WO2009152635A1

WO2009152635A1 - Gradient-based screening tools for polymorph detection and identification

Info

Publication number: WO2009152635A1
Application number: PCT/CH2009/000210
Authority: WO
Inventors: Nicolas Spencer; Eva Beurer; Sara Maria Morgenthaler; Stefan Zürcher
Original assignee: ETH Zürich
Priority date: 2008-06-20
Filing date: 2009-06-19
Publication date: 2009-12-23

Abstract

Methods, kits and crystallization templates for inducing and determining polymorphs in a compound are described herein. In one embodiment, the crystallization templates contain one or more surface-chemical and/or surface morphology gradients. The crystallization template is typically exposed to a solution or vapor containing the composition to be tested. Then the compound of interest is allowed to crystallize on the surface. The nucleation effects can be either morphological or chemical in nature. A variety of surfaces, modified either chemically or morphologically in different ways, can be used to identify the presence and types of polymorphs in a compound or composition of interest. Analysis of the crystalline structure of the tested compound may be performed by any suitable detection and analysis technique. After identifying the different polymorphs one or more polymorphs may be selected for further testing, and/or production.

Description

GRADIENT-BASED SCREENING TOOLS FOR POLYMORPH

DETECTION AND IDENTIFICATION CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Application Serial No. 61/074,300, entitled "Gradient-Based Screening Tools for Polymorph Detection and Identification" filed June 20, 2008.

FIELD OF THE INVENTION

This invention is in the field of methods and devices for detecting and identifying polymorphs. BACKGROUND OF THE INVENTION

Polymorphism in material science refers to the ability of a solid material to exist in more than one crystal form or crystal structure. Polymorphism can be found in any crystalline material including polymers, minerals, small molecules, such as drugs, proteins, and metals. Polymorphism in crystalline materials is related to allotropy, which is the ability of elemental solids to take two or more different forms in which the atoms are bonded together in a different manner. Examples of polymorphism include glycine, which is able to form monoclinic and hexagonal crystals, and silica, which is known to form several polymorphs, including α-quartz, β-quartz, tridymite, cristobalite, coesite, and stishovite.

Together with polymorphism, the complete morphology of a material can be described by other variables such as crystal habit, amorphous fraction and crystallographic defects.

Polymorphism can exist as a result of differences in crystal packing, referred to as packing polymorphism. Polymorphism can also result from the existence of different conformers of the same molecule, referred to as conformational polymorphism. In pseudopolymorphism the different crystal types are the result of hydration or solvation.

Polymorph selective crystallization on chemically modified surfaces has been shown for example with CaCO₃, anthranilic acid and 2-iodo-4- nitroaniline {see Kuther, et ah, Templated growth of calcite, vaterite and aragonite crystals on self-assembled monolayers of substituted alkylthiols on gold, Journal of Materials Chemistry, 8(3): 641-650 (1998); Carter & Ward, Directing Polymorph Selectivity During Nucleation of Anthranilic Acid on Molecular Substrates, J Amer. Chem. Soc 'y, 116(2):769-770 (1994); Hiremath, et ah, Selective growth of a less stable polymorph of 2-iodo-4- nitroaniline on a self-assembled monolayer template, Chem. Comm., 23: 2676-2677 (2004)). Chemically modified surfaces can also suppress crystallization of kinetically favored but thermodynamically less stable polymorphs by inhibiting heterogeneous nucleation on the surface {see e.g. Cox, et ah, Selective growth of a stable drug polymorph by suppressing the nucleation of corresponding metastable polymorphs. Angewandte Chemie- International Edition, 46 (23): 4333-4336 (2007)). The kinetics of the crystallization process can also be influenced by the surface properties {see e.g. Frostman, et α/. , Nucleation and Growth of Molecular-Crystals on Self-Assembled Monolayers, Langmuir, 10 (2):576- 58 (1994)). A strong interaction with the surface leads to faster nucleation, meaning that more and smaller crystals are formed. This becomes important in crystallizing proteins. To determine their crystal structure, the crystals need to be as large as possible. Pham, et al., Well-ordered self-assembled monolayer surfaces can be used to enhance the growth of protein crystals. Colloids and Surfaces B-Biointerfaces, 34, (3), 191-196 (2004), discloses surface modification of a crystallization vessel that inhibited protein interaction with the surface and allowed for the growth of large protein crystals.

The fact that compounds can form polymorphs is of particular concern to the pharmaceutical industry, where different polymorphs of the same chemical compound can display different pharmacological properties and different physical properties that may be pharmacologically relevant, such as solubility and stability. If the most stable polymorph is not the pharmacologically desired form, i.e. the product, there is a danger that the product will, over time or in the presence of other external factors, such as heat, humidity, and/or nucleation conditions, convert into a less desirable, but more stable polymorph. It is important that the manufacturer of the pharmaceutical product is aware of the polymorphs that might exist for every new product under many different conditions. Further, it is particularly important that the manufacturer is aware of those polymorphs that are not easily formed, such as those that are kinetically hindered, but are nevertheless very stable. Therefore manufacturers of pharmaceutical compositions carry out extensive screening procedures to detect polymorphs.

Currently screening for polymorphs is performed by solvent- mediated polymorphic transformation, which involves exposing solutions of the compound in many different solvents (for example, to cover a range of polarities) to a wide range of temperature conditions. Generally, both thermodynamic and kinetic (e.g. rapid cooling) conditions are explored. These screening techniques do not take into account the influence of the heterogeneous nucleation on surfaces and interfaces. This phenomenon can be explored by testing a wide variety of surfaces individually or by using surface gradients. However, the former techniques are typically time- consuming.

There exists a need for improved methods and devices to screen for polymorphism, which are less time consuming and more economical than current methods and devices.

Therefore it is an object of the invention to provide improved methods and devices to screen for polymorphs in chemical compounds, particularly in pharmaceutical compositions.

SUMMARY OF THE INVENTION Methods, kits and crystallization templates for inducing and determining polymorphs in a compound are described herein. In one embodiment, the crystallization templates contain one or more surface- chemical and/or surface morphology gradients. The crystallization template is typically exposed to a solution or vapor containing the composition to be tested. Then the compound of interest is allowed to crystallize on the surface. Next the template is analyzed to determine the various polymorphs of the compound. Analysis of the crystalline structure of the tested compound may be performed by any suitable detection and analysis technique. After identifying the different polymorphs one or more polymorphs may be selected for further testing, and/or production.

The surface of the template can influence the nucleation process; depending on the particular properties of the surface a particular polymorph may be formed. The nucleation effects can be either morphological or chemical in nature. A variety of surfaces, modified either chemically or morphologically in different ways, can be used to identify the presence and types of polymorphs in a compound or composition of interest. BRIEF DESCRIPTION OF THE DRAWINGS

Figure IA is an illustration of a crystallization template having a circular configuration. Figure IB is an illustration of a crystallization template having a rectangular configuration. The crystallization template may contain dividers, as illustrated in these Figures.

Figure 2 is a schematic of an experimental procedure for forming a hydrophilicity gradient followed by immersion in a solution of the molecule to be analyzed to from a further gradient over the immersion time.

Figure 3 is a bar graph showing % of the average crystal size (white bars) and % of the average number of crystals (dark shaded bars) as a function of immersion time (seconds) in a dodecanethiol solution for a substrate having surface-chemical wettability gradients on its surface. The values for crystal size and number of crystals were normalized by setting the average for each immersion time as 100 %. Values greater than 100 % indicate that the crystals were larger than the average crystal size, and values smaller than 100 % indicate that the crystals were smaller than average crystal size for this particular immersion time. Figure 4 is a bar graph showing % of the average crystal size (white shaded bars) and % of the average number of crystals (dark shaded bars) as a function of immersion time (seconds) in a dodecanethiol solution for a substrate having surface-chemical wettability gradients alternating with hydrophobic strips on its surface. The values for crystal size and number of crystals were normalized by setting the average for each immersion time as 100 %. Values greater than 100 % indicate that the crystals were larger than the average crystal size, and values smaller than 100 % indicate that the crystals were smaller than average crystal size for this particular immersion time. DETAILED DESCRIPTION OF THE INVENTION

I. Definitions

As used herein "gradient" refers to a graded change in the magnitude of some physical or chemical property of the surface. For example, the graded change can be in the height, width, and/or distance of structures on the surface, such as grooves or other structures or textures, which can initiate crystallization. Alternatively, the change can be a change in the density of functional groups on the surface, for example, which increase or decrease a chemical property of the surface, such as hydrophobicity, hydrophilicity, bioactivity, etc. A simple example is a continuous change in hydrophobicity from one end of a substrate to the other. This is a chemical-composition gradient, and virtually any pair of surface-bound species can be combined into a gradient. A further property that lends itself to gradient fabrication is morphology, and roughness gradients, for example, can readily be combined with surface-chemical gradients, either in parallel or orthogonally.

As used herein "graded change" refers to variations in the same increments in a given property for the template. II. Devices for Screening Polymorphs

A crystallization template (10) may be used to induce the formation of polymorphs. Representations of a circular and rectangular template are shown in Figures Ia and Ib of the template. For example, the template may induce formation of a crystal form different from the crystal form found in solution. The crystallization template contains a surface with one or more gradients of one or more properties of the surface. In one embodiment, the surface contains a chemical gradient, i.e. surface chemistry gradually changes. In another embodiment, the surface contains a morphological gradient, e.g., the surface roughness gradually changes.

The crystallization template may contain more than one gradient. In one embodiment, the crystallization template contains two gradients. In a preferred embodiment the two gradients are perpendicular to each other. In another embodiment, the gradients are parallel to each other.

Optionally, the crystallization template contains one or more dividers (12 a, b, and c) separating the template into multiple regions (see Figure IB).

In one embodiment, the surface of the crystallization template is patterned to selectively form crystals at certain locations on the surface. For example, patterned gradients can be prepared as described in Morgenthaler et ai, Biointerphases, Vol. 1, No. 4, 156-165 (2006). a. Materials The template may be formed of any suitable material. The choice of material for the surface of the template for chemical gradients is determined by the adsorbate-substrate interaction and method of analysis for polymorphs. The surface of the template does not form a covalent bond with the molecule to be tested.

In a preferred embodiment, the surface of the template allows for the formation of a variety of chemical gradients and is suitable for analysis of the substance to be tested using any suitable analysis method, such as Raman spectroscopy or x-ray diffraction. In one embodiment, the template material is an inorganic material including, but not limited to, glass, silicon or another semiconductor material, metals, metal oxides, metalloids, ceramics, and combinations thereof. For example, the surface may be the surface of a silicon wafer or other semiconductor. In another embodiment, the template is a metal, such as gold, silver, palladium, or copper. In another embodiment, the material is an organic material, such as a naturally occurring, semi-synthetic, or synthetic polymer.

If the template is non-metallic, the surface of the template may be treated to contain a metal coating, such as a layer of gold, silver, palladium, or copper. Typically the metal coating has a thickness ranging from about 50nm to about 300nm, preferably from about 80 run to about 100 nm. In a preferred embodiment, the template is formed from glass or silicon, and the surface of the template has been modified to contain a metal coating, such as gold, silver, palladium, or copper, preferably a gold coating. In another preferred embodiment, template is formed from a synthetic polymer, which is coated with a metal coating, preferably a gold coating. For example, a polyimide foil can be coated with a thin layer of gold (e.g. 80 nm).

The template can have any suitable dimensions. With respect to thickness, the thickness of the template should be sufficient to prevent deformation of the template during immersion. Typically templates are about 1 mm thick. Typical lengths range from 5 mm to 10 centimeters, or longer. In a preferred embodiment, the length of the surface is 1 cm or longer, and typically ranges from 1 cm to 5 cm. b. Gradients A wide range of different types of gradients may be created on the surface of the crystallization templates. Preferably the gradient is a chemical or morphological gradient. Morphological and chemical gradients can also be combined on a single surface. In one embodiment, morphological and chemical gradients are combined on a single surface forming 2-dimensional, orthogonal gradients, where one gradient is perpendicular to a second gradient. In another embodiment, the gradients are parallel to each other. 1. Surface Chemical Gradients

A gradual change in a physical property, such as the wettability, can be induced by a change in surface chemistry, for example a gradually changing surface composition. The surface-chemical gradients may form hydrophobicity gradients, where the hydrophobicity/hydrophilicity of the surface increases or decreases along the length (or radius) of the substrate surface, or gradients that contain bioactive molecules, where the concentration of bioactive molecule increases or decreases along the length (or radius) of the substrate surface. The gradients are typically self- assembled monolayers (SAMs).

The surface-chemical gradients typically display a high packing density, as demonstrated by the low hysteresis in dynamic contact angle and x-ray photoelectron spectroscopy (XPS) and reflection-absorption infrared spectroscopy (RAIRS) measurements. However, the surface-chemical gradient may be a packing density gradient. A packing density gradient leads to a gradual change in the order and disorder of the molecule(s) on the surface of the template as demonstrated the dynamic contact angle, RAIRS, and x-ray photoelectron spectroscopy (XPS) measurements.

Other chemical modifications can be used to separate polymorphs. For example, poly (L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) may be attached to the surface via interactions between the surface and the polylysine end to form a gradient. Preferably the polyethyleneglycol is end- functionalized with a functional group. The end-functionalized molecules may be coupled to biomolecules to form a biochemical gradient.

In one embodiment, the surface is modified by immobilizing chiral compounds on the surface. This allows for enantioselective crystallization of the compound to be tested. Methods for making Chemical Gradients

Any method that forms a surface chemistry gradient having the desired properties may be used. A number of methods for forming surface chemistry gradients are known (see e.g. Ruardy, T.G., et al., Surf. Sci. Rep. 1997, 29, 1-30; Liedberg, B. and Tengvall, P. Langmuir, 1995, 11, 3821- 3827; Efimenko K., et al., Macromolecules 2003, 36, 2448-2453; Morgenthaler et al, Soft Matter, Vol. 4, 419-434 (2008); and Genzer et al., Langmuir, Vo. 24, No. 6, 2294-2317 (2008)). For example, several methods can be used to form thiol-based chemical gradients, including (1) the cross- diffusion of two thiol solutions through a polysaccharide matrix (Liedberg, B. and Tengvall, P. Langmuir, 1995, 11, 3821-3827), (2) applying an electrochemical potential to a substrate during adsorption (Terrill R.H., et al., J.Am.Chem.Soc 2000, 122, 988-989), (3) the use of microfluidic devices (Jeon N.L., et al., Langmuir 2000, 16, 8311-8316; Dertinger S.K.W., et al., Anal.Chem. 2001, 73, 1240-1246), and (4) scanning-tunneling-microscopy- based replacement lithography (Fuierer R.R., et al., Adv.Mater. 2002, 14, 154-157). Vapor deposition methods may be used to form gradients (see e.g. U.S. Patent No. 6,770,323 to Genzer).

A preferred method for forming surface chemical gradients is provided in European Patent No. 1 610 909 to Eidgenossische Technische Hochschule Zurich. European Patent No. 1 610 909 discloses forming a surface chemical gradient using an adsorbate-containing liquid boundary that is in relative motion to the substrate. This may be accomplished through controlled immersion of a substrate into one or more solutions containing an adsorbate using a linear-motion drive to form one or more linear gradients. Alternatively a syringe and a syringe pump may be used to form a radial gradient. The speed at which the substrate is exposed to the advancing front of a solution containing an adsorbate is selected based on the absorption kinetics of the adsorbate to ensure that the adsorbate is exposed to the advancing front of the solution for a sufficient time period to adsorb the adsorbate onto the surface of the substrate and form a chemical gradient.

Any solution containing a compound that adsorbs onto the surface of the substrate can be used. Any solvent or solvent system (such as a co- solvent) can be used provided is dissolves the organic or inorganic material to be screened. The solvent can be an aqueous solvent or an organic solvent. In a preferred embodiment, the adsorbate solution contains a thiol. Suitable thiols include alkane thiols, such as methyl-terminated thiols with varying hydrocarbon chain length, such as CH₃(CHi)_nSH, where n = 4 - 18; hydroxyl-terminated thiols with varying hydrocarbon length, such as

OH(CH₂)_nSH, where n = 8 - 18, carboxylic-terminated thiols with varying hydrocarbon chain length, such as HOOC(C^)_nSH, where n = 8 -18; and lH,2H,2H,2H-perfluordecane-l-thiol ((CF₃(CF₂)₇(CH₂)₂SH).

Other suitable thiols include thiols with larger cross sectional areas, such as functionalized mercaptobiphenyl. Optionally, the alkanes are end- functionalized with reactive groups. Such reactive groups include biotin, vinylsulfone, maleimide, or N-hydroxy succinimide. These reactive groups may be coupled to biomolecules to prepare a biochemical gradient. The biomolecules may be any bioactive molecule, including for example peptides, proteins, oligosaccharides, polysaccharides, DNA, RNA, or lipids.

For example, linear and radial gradients may be produced on, for example, oxidized silicon wafers, by means of two different adsorbing polyelectrolytes, such as poly (L-lysine)-g-poly(ethylene glycol), with or without end functionalization. The end-functionalized molecules may be coupled to biomolecules to form a biochemical gradient.

The concentration of the adsorbing solution typically ranges from about 0.1 μM to about 0.1 M, preferably from about 1 μM to about 1 mM. The concentration is selected, along with the speed, to produce a surface where the concentration of the adsorbate increases from one end to the other. Thus one end contains little or none of the first adsorbate, while the other end is fully saturated, or nearly saturated with the first adsorbate. If a second adsorbate is added, it has an opposite concentration gradient to the concentration gradient of the first adsorbate.

A surface-chemical gradient film composed of a single component is a result of the varied coverage and packing of the adsorbate along the immersion axis of the substrate. Since partial monolayers are generally less ordered than full monolayers, this initial surface also displays a gradient in order. To remove this non-homogeneity to promote the formation of a complete monolayer while maintaining the surface-chemical gradient, the substrate is immersed in a second adsorbate solution in a second step. Generally, in the second step, a more concentrated adsorbate solution is used.

Optionally, the gradient may be formed using two perpendicular immersions into two separate adsorbates. This process forms a 2- dimensional, orthoganol chemical gradient.

The self-assembling monolayers (SAMs) can be functionalized in order to generate surfaces that present a range of functionalities, such as nonpolar, polar, electroactive, biologically active, etc. Methods for engineering surfaces are described in Whitesides et ah, Chem. Rev., 105, 1103-1169 (2005) and fall into three categories: (1) synthesis of functionalized thiols for forming single component or mixed SAMs by (co- )adsorption; (2) insertion of synthesized thiols into defect sites of preformed SAMs; and (3) modification of the surface composition of a preformed SAM. Covalent reaction and non-covalent interactions (van der Walls forces, hydrogen bonding, metal-ligand bonding, etc.) can be used to generate new surfaces.

Bioactive molecules can be coupled to the SAM by direct reaction with exposed functional groups on SAMs. Exposed functional groups immersed in a solution of bioactive molecules can react directly with the molecules in solution under appropriate reaction conditions. For example, bioactive molecules, such as peptides and carbohydrates can react with SAMs having maleimide functionalities on the surface. Examples of other reactive surface groups are provided in Table 1.

Table 1: Reactive surface groups and the complexes formed following reaction with a ligand

Surface Group (R₁) Ligand Complex Formed (R₂)

R

^= =J* ^■r

Methods for reacting biomolecules with the reactive surface groups in Table 1 are described in Whitesides et al, Chem. Rev., 105, 1103-1169 (2005).

Alternatively, ligands can be attached to the surface of SAMs by forming a reactive intermediate, which is then coupled to the ligand or bioactive molecules to be immobilized on the surface. Two advantages of this technique are that the common intermediate can react with a variety of ligands; and (2) it allows the spatial discrimination of active and inactive regions of the SAM, i.e., the reactivity of the regions on the surface can be turned "on" or "off. Such spatial discrimination can be used to create a gradient of bioactive molecules on the surface. Further, methods for spatial patterning, such as microcontact printing and scanning probe lithography can be used in combination with reactive intermediates to attach biomolecules in specific locations to form the desired gradient.

Examples of reactive intermediates are provided in Table 2. Table 2: Examples of surface groups, reactive intermediates, and complex formed

Surface Group (Ri) Intermediate (R₂) Ligand Complex Formed (R,)

Functional groups on the surface of the SAM may be converted to reactive intermediates by chemical reaction and/or by the application of external stimuli, such as electrochemical potentials, photoradiation, ultrasound, and combinations thereof.

In still another embodiment, reactive functional groups may be introduced onto the surface of the SAM by cleaving covalent bonds of surface functional groups to generate a reactive functional group. In yet another embodiment, bioactive molecules can be immobilized on the surface of the SAM via a linker. Suitable linkers include small organic molecules, oligomers, and polymers. For example, a polymer can be grafted to the surface of the SAM and the bioactive molecule(s) can be coupled to the grafted polymer. A list of exemplary polymers that can be grafted to SAMs is provided in Table 3 along with a corresponding mechanism for attachment. Table 3: Examples of Polymers Grafted to SAMs via Surface Initiation polymer rβf polystyrene phσtoicitlated radical poϊymerfzattαn 379 thermal radical pdymeriiation 380 ifvfmg anionic poljnierizaticn 381 polyacrylonitrflβ pltotoiniiiatect radical poiynioriZatico 382 polyacrylαmide ATRP 383 polydiorbαrnens) ring-opening metathesis 384 poiy(methYl mβthacrylaie) ATRI* 385 poljtBiycidyl mβthacrylatβ) ATRP 385 ρoly( butyl mathacrylale) ATRP 385 po!y(2-hydroryetliyl methacrylate) ATRP 385 polylactlds ring-opening polymerization 386 poly(p-diαianone) ring-opening polymerization 387 enzymatic polymerization 388 poly(3-hy<lrojcyt>utjτate) enzymatk polymerizatioii 389 polyethylene glycol dimethacrylate) ATRP 390 polyCe-caprolactane) 388 ring-opening polymeri∑atton 386

' ATBP = Atom transfer radical polymerization.

The surface of the SAM may be modified non-covalently by using the intrinsic properties of the surface (e.g., hydrophobicity, electrostatics, etc.) or selective interactions with preformed chemical functional groups on the surface to promote absorption of materials on the surface. Suitable classes of molecules that can absorb onto the exposed surface of a SAM include, but are not limited to, surfactants, polymers, polyelectrolytes, proteins, organic dyes, and colloidal particles. For example, hydrophobic SAMs readily absorb amphiphilic molecules (e.g., surfactants), some polymers, and most proteins. 2. Surface morphology gradients

The morphology on the surface of the crystallization template can be controlled, such that it changes over a given distance, creating a morphological gradient. The morphological gradient can be any physical change or alteration to the surface, such as smooth to rough. Typically changes in the surface morphology are on the nanometer scale. The imperfections or rough areas of the surface may serve as nucleation sites and thereby influence crystallization and the formation of polymorphs. Usually nucleation starts heterogeneously from a surface or an interface, such as the surface of the crystallization template. Morphology gradients can be fabricated using a variety of techniques known in the art. For example, morphology gradients can be fabricated using a two-step roughening and smoothening process. In a first step, the template is bead blasted to form a homogeneous roughness on the surface of the template. In a subsequent chemical polishing process, the substrate is immersed into a polishing solution, such as a hot acidic solution (e.g. a combination of phosphoric, nitric and sulphuric acid), and continuously withdrawn by means of a linear motion drive. The polishing solution, depending on the residence time of a specific surface location, preferentially removes features with a small radius of curvature and thus leads to the smoothing out of the surface topography and resulting in a roughness gradient. This method is particularly preferred for forming a morphology gradient on a metal surface.

Other suitable techniques for forming morphology gradients include, but are not limited to, lithography; such as photolithography; chemical vapor deposition (e.g., followed by solvent vapor exposure); crystallization of breath figures (i.e. spherical cavities); pulsed laser ablation; etc. III. Methods for screening polymorphs

The templates and methods described herein can be used to identify a variety of polymorphs for a given compound. The templates and methods described herein can also be used to determine the most thermodynamically favorable polymorph or one or more metastable polymorphs. A metastable pharmaceutical solid form can change crystalline structure or solvate/desolvate in response to changes in environmental conditions, processing, or over time.

Polymorphs of a compound may be induced to crystallize on a surface by exposing the surface of a crystallization template to a solution containing the compound to be tested. The entire surface may be exposed to the solution, hi another embodiment, only portions of the surface are selected and exposed to the solution, where each portion of the surface is separated from the other portions by one or more dividers.

In one embodiment, the method by which the template is exposed to the compound to be tested also creates a gradient. For example, the entire surface of the template may be exposed to the solution or vapor containing the compound to be tested and then slowly withdraw from the solution or vapor containing the compound to be tested. This creates an immersion time or exposure time gradient with respect to the compound to be tested. Suitable means of exposure include immersing the gradient in a solution of the compound to be crystallized, or painting, spraying, or otherwise applying a solution of the compound to be crystallized to the gradient. In one embodiment, the surface of the crystallization template is exposed to a vapor containing the compound to be tested. The compound to be tested sublimates and is thereby deposited on the surface of the template.

In another embodiment, the solution or vapor containing the compound to be analyzed is oversaturated with the compound. Oversaturation can be achieved by lowering the temperature of the solution to be analyzed, reducing the amount of the solvent in the solution to be analyzed, and/or lowering the temperature of the gradient.

In some embodiments, parameters, such as temperature, pressure and/or humidity of the environment, may be controlled to ensure that one or more polymorphs are formed and detected.

Following deposition on the surface, the compound crystallizes and forms one or more polymorphs depending on the properties of the portion of the surface to which the compound is exposed.

Optionally, surface gradient separation, as described above, can be combined with solvent-mediated polymorphic transformation and temperature programs in order to increase the probability that all polymorphs have been identified for a particular compound. It is known that unstable polymorphic forms have a greater solubility than the metastable forms in a particular solvent and that monotropic forms have a lower melting point than enantiotropic forms. These observations have been related to the phenomenon of supersaturation and supercooling in Ostwald's Rule of Steps or Law of Successive Reactions.

Solvent-mediated polymorphic transformation can be used to obtain the most stable polymorph of a material. For example, the most stable polymorph can be obtained by (1) dissolving the metastable phase to form a solution which is supersaturated with respect to the more stable phase; (2) nucleation of the more stable phase; and (3) growth of the more stable phase.

Analysis of the crystalline structure of the tested compound may be performed by any suitable technique. Suitable techniques include, but are not limited to, X-ray powder diffraction (XRPD), Raman spectroscopy, differential scanning calorimetry (DSC), infrared (IR) spectroscopy, solid stated nuclear magnetic resonance (NMR), and/or optical microscopy {see e.g. Kamat et al, Pharm. Res., 5(7): 426-429 (1988); Pan, et al, AAPS Pharm Sci Tech, 7(l):Article 11, pages E1-E7, El, right col., (2006)).

Following crystallization and analysis of the polymorph(s) formed on the template, one or more polymorphs can be selected for further testing, production and/or scale up, based on a variety of characteristics, such as melting point, chemical reactivity, apparent solubility, dissolution rate, optical and electrical properties, vapor pressure, density, and combinations thereof. These properties can directly impact the processability of drug substances and the quality/performance of drug products, such as stability, dissolution, and bioavailability.

Compounds to be analyzed Any compound may be tested using the above-described crystallization template and method. In a preferred embodiment the compound to be tested is a bioactive molecule, such as a molecule having therapeutic, prophylactic or diagnostic properties.

The method and device described herein may be used to test polymorphs in a variety of compounds, which may be inorganic or organic compounds. Additionally, the method and device may be used to test polymorphs in a composition containing more than one compound. However, the method and device described herein are particularly preferred for use in detecting polymorphs in pharmaceutical compositions and/or pharmaceutical compounds. The detection of polymorphs in a given composition can aid in selecting storage containers and conditions for the composition of interest.

Exemplary classes of therapeutic agents that can be tested using the method and device described herein include, but are not limited to, analeptic agents; analgesic agents; anesthetic agents; antiasthmatic agents; antiarthritic agents; anticancer agents; anticholinergic agents; anticonvulsant agents; antidepressant agents; antidiabetic agents; antidiarrheal agents; antiemetic agents; antihelminthic agents; antihistamines; antihyperlipidemic agents; antihypertensive agents; anti-infective agents; anti-inflammatory agents; antimigraine agents; antineoplastic agents; antiparkinsonism drugs; antipruritic agents; antipsychotic agents; antipyretic agents; antispasmodic agents; antitubercular agents; antiulcer agents; antiviral agents; anxiolytic agents; appetite suppressants (anorexic agents); attention deficit disorder and attention deficit hyperactivity disorder drugs; cardiovascular agents including calcium channel blockers, antianginal agents, central nervous system ("CNS") agents, beta-blockers and antiarrhythmic agents; central nervous system stimulants; diuretics; genetic materials; hormonolytics; hypnotics; hypoglycemic agents; immunosuppressive agents; muscle relaxants; narcotic antagonists; nicotine; nutritional agents; parasympatholytics; peptide drugs; psychostimulants; sedatives; sialagogues, steroids; smoking cessation agents; sympathomimetics; tranquilizers; vasodilators; beta-agonist; and tocolytic agents.

Exemplary therapeutic agents that can be tested using the method and device described herein include, but are not limited to, ceclofenac, acetaminophen, adomexetine, almotriptan, alprazolam, amantadine, amcinonide, aminocyclopropane, amitriptyline, amolodipine, amoxapine, amphetamine, aripiprazole, aspirin, atomoxetine, azasetron, azatadine, beclomethasone, benactyzine, benoxaprofen, bermoprofen, betamethasone, bicifadine, bromocriptine, budesonide, buprenorphine, bupropion, buspirone, butorphanol, butriptyline, caffeine, carbamazepine, carbidopa, carisoprodol, celecoxib, chlordiazepoxide, chlorpromazine, choline salicylate, citalopram, clomipramine, clonazepam, clonidine, clonitazene, clorazepate, clotiazepam, cloxazolam, clozapine, codeine, corticosterone, cortisone, cyclobenzaprine, cyproheptadine, dapoxetine, demexiptiline, desipramine, desomorphine, dexamethasone, dexanabinol, dextroamphetamine sulfate, dextromoramide, dextropropoxyphene, dezocine, diazepam, dibenzepin, diclofenac sodium, diflunisal, dihydrocodeine, dihydroergotamine, dihydromorphine, dimetacrine, divalproxex, dizatriptan, dolasetron, donepezil, dothiepin, doxepin, duloxetine, ergotamine, escitalopram, estazolam, ethosuximide, etodolac, femoxetine, fenamates, fenoprofen, fentanyl, fludiazepam, fluoxetine, fluphenazine, flurazepam, flurbiprofen, flutazolam, fluvoxamine, frovatriptan, gabapentin, galantamine, gepirone, ginko bilboa, granisetron, haloperidol, huperzine A, hydrocodone, hydrocortisone, hydromorphone, hydroxyzine, ibuprofen, imipramine, indiplon, indomethacin, indoprofen, iprindole, ipsapirone, ketaserin, ketoprofen, ketorolac, lesopitron, levodopa, lipase, lofepramine, lorazepam, loxapine, maprotiline, mazindol, mefenamic acid, melatonin, melitracen, memantine, meperidine, meprobamate, mesalamine, metapramine, metaxalone, methadone, methadone, methamphetamine, methocarbamol, methyldopa, methylphenidate, methylsalicylate, methysergid(e), metoclopramide, mianserin, mifepristone, milnacipran, minaprine, mirtazapine, moclobemide, modafinil (an anti- narcoleptic), molindone, morphine, moφhine hydrochloride, nabumetone, nadolol, naproxen, naratriptan, nefazodone, neurontin, nomifensine, nortriptyline, olanzapine, olsalazine, ondansetron, opipramol, orphenadrine, oxaflozane, oxaprazin, oxazepam, oxitriptan, oxycodone, oxymorphone, pancrelipase, parecoxib, paroxetine, pemoline, pentazocine, pepsin, perphenazine, phenacetin, phendimetrazine, phenmetrazine, phenylbutazone, phenytoin, phosphatidylserine, pimozide, pirlindole, piroxicam, pizotifen, pizotyline, pramipexole, prednisolone, prednisone, pregabalin, propanolol, propizepine, propoxyphene, protriptyline, quazepam, quinupramine, reboxitine, reserpine, risperidone, ritanserin, rivastigmine, rizatriptan, rofecoxib, ropinirole, rotigotine, salsalate, sertraline, sibutramine, sildenafil, sulfasalazine, sulindac, sumatriptan, tacrine, temazepam, tetrabenozine, thiazides, thioridazine, thiothixene, tiapride, tiasipirone, tizanidine, tofenacin, tolmetin, toloxatone, topiramate, tramadol, trazodone, triazolam, trifluoperazine, trimethobenzamide, trimipramine, tropisetron, valdecoxib, valproic acid, venlafaxine, viloxazine, vitamin E, zimeldine, ziprasidone, zolmitriptan, Zolpidem, zopiclone and isomers, salts, and combinations thereof.

For example, polymorphs of theophylline can be identified using a hydrophobic/hydrophilic gradient. Hydrophobic/hydrophilic gradients can be prepared by controlled immersion of a gold-coated glass slide with a chromium adhesive layer into a dilute solution (e.g. 0.005 mM) of a thiol- terminated molecule, such as dodecanethiol. The chemical gradient can be completed by immersing the whole substrate into an 11-mercaptoundecanol solution (e.g. 0.01 mM) over night. The presence of polymorphs in a drug, such as theophylline (also known as dimethylxanthine), can be tested by exposing the gradient to a solution of theophylline (nearly saturated). Pseudopolymorph-selective crystallization can occur during evaporation of the solvent. Similar hydrophobic/hydrophilic gradients can be used to identify polymorphs of anthranilic acid. Polymorphs of other compounds can be identified using hydroxyl/acid gradients. Such gradients can be prepared by controlled immersion of a gold-coated glass slide with a chromium adhesive layer into a dilute solution (e.g. 0.005 mM) of a thiol- terminated molecule, such as 11- mercaptoundecanol. The chemical gradient can be completed by immersing the whole substrate into an 11-mercaptoundecanoic acid solution (e.g. 0.01 mM) overnight. The presence of polymorphs in a drug, such as carbamazepine, can be tested by immersing the gradient into a solution of carbamazepine (nearly saturated). Phase-selective crystallization occurs during evaporation of the solvent. The gradients described herein can also be used to induce enantionselective crystallization. For example, for the preparation of a R- leucin/S-leucin gradient a full monolayer of 11-mercaptoundecanoic acid can be formed on a gold-coated glass slide with a chromium adhesive layer. The monolayer can be immersed into a 0.2 M N-hydroxysuccinimide solution in the presence of 0.8 M water-soluble carbodiimide ( 1 -ethyl-3 -(3 - dimethylaminopropyl)-carbodiimide hydrochloride). Gradient formation can be achieved by controlled immersion of the substrate into a saturated R- leucine solution containing 10 mM Tris-HCl and 0.2 M NaCl (pH 7.8) for the covalent immobilization of R-leucine on the mercaptoundecanoic acid. The formation of the gradient can be completed by immersing the whole substrate into a saturated L-leucin solution overnight. The gradient can then be immersed into a solution of a racemic product (nearly saturated); and enantioselective crystallization can occur during evaporation of the solvent. IV. Kits The gradients can be packaged in a kit. In one embodiment, the finished template (with one or more gradients already formed on the surface) is packaged and incorporated into a kit. In another embodiment, the kit contains the substrate on which the gradient will be formed, along with one or more containers containing reagents for forming the gradient.

The kit optionally contains directions for preparing and/or using the template.

Example Example 1. Crystallization of Carbamazepine

Experimental Procedure Preparation of substrate

Two different types of screening tools were prepared: Type A. Surface-chemical wettability gradients created by a dodecanethiol/ mercaptoundecanethiol surface-concentration ratio gradient prepared with immersion technique (S. Morgenthaler, et al, Langmuir, Vol. 19, No. 25, 10459-10462 (2003)).

Type B. 1 mm thick stripes of surface-chemical gradients of the same type as described above alternating with 1 mm thick hydrophobic stripes (hexadecanethiol) along the gradient.

Screening Tool Type B was been prepared in order to create a chemical contrast.

Surface-chemical wettability gradients (Type A) A 2 x 2 cm silicon- wafer was coated with a 10 nm thick chromium adhesion layer and 80 nm of gold. Prior to gradient preparation, the sample was ultra-sonicated in ethanol for 10 minutes to remove particles from the surface. Then, the wafer was air-plasma cleaned for 30 seconds to oxidize organic contamination. Next, the wafer was immersed in ethanol for 10 minutes to reduce the gold-oxide on the surface. Finally, the wafer was blow-dried with nitrogen.

A surface-chemical gradient was prepared by immersing the wafer into a 0.005 mM dodecanethiol ethanol solution by means of a linear motion drive with a speed of 0.0375 mm/s (see e.g. Step 1 illustrated in Figure 2). After complete immersion, the wafer was withdrawn from the solution, rinsed with ethanol and blow-dried with nitrogen. Then, the wafer was immersed into 0.01 mM 11-mercaptoundecanethiol solution over night (about 15hours), rinsed with ethanol and blow-dried with nitrogen.

Surface chemical wettability gradients having alternating hydrophobic strips along the gradient (Type B) A 2 x 2 cm silicon- wafer was coated with a 10 nm thick chromium adhesion layer and 80 nm of gold. Prior to gradient preparation, the sample was ultra-sonicated in ethanol for 10 minutes to remove particles from the surface. Then, the wafer was air-plasma cleaned for 30 seconds to oxidize organic contamination. A poly(dimethylsioxane) (PDMS) stamp, 1 mm wide thick grid with 1 mm wide gaps), was placed onto the surface with 1 mM hexadecanethiol ethanol-solution. The stamp was dried in open air, until the surface of the wafer appeared to be dry. The stamp was placed on the surface of the wafer and, initially, a soft force was manually applied to the stamp with a fingertip for a very short period of time, e.g., a fraction of second to a second. After 60 seconds, the stamp was removed, the wafer was rinsed with ethanol and blow-dried with nitrogen.

A surface-chemical gradient was prepared by immersing the wafer into a 0.005 mM dodecanethiol ethanol solution by means of a linear motion drive with a speed of 0.0375 mm/s. After complete immersion, the wafer was withdrawn from the solution, rinsed with ethanol and blow-dried with nitrogen. Then, the wafer was immersed into 0.01 mM 11- mercaptoundecanethiol solution over night (about 15 hours), rinsed with ethanol and blow-dried with nitrogen.

Figure 2 is a schematic of a portion of the above-described experimental procedure. A computer is electrically connected to the linear motion drive. Step 1 represents the immersion of the substrate in diluted dodecanethiol solution. The subsequent backfilling process by total immersion of the substrate into mercaptoundecanol solution is not illustrated in Figure 2. Crystallization

The crystallization process is illustrated in Step 2 of Figure 2. The sample is slowly withdrawn from an oversaturated solution of the compound to be crystallized, which creates a gradient of the compound to be tested, and analyzed for polymorphs. In a second step, substrates of Type A and B were immersed in an oversaturated Carbamazepine solution.

25 mM solution of Carbamazepine in toluene was stirred for about 30 minutes at 55 °C. The solution was filtrated with a paper filter into a 25 ml clean glass-beaker. The sample was completely immersed immediately by means of a linear motion drive. After 1 minute of total immersion time, the sample was slowly withdrawn from the crystallization solution (for 29 minutes) in order to create an immersion time gradient from 1 minute to 30 minutes. Analysis

The crystals formed on the surface were recorded with optical microscopy and their average size and their distribution density on the surface was analyzed. Raman spectroscopy can be used, using standard procedures, to determine the polymorphic forms of the crystal on the surface. The crystals on the substrate surface were imaged with a 4 x enlarging microscope. The whole substrate surface was mapped (10 x 10 images). The images were analyzed with the software ImageJ to determine an average crystal size, and the number of crystals for each image. From these images, an overall average crystal size and an average number of crystals for a given immersion time were calculated. The values were normalized by setting the average for each immersion time as 100 %. Therefore, values greater than 100 % indicate that the crystals were larger than the average crystal size, and values smaller than 100 % indicate that the crystals were smaller than average crystal size for this particular immersion time. These normalized values were summarized for all immersion times.

The average crystal size for immersion time (D) in dodecanethiol and immersion time(C) into carbamazepine solution were calculated using Formula I:

Formula I

where CS_DC~ average crystal size for immersion time (D) into dodecanethiol and for immersion time (C) into carbamazepine solution,

CS_D= average crystal size for immersion time (D) into dodecanethiol N_D= number of analysis points for immersion time (D) into dodecanethiol solution, and Nc= number of analysis points for immersion time (C) into

Carbamazepine solution.

The number of crystals for immersion time (D) in dodecanethiol and immersion time (C) in carbamazepine solution can be calculated using

Formula II:

NC_n = ^^ N i^y D

Formula II where

NC_DC ⁼ number of crystals for immersion time (D) into dodecanethiol and for immersion time (C) into carbamazepine solution, NC_D= number of crystals for immersion time (D) into dodecanethiol,

N_D= number of analysis points for immersion time (D) into dodecanethiol solution, and

Nc= number of analysis points for immersion time (C) into Carbamazepine solution Discussion

The results for Type A substrates (dodecanethiol vs. 11- mercaptoundecanethiol gradient) are shown in Figure 3. For Type A substrates, the number of crystals and crystal size increased with increasing hydrophilicity.

The results for Type B substrates (dodecanethiol vs 11- mercaptoundecanethiol gradient, including μCP stripes) are shown in Figure 4. For Type B substrates the number of crystals decreased with increasing hydrophilicity while crystal size increased with increasing hydrophilicity The differences in crystal distribution and crystal size along the gradient indicate that different kinetic processes take place on the substrate for surfaces with different surface chemistries. Thus, for some crystal systems the small changes in the crystallization kinetics may lead to different polymorphic systems. By testing different samples in different experiments, fluctuations in the crystallization conditions cannot be avoided and therefore the differences from sample to sample can easily be misinterpreted. However, tests for a given sample on a substrate containing one or more surface gradients allow for varying surface properties to be tested for the crystallization process in one experiment. If a difference is observable on the gradient it must arise from the different surface chemistry since the crystallization solution is the same for the whole gradient.

Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

Claims

We claim:

1. A method for crystallizing and identifying polymorphs in a compound comprising

(a) exposing a crystallization template comprising a surface with one or more gradients selected from the group consisting of chemical gradients, morphological gradients and combinations thereof to a solution or a vapor comprising the compound to be tested,

(b) allowing the compound to crystallize on the surface of the template, and

(c) analyzing the crystals formed on the surface of the template for polymorphs of the compound.

2. The method of claim 1 , wherein the template is exposed to a solution comprising the compound to be tested and wherein the solvent evaporates from the solution.

3. The method of claim 1 , wherein the template is exposed to a vapor comprising the compound to be tested and wherein the compound sublimates.

4. The method of any one of claims 1 to 3, wherein the solution or vapor is supersaturated with the compound to be tested.

5. The method of any one of claims 1 to 4, wherein the template comprises one or more dividers and wherein the solution or vapor is exposed to only a portion of the template.

6. The method of any one of claims 1 to 5, wherein the compound to be tested is an inorganic compound.

7. The method of any one of claims 1 to 5, wherein the compound to be tested is an organic compound.

8. The method of any one of claims 1 to 5, wherein the compound to be tested is a bioactive compound.

9. The method of any one of claims 1 to 8, further comprising after step°(c), selecting one or more polymorphs for further testing or production.

10. A crystallization template for use in the method of claim 1 , comprising a surface with one or more gradients selected from the group consisting of chemical gradients, morphological gradients and combinations thereof.

11. The crystallization template of claim 10, wherein the one or more gradients are chemical gradients.

12. The crystallization template of claim 10, wherein the one or more gradients are morphological gradients.

13. The crystallization template of claim 10, wherein the one or more gradients are a combination of at least one chemical gradient with at least one morphological gradient.

14. The crystallization template of claim 13, wherein the template comprises one chemical gradient and one morphological gradient, and wherein the chemical gradient is perpendicular to the morphological gradient.

15. The crystallization template of claim 13 , wherein the template comprises one chemical gradient and one morphological gradient, and wherein the chemical gradient and morphological gradient are in the same direction.

16. The crystallization template of any one of claims 10 to 15, wherein the template further comprises one or more dividers that separate the surface of the template into sections.

17. A kit comprising the crystallization template of any one of claims 10 to 16.

18. A kit comprising a substrate and one or more reagents for preparing a gradient on the surface to from the crystallization template of any one of claims 10 to 16.

19. The kit of claim 17 or 18, further comprising instructions for preparing and/or using the crystallization template.