在晶格空间排列不同，形成不同的晶体结构。同一物质具有两种或两种以上的空间排列和晶胞参数，形成多种晶型的现象称为多晶现象(polymorphism)。虽然在一定的温度和压力下，只有一种晶型在热力学上是稳定的，但由于从亚稳态转变为稳态的过程通常非常缓慢，因此许多结晶药物都存在多晶现象。固体多晶型包括构象型多晶型、构型型多晶型、色多晶型和假多晶型。 同一药物的不同晶型在外观、溶解度、熔点、溶出度、生物有效性等方面可能会有显著不同，从而影响了药物的稳定性、生物利用度及疗效,该种现象在口服固体制剂方面表现得尤为明显。药物多晶型现象是影响药品质量与临床疗效的重要因素之一，因此对存在多晶型的药物进行研发以及审评时，应对其晶型分析予以特别的关注。目前鉴别晶型主要是针对不同的晶型具有不同的理化特性及光谱学特征来进行的，现将几种常用且特征性强、区分度高的方法介绍如下，以供参考。 1 X-射线衍射法（X-ray diffraction） X-射线衍射是研究药物晶型的主要手段，该方法可用于区别晶态和非晶态，鉴别晶体的品种，区别混合物和化合物，测定药物晶型结构，测定晶胞参数（如原子间的距离、环平面的距离、双面夹角等），还可用于不同晶型的比较。X-射线衍射法又分为粉末衍射和单晶衍射两种，前者主要用于结晶物质的鉴别及纯度检查，后者主要用于分子量和晶体结构的测定。 1.1 粉末衍射粉末衍射是研究药物多晶型的最常用的方法。粉末法研究的对象不是单晶体，而是众多取向随机的小晶体的总和。每一种晶体的粉末X-射线衍射图谱就如同人的指纹，利用该方法所测得的每一种晶体的衍射线强度和分布都有着特殊的规律，以此利用所测得的图谱，可获得出晶型变化、结晶度、晶构状态、是否有混晶等信息。该方法不必制备单晶，使得实验过程更为简便，但在应用该方法时，应注意粉末的细度，而且在制备样品时需特别注意研磨过筛时不可发生晶型的转变。 1.2 单晶衍射单晶衍射是国际上公认的确证多晶型的最可靠方法，利用该方法可获得
体内药物分析 体内药物分析是通过分析的手段了解药物在体内（包括实验动物等机体）数量与质量的变化，获得各种药物代谢动力学的各种参数和转变、代谢的方式、途径等信息。从而有助于药物生产、实验、研究、临床等各个方面对所研究的药物作出估计与评价，以及对药物的改进和发展作出贡献。 体内药物分析任务和对象的特点： 1、被测定的药物和代谢物的浓度或活性极低； 2、样品中存在各种直接或间接影响测定结果的物质，大多需要分离和净化； 3、样品量少，尤其是连续测定时，很难再度获得完全相同的样品； 4、工作量较大，随着工作的深入开展，会成倍地甚或按指数级数增加； 5、往往要求很快地提供结果，尤其在毒物检测工作中； 6、实验室应有多种检测手段，可进行多项分析工作； 7、测定数据的处理和阐明有时不太容易。 样品的种类、采集和储存 一、样品的种类和选取原则：（一）血样：血浆(plasma)和血清(serum)是体内药物分析最常采用的样本，其中选用最多的是血浆。因血浆中的药浓可反映药物在体内（靶器管）的状况。而且血浆中药物浓度的数据报道较多，可供借鉴。血浆是全血(whole blood)在加肝素、枸橼酸、草酸盐等抗凝剂的全血经离心后分取，量约为全血的一半。血清则是在血液中纤维蛋白元等影响下，引起析出血块，离心取得。血块凝结时往往易造成药物吸附损失。全血也应加入抗凝剂混匀，以防凝血。对大多数药物来说血浆浓度与红细胞中的浓度成正比，所以测定全血也不能提供更多的数据，而全血的净化较血浆与血清麻烦，尤其是溶血后，血色素等可能会给测定带来影响。但是一些可与红血球结合或药物在血浆和血球的分配比率因不同病人而异的情况下，则宜采用全血。血样采取量会受到一定的限制，血样取样时间间隔问题也常随测定目的不同而异。目前大都是测定原型药物总量。当药物与血清蛋白结合率稳定时，血药总浓度可以有效表示游离药物的浓度。但对低蛋白症或尿毒症患者，药物结合率降低，则在通常安全有效的血药总浓度中，游离型药物浓度可显著增加。 （二）尿样(urine)：尿样测定主要用于药物剂量回收研究、药物肾清除率和生物利用度等研究，以及测定代谢物类型等。体内药物清除主要是通过尿液排出，药物可以原型（母体药物）或代谢物及其缀合物形式排出。尿液药物浓度较高，收集量可以很大，但尿液浓度通常变化较大，所以宜测定一定时间内尿中药物的总量（如8、12、24小时内的累计量），需记录排出尿液体积及尿药浓度。尿药浓度改变不直接反映血药浓度，受试者肾功能将影响药物的排泄。尿中药物大多呈缀合状态，测定前要将缀合的药物游离。此外，采集尿液不可能在较短时间内多次取样，排尿时间较难掌握（尤其是婴儿），同时也具有不易采集完全的缺点。（三）唾液(saliva)：唾液中的药物浓度通常与血浆浓度相关。样品易得，取样无损害，尤易为儿童接收。有些可从药物唾液浓度推定血浆中游离药物浓度。但有些蛋白结合率较高的药物在唾液中的浓度比血浆浓度低得多，需高灵敏度的方法才能检测。唾液pH值6.9±0.5，每日分泌量1～1.5L，含有的主要电解质有Na+、K+、Cl-、HCO3-等，主要有机成分是粘液质和淀粉酶。采样一般是在漱口后15分钟，收集口内自然流出或经舌在口内搅动后流出的混合唾液（吸管内吸附的少量唾液用稀释液洗出），用2000～3000rpm离心15分钟，小心吸取上清液，进一步分离、净化。也可采用物理（嚼石蜡片、小块聚四氟乙烯或玻璃大理石）或化学（酒石酸、维生素C）的方法刺激，在短时间内可得到大量唾液，但药浓也可能会受到影响。