Ana Cameirão

Fabienne Espitalier

René David

Loïc Rouleau

Validation of a Model for Multiple Agglomeration

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Introduction

Several crystallization products exhibit multiple levels of agglomeration: basic crystallites are embedded in primary agglomerates which, in turn, are reagglomerated in secondary agglomerates. The structure properties like porosities or sizes of the different levels of these particles influence their properties of use like catalytic performances or flowability.

A previous work [1] dealt with the hydrothermal autoclave crystallization of a zeolite from a gel containing amorphous particles. After pouring the reactants into the autoclave vessel, heating up to 150-200 °C was started. Then, the transformation into crystallites and the consecutive growth and agglomeration of these crystallites required about 72 hours to be completed. Chemical composition, stirring speed, temperature and ripening of the gel were varied. Final crystal size distributions, the desupersaturation profile and the crystallinity index against time were monitored.

Generation of Crystallites

Crystallites were supposed to be generated from amorphous particles with size L 0 and volume v 0 from surface contamination by a shrinking core model. This process starts at t 0 . The conversion into a crystallite is completed within t con . The amorphous volume v a and the amorphous surface A a are related by 1) :

dv a dt À L 0 2t con A a 1
By integration of Eq. ( 1) one obtains the crystalline volume v c:

if: t < t 0 : v c = 0 (2) if t 0 ≤ t ≤ t 0 + t con: v c v 0 1 À 1 À t À t 0 t con 3 3 if t > t 0 + t con : v c = v 0 ( 4
)
t 0 is different for each amorphous particle and is assumed to follow a Gaussian distribution with the mean value t 0 and the standard deviation r.

pt 0 1 r 2p p exp À t 0 À t 0 2 2r 2 ! 5
The ratio between the total crystalline volume V c and the initial amorphous volume V 0 is:

V c t V 0 Z t 0 pt 0 v c v 0 t; t 0 dt 0 6
The crystallinity index V c /V 0 was experimentally monitored against the time from the samples by X-ray diffraction (see Fig. 1). As an amorphous particle with an external crystalline layer can start to agglomerate, the nucleation rate of crystallites will be proportional to the surface contamination and the initial concentration of amorphous particles.

r N = N A0 p(t) (7) t 0 and t con were calculated from the starting time t start of the experimental curve V c /V 0 (see Fig. 1): no amorphous

1)

List of symbols at the end of the paper.

particle begins to transform before t 0 -2r; thus, for the entire suspension it is assumed that:

t 0 = t start + 2 r (8)
The half-life time t 50 of an amorphous particle is derived from Eq. (3): t 50 t 0 t conso 4:85 9

For the complete set of amorphous particles, the half-life time is approximated as: t 50 t 0 t conso 4:85 10

Then, by fitting r it is possible to simulate the crystallinity index V c /V 0 against the time via Eqs. (2-6), (8), and (10); t con was found identical for all experiments and equal to 3.1 h. Values of t 0 and r are reported in Tab. 1 for some typical runs. Runs 1 and 2 were done under the same chemical and stirring conditions, except a ripening period after reactant mixing, but before heating for run 2. Run 3 was conducted under different chemical (alkalinity) and stirring conditions as runs 1 or 2 and without ripening. All the runs were done at the same temperature. t 0 is influenced by alkalinity and ripening and by stirring speed, whilst r is very sensitive to ripening and alkalinity and less to the stirring speed. The crystallinity curve of the particulate mass against time (see Fig. 1) fits experimental results extremely well when using this assumption.

Agglomeration Model

A complete model [2] of the multiple agglomeration of the zeolite was set up. Crystal agglomeration starts when the particle surface is crystalline and follows a three-step process (see Fig. 2); First, collision of agglomerates or individual particles takes place and results in a labile aggregate, which may undergo either disruption or consolidation by crystal growth. It is generally accepted that the collision rate r col is size-dependent and linked to the concentrations of colliding species:

r col = K col (S i , S j ) N i N j ( 11
)
where K col depends on the collision regime (Brownian if mother crystal sizes < Batchelor scale, or laminar shear if Kolmogoroff scale > mother crystal sizes > Batchelor scale) encountered by the colliding particles:

K col,B = 2kT(S i + S j ) 2 /(3l S i S j ); K col,L = 0.16 (P/m) 1/2 (S i + S j ) 3 (12)

Note that K col,L increases with the stirring power whereas K col,B is not stirring power dependent. If both particles to agglomerate are of the same size S, K col,L varies as S 3 and K col,B is size-independent. When keeping the largest size S i constant one can observe that K col,B has a minimum for S j = S i , whereas K col,L reaches its maximum value for S j = S i . If the disruption of the aggregate and the crystal growth are independent processes and of first order with respect to the aggregate number concentration (i.e., not consecutive to further collisions), the agglomeration rate is the collision rate times an efficiency factor g [2].

r aggl = r col /(1 + k d /k c ) = g r col ( 13
)
If the crystal growth rate is high, this efficiency factor is close to 1. By contrast, with low growth rates:

g ∼ G/(k d S j ) (14)
A reasonable assumption for g B would be to consider k dB as constant. Recently, Liew et al. [3] proposed an expression for k dL :

k dL ∼ q L PS i /(r*L*) ( 15
)
which leads to:

g L = AG/(PS i S j ) ( 16
)
where the constant A stands for both, liquid and solid properties. Table 1. Values of parameters obtained for three typical runs at the same temperature (for the entire sets of experimental conditions see [1] and [2]).

Run Summarizing, for a given growth rate r aggl,B is not dependent on P, it is proportional to the common size S -1 , and it is minimum for agglomeration of particles of the same size. During size enlargement, agglomerations of small and large particles are thus favored (snowball effect), but the rate of agglomeration diminishes with size increase.

Conversely, r aggl,L is a decreasing function of P and is proportional to the common size if S j = S i . At constant S i it is maximum for S j = k B , minimum for agglomeration of particles of S j = S i /3, and reaches a weak secondary maximum at S j = S i . During size enlargement, if S i >> k B , agglomerations of particles of very different sizes are thus favored but, if S i is of the same order of magnitude as k B , the rate of agglomeration becomes quasi-independent of the particle size. In several experiments size-independent agglomeration was observed [4]. Globally, the agglomeration rate is enhanced by size increase. Anyway, the agglomeration rate depends on supersaturation via G. When the supersaturation decreases, agglomeration, which is then mainly is laminar, becomes more and more difficult. It is thus expected that agglomerates formed under Brownian and laminar shear regimes show different morphologies.

Agglomeration is modeled like a set of stoichiometric chemical reactions between particle classes [2]. The stoichiometric coefficients are designed in order to account for both mass conservation and disappearance of one particle per agglomeration.

However, the method described in [2] was improved in this paper by including a liquid volume v l into the apparent volume of the newly formed agglomerate:

a v j + b v i + v l → c v m ( 17
)
This allows one to predict the external porosity of the agglomerates, which was observed experimentally (see Fig. 3). The coefficients a, b, c, and v l are given in Tab. 2.

Then, the porosity is calculated as a function of time: It is the ratio of the mean trapped liquid volume v l per unit of suspension volume to the total apparent solid volume concentration.

v l is calculated by integrating Eq. ( 18) for all interactions (i, j) and their consequences for particle classes ranging from n = 1 to n c :

dv l dt X n c n1 X n c i1 X i j1 v l r aggl i; j; n; t 18
All other equations of the model can be found in [2].

Results and Discussion

The calculations of the model developed above were done with the same parametric values as in [2]. The agreement obtained with the experimental agglomerate size distribution is better (see Fig. 4). Note that the laser particle sizer measures apparent external dimensions of the agglomerates including external porosity between the primary agglomerates (see Fig. 3). Two codes, both based on backward differentiation, were used: LNAG did not use a numerical Jacobian matrix, whilst LSODE does, yielding similar results. The quality of the agreement is much better than the one resulting from a previous model [2], but the model does not completely fit the experimental size distribution. In fact, Tab. 2 is a very crude approach of external porosity: a finer tuning of the daughter classes of the agglomerates is necessary.

l = cv m -av j -bv i 1/2 i 1/2 1/2 i +1 i + 2 v i 1 i-1 1/2 1/2 i +1 i + 1 0 4/3 i-2 1 4/3 i i + 1 4v i /3 8/7 i-3 1 8/7 i i + 1 8v i /7 v i /(v i -v j ) j < i-3 1 v i /(v i -v j ) i i 0

Conclusions

The agglomeration process during crystallization is far from being completely understood. The crystalline growth is a key feature of this process, which makes the major difference with colloidal aggregation by playing the role of sticking particles. In some cases like the present one, the agglomeration becomes the dominant size enlargement process, more than crystal growth. The building-up of the final particle structure from the basic crystallites is often made of several agglomeration steps. Finally, efficient models of agglomeration must consider the importance of taking external porosity, especially for secondary agglomerates, when applicable, into account.

Figure 1 .

 1 Figure 1. Cristallinity index against time for run 3.

Figure 3 .

 3 Figure 3. SEM picture of synthesized zeolite.

Figure 4 .

 4 Figure 4. Apparent experimental and calculated particle size distributions with porosity modeling for run 3 using two different codes.

Table 2 .

 2 Stoichiometric coefficients and included liquid volume (see Eq. (17)) as functions of agglomeration classes i and j ≤ i.

	a	j	b	c	Class m of	Class m	v
					agglomerate	agglomerate
					without porosity	with porosity

Acknowledgement

The authors wish to thank the Institut Français du Pétrole for the financial support of this work.

Symbols used

[s] half-life time of an amorphous particle


M G Cares-Pacheco

R Calvet

G Vaca-Medina

A Rouilly

F Espitalier

Inverse gas chromatography a tool to follow physicochemical modifications of pharmaceutical solids: Crystal habit and particles size surface effects

Keywords:

Powders are complex systems and so pharmaceutical solids are not the exception. Nowadays, pharmaceutical ingredients must comply with well-defined draconian specifications imposing narrow particle size range, control on the mean particle size, crystalline structure, crystal habits aspect and surface properties of powders, among others. The different facets, physical forms, defects and/or impurities of the solid will alter its interaction properties. A powerful way of studying surface properties is based on the adsorption of an organic or water vapor on a powder. Inverse gas chromatography (IGC) appears as a useful method to characterize the surface properties of divided solids.

The aim of this work is to study the sensitivity of IGC, in Henry's domain, in order to detect the impact of size and morphology in surface energy of two crystalline forms of an excipient, D-mannitol. Surface energy analyses using IGC have shown that the a form is the most energetically active form. To study size and shape influence on polymorphism, pure a and b mannitol samples were cryomilled (CM) and/or spray dried (SD). All forms showed an increase of the surface energy after treatment, with a higher influence for b samples (g d s of 40-62 mJ m À2 ) than for a mannitol samples (g d s of 75-86 mJ m À2 ). Surface heterogeneity analysis in Henry's domain showed a more heterogeneous b-CM sample (62-52 mJ m À2 ). Moreover, despite its spherical shape and quite homogeneous size distribution, b-SD mannitol samples showed a slightly heterogeneous surface (57-52 mJ m À2 ) also higher than the recrystallized b pure sample ($40 mJ m À2 ).

Introduction

Pharmaceutical solids must comply with well-defined specifications in terms of bioavailability, solubility, toxicity and stability. Nowadays, the requirements are more and more draconian, imposing narrow particle size range, control on the mean particle size, crystalline structure, crystal habits aspect and surface properties of powders, among others. A large set of operations is developed to answer these requirements. The processes for producing fine powders (around micrometer) are varied as melt quenching, grinding, freeze-drying, spray drying, crystallization, antisolvent precipitation, milling, and supercritical fluids. Depending upon the nature of the active pharmaceutical ingredients (APIs), it is known that the preparation method influences the physical stability and crystallization behavior. The impact of these processes on solid phase transformations may lead to the formation of a metastable or an amorphous form, or a mixtures of various crystalline forms including other hydrates (or solvates). These changes are desired for certain stages of the formulation or for some use properties of the API, but sometimes can also have undesirable effects on the solid.

Inverse gas chromatography (IGC) appears as a tool to study the changes on surface properties in order to highlight process influences to assess drug delivery systems performance. Mechanical operations are the most studied processes to determine pharmaceutical solids surface's behavior. Most authors described an increase of the dispersive component of the solid surface energy, g d s , after mechanical grinding (Table 1). This increase is generally attributed to the exposure of specific crystal facets that present different chemical groups, the formation of higher energy zones such as crystal defects, dislocations and/or to solids state transformations [START_REF] Chamarthy | The nature of crystal disorder in milled pharmaceutical materials[END_REF][START_REF] Feeley | Processing effects on the surface properties of a-lactose monohydrate assessed by inverse gas chromatography (IGC)[END_REF][START_REF] Feng | Process induced disorder in crystalline materials: differentiating defective crystals from the amorphous form of griseofulvin[END_REF][START_REF] Heng | The effects of milling on the surface properties of form I paracetamol crystals[END_REF][START_REF] Ho | Effect of milling on particle shape and surface heterogeneity of needle-shaped crystals[END_REF][START_REF] Newell | Inverse gas chromatography: investigating whether the technique preferentially probes high energy sites for mixtures of crystalline and amorphous lactose[END_REF].

Surface energy, particles size and surface chemistry

Due to the anisotropic nature of powders, the exhibition of new crystal faces under milling can change the acidic/basic character of the solid depending on the functional groups present in the exposed facets. [START_REF] Heng | The effects of milling on the surface properties of form I paracetamol crystals[END_REF] highlighted the anisotropic nature of form I paracetamol crystals. The confrontation of sessile drop and IGC, allowed them to conclude that grinding leads to a fragmentation of the facet (0 1 0), which possesses the weakest attachment energy and exhibits the large g d s . Thus, after milling the g d s of the samples increased, showing a more hydrophobic surface, with decreasing particle size. [START_REF] York | Characterisation of the surface energetics of milled DL-propranolol hydrochloride using inverse gas chromatography and molecular modelling[END_REF] were interested in the milling of DLpropranolol hydrochloride. The evolution of g d s showed to depend on the particles size. During milling the surface becomes increasingly more energetic (g d s from 45 to 61 mJ/m 2 for particles 75-16.5 mm) until reached a plateau followed by a small fall in g d s for the finest powder (<10 mm). The authors concluded that the increases in g d s , and in Dg ads using CH 2 Cl 2 as probe molecule, are due to a fragmentation releasing the dominant crystal face, which posses the lowest attachment energy and is rich in naphthalene groups. Attrition might become significant as milling intensity increase releasing faces having OH groups, naphthalene and Cl À ions. [START_REF] Trowbridge | Influence of milling on the surface properties of acetaminophen[END_REF] highlighted that acetaminophen size reduction from 30 to 10 mm leaded to an increase of g d s from 50.9 to 61.3 mJ/m 2 and to an increase of Dg ads from 327 to 506 J/mol, using chloroform as probe molecule. Milling also increases the hydrophobic and basic character of acetaminophen surface. This results are in agreement with those obtained by molecular modeling which established that milling leads to the exposure of the crystal facet (0 1 0) which contain an hydrophobic methyl group, a benzene ring and a carboxyl group, both basic. [START_REF] Ohta | Determination of the changes in surface energetics of cefditoren pivoxil as a consequence of processing induced disorder and equilibration to different relative humidities[END_REF] studied surface energetic changes of cefditoren pivoxil, a cephalosporin antibiotic, as consequence of milling. After grinding in a vibration mill, the authors found a decrease in the g d s according to the grinding time, from 52.3 mJ/m 2 before milling to 45.8 mJ/m 2 after 30 min of grinding with a decrease in solid crystallinity. In addition, the authors have shown a decrease in the solid acidic character with an increased on its basicity. These effects are attributed to the exposure of carbonyl groups, which have an electron donating nature. [START_REF] Luner | Evaluation of milling method on the surface energetics of molecular crystals using inverse gas chromatography[END_REF] studied by IGC the impact of high shear wet milling (HSWM) and dry milling (DM) on the surface properties of two pharmaceutical compounds, succinic acid and sucrose. Physicochemical characterization of both samples showed that bulk properties were unaffected by wet and dry milling while surface properties analyses showed an increase of solids dispersive surface energy after DM and HSWM. Succinic acid samples, g d s = 35 mJ/m 2 , exhibit minor differences between dry milled and wet milled samples, 40 AE 2 mJ/m 2 , attributed to minimal impact of cleavage and the exposure of crystal facets with similar atomic surface arrangements. For HSWM sucrose, the polarity of the solvents used during wet milling influenced g d s of the milled samples from 55 to 71 to 91 mJ/m 2 , for hexane, methyl tertiary butyl ether and ethanol respectively while dry milled samples exhibit a g d s of 44 mJ/m 2 . Differences between dry and wet milling processes were attributed to the attrition mechanism in presence of solvent.

Reducing particle size may also be necessary for the API to reach the target organ, particularly when the drug administration is by inhalation. Nowadays, dry powder inhalers (DPIs) are of great interest thanks to the absence of propellant and the stability of the formulation as a result of the dry state. A successful drug delivery will depend on the interaction between the powder formulation and the device performance. During inhalation, the API detaching from the carrier by the energy of the inspired airflow that overcomes the adhesion forces between the API and the carrier.

Over the past few years, lactose has been considered as the excipient of choice in several solid oral dosage forms and so many studies has been carried on lactose, most of them based on the study of its physical-chemical properties as function of the manufacturing process [START_REF] Pilcer | Lactose characteristics and the generation of the aerosol[END_REF]. Thus, why lactose is also the most studied organic solid by IGC. [START_REF] Feeley | Processing effects on the surface properties of a-lactose monohydrate assessed by inverse gas chromatography (IGC)[END_REF] and [START_REF] Shariare | The impact of material attributes and process parameters on the micronisation of lactose monohydrate[END_REF] studied the influence of milling on the surface properties of lactose. IGC analyses showed a low sensitivity of g d s to milling. However, the study of basic and amphoteric probes showed changes of the hydroxyl groups presented in lactose surface. [START_REF] Shariare | The impact of material attributes and process parameters on the micronisation of lactose monohydrate[END_REF] also highlighted that the acidic or basic character of the powder seems to be related to the size of starting particles. For sample, the micronization of larger particles, 50-100 mm, results in an increase of the specific component of solid surface energy, g sp s , measured with THF (basic probe). While the micronization of finer particles, <20 mm, leads to an increase of g sp s using amphoteric probes molecules such as acetone.

1.2. Surface energy and crystalline solid state [START_REF] Tong | Characterization of two polymorphs of salmeterol xinafoate crystallized from supercritical fluids[END_REF][START_REF] Tong | Predicting the aerosol performance of dry powder inhalation formulations by interparticulate interaction analysis using inverse gas chromatography[END_REF] studied the influence of solid-solid interactions on the aerosol performance of salmeterol xinafoate (SX) polymorphs and lactose carrier by IGC. SX is a highly selective bronchodilator, known to exist in two crystalline forms. Three active batches of SX were generated: the two polymorphs, SXI and SXII, crystallized using solution enhanced dispersion by supercritical fluids from methanol solution (SEDS) and a form called MSXI generated by the micronisation of the SXI form. First of all, dispersive surface energy analysis by IGC exhibits a more active MSXI sample (g d SXI $33 mJ/m 2 ,g d SXII $29 mJ/m 2 and g d MSXI $39 mJ/ m 2 ). IGC was also applied to calculate the cohesion between SX samples and the adhesion between the samples of SX and lactose. The study of the strength drug-drug cohesion and drug-carrier adhesion suggests that the active particles of SX bond more strongly to the carrier particles of lactose that to those of their own species, except for SXII-lactose: SXI-SXI (190.7 MPa), SXII-SXII (67.3 MPa), MSX-MSX (245.0 MPa), SXI-lactose (212.6 MPa) SXIIlactose (47.5 MPa), . The use of lactose as a carrier improves aerosol performance about 25% for the batch SXI and 140% for the batch MSXI. [START_REF] Traini | The influence of lactose pseudopolymorphic form on salbutamol sulfate-lactose interactions in DPI formulations[END_REF] studied by IGC the lactose-salbutamol sulfate interactions in DPI formulations. The aim was to investigate lactose pseudo-polymorphs, a-anhydrous, a-monohydrate and b-anhydrous, in terms of carrier functionality. In this work, a-monohydrate form exhibits best aerosol performance, indicating that carrier surface chemistry plays a dominating role in DPIs. The authors also highlighted an inverse relationship between surface energy and aerosol efficiency.

More recently studies has been carried out in order to provide a substitute for a-lactose monohydrate as carrier in DPI formulations. Indeed, lactose possesses several drawbacks due to its bovine origin, its incompatibility with amino groups like peptides and proteins and its tendency to become amorphous after mechanical treatment. Nowadays, mannitol appears to be an adequate substitute for lactose because it does not carry reducing groups that may cause chemical interaction with proteins and is highly crystalline even upon spray drying [START_REF] Maas | The impact of spray drying outlet temperature on the particle morphology of mannitol[END_REF].

Surface energy analysis of D-mannitol has been focused on the stable b form. IGC has highlighted its acidic nature, attributed to the high density of hydroxyl groups at mannitol surface [START_REF] Saxena | Application of molecular modelling to determine the surface energy of mannitol[END_REF]. More recently research were focused on the effect of milling and/or surface energy heterogeneity of the stable b form [START_REF] Ho | Determination of surface heterogeneity of D-mannitol by sessile drop contact angle and finite concentration inverse gas chromatography[END_REF][START_REF] Ho | Effect of milling on particle shape and surface heterogeneity of needle-shaped crystals[END_REF]. Our previous research work, [START_REF] Cares-Pacheco | Physicochemical characterization of D-mannitol polymorphs: the challenging surface energy determination by inverse gas chromatography in the infinite dilution region[END_REF], was focused on the study of D-mannitol polymorphs. Dmannitol pure polymorphs exhibit a more active and highly heterogeneous a form (74.9 to 45.5 mJ/m 2 ) with a less active and quite homogeneous stable b mannitol ($40-38 mJ/m 2 ), which also behave similarly to the instable d form. Mannitol particles, generated by SD, have been used commercially in bronchial provocation test (Aridol). [START_REF] Tang | Characterisation and aerosolisation of mannitol particles produced via confined liquid impinging jets[END_REF] studied the impact of different generation processes in particles to improve aerosol performance. Three powder samples were prepared: by confined liquid impinging jets (CLIJs) followed by jet milling (JM), SD and JM. These processes generated quite different powder samples, needle-shape for CLIJs samples, spherical for SD samples and orthorhombique for the JM samples. Dispersive surface energy analysis by IGC exhibits a more active CLIJs sample attributed to the presence of a mannitol (g d CLIJs $85 mJ/m 2 , g d SD $60 mJ/m 2 and g d JM $48 mJ/m 2 ). Particles shape showed to be an important contributor to the aerosol performance of mannitol powders as it affects the surface energy and particles dynamics.

Yamauchi et al. ( 2011) studied physicochemical differences with emphasis on the surface properties, by IGC and DVS, of niclosamide and naproxen sodium and their respective anhydrate and dehydrated hydrated forms. The naproxen sodium anhydrate form showed a higher g d s and higher rate of moisture sorption than the dehydrated hydrated form, whereas the opposite was observed for niclosamide.

Surface energy, defects and amorphization

Many APIs are poorly soluble in water, which limits their bioavailability. Their rate of dissolution may improve by using an amorphous phase of the API. Indomethacin (IDMC) is a good example. IDMC is an antipyretic and anti-inflammatory drug used in many pharmaceutical formulations, it has four polymorphs and an amorphous. According to the grinding temperature, below or higher than its glass transition temperature (T g ), and grinding intensity the most stable form at ambient conditions, g-IDMC, can transform to the metastable a form or lead to an amorphous state [START_REF] Crowley | Cryogenic grinding of indomethacin polymorphs and solvates: assessment of amorphous phase formation and amorphous phase physical stability[END_REF][START_REF] Desprez | Transformations of glassy indomethacin induced by ball-milling[END_REF]. As IDMC is highly insoluble in water (0.02 mg/mL), the bioavailability of the product and its absorption by the gastrointestinal tract can be improved by using an amorphous form of the drug [START_REF] Imaizumi | Stability and several physical properties of amorphous and crystalline forms of indomethacin[END_REF]. [START_REF] Planinsek | Structural evolution of indomethacin particles upon milling: time-resolved quantification and localization of disordered structure studied by IGC and DSC[END_REF] studied the crystalline fraction that transforms into amorphous forms upon intensive milling. IGC was used to detect surface changes caused by milling crystalline g-IDMC while DSC was used to determine the mass (or equivalent volume) fraction of the samples transformed. The authors determined that the dispersive energy of the stable g-IDMC was 32.2 mJ/m 2 and that of the amorphous, generated in a ball mill for 120 min, of 43.3 mJ/m 2 . Studies on the influence of milling time, allows the authors to illustrate the sensitivity of the IGC-ID to detect crystal surface defects as high-energy sites (g

d 30min = g d 60min = g d amorphe )
. By comparing time evolution of the amorphous fraction of the surface by IGC and volume by DSC the authors showed that surface transforms at higher rate (an order of magnitude) than the bulk phase. IGC seems to be a powerful tool to quantify amorphous rate and to locate it by the confrontation with bulk (or volume) measurement techniques [START_REF] Brum | Quantification of surface amorphous content using dispersive surface energy: the concept of effective amorphous surface area[END_REF].

The development of cost-efficient technologies for the generation of drug powders with desired physicochemical properties is still a challenge for pharmaceutical companies. To actually define a successful protocol it seems necessary to study surface energy as a function of polymorphism, surface chemistry of the exposed facets, particles size and shape (crystal habits). Nevertheless, as can be depicted, it is quite difficult to actually distinguish each effect influence. The objective of this work is to quantify the effect of size and crystal habits in surface energy of an API, D-mannitol.

To study particles size and shape influence on surface energetics of D-mannitol polymorphs two techniques are used: spray drying and/or cryomilling. As mannitol is known to be highly crystalline even during milling or spray drying, size reduction processes used in this work were established in ways that polymorphs solids samples do not undergo any phase transformation. IGC at infinite dilution (IGC-ID) is used to study solid surface anisotropy at low surface coverage, while DVS will be used to have a more global view of the interaction potential. The dual aim of this work is to highlight the impact of size and crystal habits on the adsorption behavior of different anhydrous forms of D-mannitol and to compare the surface energy determination techniques used pointing out their relevance considering the physical sense of the measure.

Materials and methods

Materials

D-mannitol was generously provided by Roquette (France). The batch, Pearlitol 160C, is composed of 99% of b mannitol mass percentage and only 1% of sorbitol. High purity deionized water (18 MV cm) was obtained from a laboratory purification system. The non-polar probes octane, nonane and decane were purchased from Sigma, assay >99%.

Generation and formulation protocols

Spray drying (SD)

The experiments were performed using a laboratory mini spray dryer Büchi B-290 equipped with a two-fluid nozzle of 0.5 mm diameter. The flow rate of the aqueous mannitol solutions, 10 g/L, is set at 6 mL/min and the atomizing air rate at 600 NL/h. Air inlet temperature is set to 120 C while outlet temperature was measured by a temperature sensor in the air exit point, noted 87 C. To recover the powder the aspiration is set to 35 m 3 /h (maximum level). Samples were stored at room temperature under vacuum in glass desiccators containing silica gel (relative humidity of 6%).

Cryomilling (CM)

A Retsch cryogenic impact mill was used for milling. Samples of around 500 mg were placed in stainless steel grinding jars of 5 mL containing one stainless steel grinding ball of 5 mm diameter. The samples were milled for 4 cycles in total, each consisting of a 10 min grinding time at 25 Hz followed by intervals of 0.5 min at 5Hz. The samples were continually cooled with liquid nitrogen before and during the grinding process.

Particles physicochemical characterization

Chemical purity

The chemical purity of each sample was checked by HPLC using a Hi-Plex Ca ligand exchange column coupled with a refractive index detector (analysis conditions: mobile phase HPLC grade water, flow rate 0.3 mL min À1 , column temperature 45 C).

Polymorphs finger prints

The mannitol powder samples were analyzed using different techniques to confirm the identity of the different polymorphs. Xray powder diffraction patterns (XRPD) were obtained using a PANanalytical X'Pert Pro MPD diffractometer (set-up Bragg-Brentano). Diffraction data is acquired by exposing the powders samples to Cu-Ka radiation at a voltage of 45 kV and to a current of 40 mA. The data were collected over a range of 8-50 2u& at step size of 0.03 . Quality diffraction patterns were obtained in a relatively short exposure time (20 min). Data analysis is done with the X'Pert Data Collector software while phase identification is made with PANalytical High Score More software and databases "ICDD Powder Diffraction File 2" and "Crystallography Open Database".

FT-Raman spectra were recorded with an atomic force microscope equipped with a confocal Raman imaging upgrade, Alpha 300 from WITec. A magnification of 50Â and an excitation source of Nd:YAG (532 nm) were used. The Raman data were acquired using WITec Project Plus software that has a 16 bits resolution and a sampling rate of 250 Hz.

Thermal analysis was performed with a DSC Q200 from TA Instrument. Samples amount of about 3-5 mg are placed in nonhermetic aluminum panels, in the temperature range of 20-200 C at a heating rate of 5 C/min under nitrogen atmosphere. Nitrogen flow was adjusted at 50 mL/min. All data measurements are averages of at least 3 measures on 3 different samples. Heat of fusion values were determined using a sigmoid baseline with a standard deviation of less than 0.5% (6 measures average).

Size and particle shape analysis

Particle shape analyses were examined using a XL30 SEM, scanning electron microscopy, with a field emission gun (FEG) operating at 15 or 20 kV. Samples were placed onto doubled-sided adhesive and coated with Platinum for 4 min using an auto sputter coater SC7640 under argon gas purge.

The particle size analysis was performed by image analysis using an optical banc PharmaVision System PVS 830 from Malvern Instruments. To separate the particles, a pressure of 6 bar was set (SPD 1300). The number of particles to be analyzed was set at 70,000. Two objectifs were used "zoom 3" which can analyze a particle size range between 310 and 7.5 mm, with a resolution of 7 mm and "zoom 5" which allows to analyze a particle range between 100 and 1.7 mm with a resolution of 3 mm.

Surface analysis techniques

Specific surface area and surface energy analyses of D-mannitol polymorphs were carried out using an IGC-Surface Energy Analyser (IGC-SEA) from SMS. Each powder sample was packed into pre-silanised columns of 300 mm length and 4 mm internal diameter, plugged with silanised glass wool. Prior to analysis, each column was conditioned at 50 C for 2 h at 0% RH. Helium was used as a carrier gas at 10 sccm and the column temperature was fixed at 30 C during the analysis, all experiences were undertaken in identical conditions. The retention times of probe molecules and methane were determined using a flame ionization detector (FID). Dead volume was determined by methane injections and dispersive surface energy determination by a homologous series of n-alkanes (n-decane, n-nonane and noctane). Dispersive surface energy profiles were calculated using Cirrus Plus software (version 1.2.3.2 SMS, London) following Dorris-Gray approach. The corresponding surface coverage, u s , at each injection concentration is the ratio between the desorbed amount, n, determined from peak area and the monolayer capacity of the solid, n m (u s = n/n m ). In our experiments, n m was determined from the specific surface area value obtained by IGC-SEA using nnonane as probe molecule. Full details of the experimental procedure and analysis by IGC have been published previously [START_REF] Cares-Pacheco | Physicochemical characterization of D-mannitol polymorphs: the challenging surface energy determination by inverse gas chromatography in the infinite dilution region[END_REF]. Pearlitol 160C and the b pure form were found, the differences between patterns peaks intensity can be attributed to morphology differences between both samples. Indeed, sorbitol presences in the commercial powder P.160C is not detected by XPRD but is detected by DSC (Table 2). As shown in Fig. 2, the first endothermic peak at 81.4 C, is consistent with the melting point of a-sorbitol [START_REF] Nezzal | Polymorphism of sorbitol[END_REF] which decreases both heat of fusion and melting point of b polymorph.

Results

3

It should be noted that the detection of small quantities is complex and may depend on the location of sorbitol within the powder, near to the surface or within the bulk of the crystals.

Particles size and habit

As described in our previous work, the commercial sample Pearlitol 160C (P160C) is composed by irregular sticks with a median volume diameter D[y, 0.5] of 64 mm and a D[y, 0.9] of 250 mm (Fig. 3a). The b form generated by antisolvent precipitation is formed by better-defined sticks with smoother surfaces (Fig. 3b), and it has a D[y, 0.5] of 26.7 mm and a D[y, 0.9] of 81.7 mm. It seems that for both samples, the smaller particles get stuck on the surface of the largest ones.

The a form is the finest one with needle-shape morphology and a D[y, 0.5] of 25.3 mm and a D[y, 0.9] of 42.4 mm (Fig. 3c). It should be notice that particles size analysis using PVS 830 seems to be not well adapted for the a form analysis. In fact SEM analysis showed a narrower size distribution. It is possible that PVS 830 characterizes particles agglomerations due to higher cohesion forces between these particles.

β (2) α (3) (1) (2)
(3)

Powder crystallinity and purity

XPRD patterns revealed that the two SD samples are composed of the stable b form (Fig. 4). The small peak present at 17.3 can be attributed to the presence, in really small quantities, of a mannitol.

Nevertheless not other characteristic peaks from the a form were found in the patterns. Moreover, DSC and Raman spectroscopy analysis do not corroborate the presence of a mannitol in the samples. The differences between the patterns peaks intensity can be attributed to the morphology differences.

Some protocols to spray dry D-mannitol aqueous solutions can be found in the literature, differentiating in polymorphs generation. For example, [START_REF] Lee | e of polymorphism in spray-dried mannitol[END_REF] obtained only the stable b form after SD while [START_REF] Maas | The impact of spray drying outlet temperature on the particle morphology of mannitol[END_REF] showed that outlet temperatures at/below 90 C generated mainly b mannitol and small amounts of a mannitol (around 5%). Outlet temperatures up to 140 C increased a mannitol concentration within the samples (between 5 and 15%). Nevertheless, in our experiences, with an outlet temperature of 87 C, DSC and Raman spectroscopy analysis do not detected the presence of the a form in the SD samples.

Particles size and habit by scanning electron microscopy (SEM)

Image analysis obtained with PVS 830 cannot be used for the analysis of spray dried samples. In fact the most powerful camera lens "zoom 5" does not measure particles smaller than 3 mm, which seems to be a quite important population according to SEM analysis. In addition, the dispersion protocol used with the SPD 1300 does not seem to actually disperse the particles. Finally, crystal habits and size analysis has been exploited by SEM.

From a microscopic point of view (10-20 mm), the powders obtained after spray drying are homogeneous in size and morphology. SEM micrographs show quite spherical particles with diameters between 300 nm and 10 mm and a "quite" smooth surface (Figs. 5 and6). These values are smaller than those obtained by [START_REF] Maas | The impact of spray drying outlet temperature on the particle morphology of mannitol[END_REF] which despite the observed differences in particle formation, the particles size distributions obtained at different outlet temperatures (60, 90 and 120 C) are very closed, with a mean diameter of 13 mm. The authors also highlighted that the spray drying of mannitol at different outlet temperatures modifies particle surface topography. In their case drying at 60 C leads to the formation of acicular crystal with a smooth particle surface. In contrast, at higher temperatures, the droplets evaporate much faster and do not recrystallize immediately because of the low nucleation rate at this temperature. Even if there is expected that water evaporate prior crystallization, no difference in particle size were obtained. Thus, the authors assumed that the nucleation and crystal growth also occurs in a rather early drying stage due to the high dynamic in the spray dryer and the presence of crystalline seeds that may trigger earliest crystallization. In fact, since the crystals grow larger in higher temperatures and because of the fast solvent evaporation, the forming shell becomes quickly very rigid and the openings of the hollow particles can be seen. In our case, with an outlet temperature of 87 C, some of the particles exhibit a rough surface and some other showed to be hollow with a well visible orifice in their shell (Figs. 5 and6). SEM analyses were limited due to samples degradation for a magnification higher than 6400Â. Finally, no differences in size and particles shape were found between b-SD (Fig. 5) and P.160-SD (Fig. 6) samples.

Surface energy analysis

After spray drying b-SD and P160C-SD presented an increment of their specific surface area and so an increase of the vapor probes adsorbed amount (Table 3) allowing DVS analysis. Thus, both SD samples, exhibit a similar adsorption behavior with a type II isotherm (Fig. 7). Surface energy values obtained by DVS were calculated taking into account contact angle values obtained by capillary rise [START_REF] Cares-Pacheco | Physicochemical characterization of D-mannitol polymorphs: the challenging surface energy determination by inverse gas chromatography in the infinite dilution region[END_REF]. The error associated to the measure is 1 mJ m À2 .

The g d s values obtained by DVS of b-SD and P160C-SD samples are similar to those obtained by IGC for the b pure form (Table 3).

IGC-ID analyses show that b-SD and P160C-SD samples have similar adsorption behavior (Table 3). Moreover, g d s values of SD samples are bigger than those of the b pure form but smaller than those obtained for the commercial powder P160C (Fig. 7). This tendency of the samples to have similar adsorption behavior after spray drying can be explained by the evolution of the particles to the same size and crystals habit. It seems important to highlight that the g d s of P160C-SD is approximately 5 mJ m À2 bigger than the one of b-SD for all surface coverage studied (0.0004 < n/n m < 0.01). This difference, superior to the experimental error (<1.3 mJ m À2 ), can be attributed to the sorbitol presence in the commercial powder P160C, which one is conserved after spray drying the sample. It appears that small quantities of sorbitol in the powders (less than 1%) increase the strength of dispersive interactions of the solid dispersion.

After spray drying the samples we can found a region where the retention time does not depend on the injection volume (Fig. 8, dotted line n/n m < 0.02).

Cryogenic milled samples (CM)

The a and b D-mannitol polymorphs generated by successive precipitation protocols were cryogenic milled in order to change its size and habit structure without changing their solid-state form.

The abbreviations b-CM and a-CM are used in this manuscript to identify the cryomilled powders.

Powder crystallinity and purity

XRPD patterns were used to confirm polymorphism and to ensure the physical form of the samples. a mannitol has characteristic reflection peaks at 2u positions of 13.7 , 17.3 and 19.9, while for b mannitol are located at 14.7 , 18.8 and 23.6 . As shown in Fig. 8, both a and b pure samples keep their polymorphic form after cryomilling. Particle habit and size changes after CM can be depicted as a decrease of the characteristic peaks intensity.

Particles size and habit by scanning electron microscopy (SEM)

SEM micrographs of the CM samples are shown in Figs. 10 and 11. The a-CM samples are formed by agglomerates of approximately 50 mm composed by small needle-like particles, and also by agglomerates of bigger particles always in needle-like shape as illustrated in Fig. 9. The closer zooms show that for a-CM samples the direction of the fracture follows particles geometry. In fact, it seems that crystals fracture follows a plane perpendicular to the longitudinal axis of the needle.

The SEM analysis of the b-CM samples showed particles agglomerations. The powder seems to be composed by quite irregular particles habits between some micrometers and 80 mm. A large quantity of small particles seems to be stick around the bigger ones. For this polymorph the fracture behavior after cryogenic milling is more complex and the solid seems to have a more brittle behavior (Fig. 11).

The results obtained by image analysis using the PVS830

(1) α -CM (2) β (3) β -CM (4) (3) (1) (4)
(2)

Fig. 9. X-ray patterns of a and b mannitol samples before and after cryomilling.

differences were detected between the samples using this technique. The image analysis by SEM and by PVS830 differs, most of all due to particles agglomeration.

Surface energy analysis

After cryogenic milling both samples, b-CM and a-CM, increased their specific surface areas. This increment allowed to analyze by DVS the b-CM samples thanks to an increment of the mass up-take at really low partial pressures, revealing a type II adsorption behavior (Fig. 12). This point is quite important, indeed if solid/probe molecules interaction showed a type III isotherm, probe-probe molecules interactions will be stronger that probe-solid interactions and so the retention time measure by IGC will not be representative of the adsorption phenomenon. Due to the low amount of experimental data (adsorbed amount as function of P/P sat ranged between 0 and $0.2), between 3 and 4, and the low correlation coefficients obtained with the DVS data, the specific surface area calculations have been carried out using IGC.

As shown in Table 3, an increase in the dispersive surface energy of b and a cryogenic milled samples, at low surface coverage, has been observed. Nevertheless, for the a-CM sample this difference, with the a pure sample, tends to decrease rapidly with increasing the injected amount. Indeed, for surface coverage superiors than 0.1% both solids, a and a-CM, have similar adsorption behavior. The b-CM sample has a similar but more energetic adsorption behavior than the b pure form. The region where g d s does not depend on injection volume correspond to a radio n/n m lower than 0.002. Nevertheless, The b-CM sample is more heterogeneous, at low surface coverage, than the b pure sample. This more heterogenic adsorption behavior can be attributed to the anisotropic morphology of the b-CM particles (Fig. 13).

Discussion

Henry's domain determination by IGC

The first point to highlight is that after treatment, spray drying and cryogenic milling, the Henry's domain of each sample is reduced to really low surface coverage's (u s < 1%). Thus, surface energy solid analyses do not allowed to represent the whole solids surface but allowed us to differentiate batches behavior.

It should be notice that the linearity of the isotherm, indicated by its correlation coefficient, plays a key role over the determination of the maximum u s that define Henry's domain, and should be well defined in this type of analysis. For example, for the b-SD samples, if we accept the linearity of the isotherm for R 2 > 0.99, a R 2 = 0.998corresponds to a P = 40 Pa and so to a u s = 10% (Fig. 14a) while a R 2 = 0.999corresponds to a P = 140 Pa and so to a u s = 40% (Fig. 14b). Nevertheless, we can see that even if R 2 is higher taking account higher pressures, the data fit used does not represent quite well the first points. Thus, in this case, pressures smaller than 40 Pa are used to describe Henry's domain.

As g d s is determinate by the injection of a series of alkanes noctane, n-nonane and n-decane, Henry's domain will be determinate by the probe having the narrowest Henry's domain. As expected, the longer the chains of alkanes, the smaller the linear region of the adsorption isotherm. So in these experiences, the limit surface coverage to study surface energy by IGC in the infinite dilution region will be determined by the analysis of n-decane.

A high control of the injection size as an important number of experimental data points it is fundamental to determine with precision Henry's domain.

Dispersive surface energy

It is quite difficult to link crystal habit, size and agglomeration state to surface energy. Generally, surface energy differences after physical treatment such as milling, micronization, CM even SD amount others, are generally attributed to changes in particles size (surface area), morphology (habits), polymorphism (or amorphization) and to the exposure of more energetically active planes. During our experiences we took care to not change the solid state of the samples, thus no solid/solid transformations were obtained.

Moreover it has been determined that for b mannitol, the most energetically active plane, in terms of dispersive surface energy, has a g d s of 44.1 AE 0.6 mJ m À2 [START_REF] Ho | Determination of surface heterogeneity of D-mannitol by sessile drop contact angle and finite concentration inverse gas chromatography[END_REF]. All this indicates that surface energy increments after CM and SD cannot be attributed to the exposure of new and more active phases or to solid-state transformations after treatment. Thus, crystal size and morphology changes seem to be responsible of surface energy increments in b mannitol samples. Nevertheless, we cannot separate particles size effect of crystals habit effect over the solid surface energy because CM and SD influence at the same time these two parameters.

As shown in Table 3, after cryogenic milling and spray drying treatment, a and b mannitol samples presented an augmentation of its dispersive surface energy, at really low surface coverage. For the b form, three batches of different size and shape were generated. The first one obtained by antisolvent precipitation is composed by well defined sticks with smooth surfaces D[y, 0.5] = 26.7 mm and D[y, 0.9] = 81.7 mm (Fig. 3b). The b-SD sample is composed by well defined spheres between 1 and 4 mm (Fig. 5a) while the batch b-CM is composed by agglomerations of quite irregular particles, of few micrometers to 90 mm, with small particles stuck on the surface of the larger ones (Fig. 10). IGC analysis revealed that these three batches have different surface behavior. After CM and SD the samples are more energetically active due to a change in the crystal habits. The b-recrystallized form appears as a quite homogeneous solid ($40 mJ m À2 ) while the b-CM batch and the b-SD batch showed a more heterogeneous surface, energetically speaking. It should be noted that despite its spherical shape and quite homogeneous size distribution, b-SD samples showed a slightly heterogeneous surface (57.3-51.7 mJ m À2 ). This confirms that the particles surface is not as smooth as depicted by SEM analysis.

Surface energy analyses at really low surface coverage, allow us to differentiate the CM samples of the SD samples: for b-CM a g d s of 61.9 mJ m À2 while for b-SD a g d s of 57.2 mJ m À2 at u s = 0.04%. The b-CM particles are quite damaged and seem to have surface defects (Fig. 11). These surface irregularities can be seen as privileged sites where the probe molecules can be adsorbed by undergoing interactions from more than one surface. Surface energy analyses at really low surface coverage emphasize these differences. Indeed, if the injection volume is incremented, u s > 0.1%, the surface energy differences between b-CM and b-SD samples tend to disappear and both solids tend to similar adsorption behavior ($52 mJ m À2 ). Nevertheless, at this surface coverage both samples have a higher g d s than the depart b form (52 to 40 mJ m À2 , respectively). This difference can be still attributed to the influence of more energetically active sites related to size and crystal habit changes after SD and CM (Figs. 5 and11).

It should be noted that if IGC-ID were defined for P/P sat $0.03, as most authors do it, we would worked outside Henry's domain and we would also mislead the effect of particles habits over solid's surface energy.

DVS analyses, which provided a mean value of the solid surface energy, showed that the g d s of b-CM samples (47 mJ m À2 , pink dotted line) is higher than the g d s of b-SD samples (40 mJ m À2 , purple dotted line). These results with surface energy analyses by IGC-ID show the contribution of more active sites to the solid average surface energy (Fig. 15). Moreover, IGC-DVS confrontation clearly shows us that if the more active sites are not numerous enough or not sufficiently higher than the most energetic active crystalline face, its influence can be misled by the techniques which provides a mean value of the solid surface energy like DVS.

In fact, mean surface energy analyses can mislead to differentiate batches and thus mislead end-use properties.

For the a form, two batches of different sizes were generated but with its morphology partially conserved. coverage, indeed, for u s < 0.005 both samples showed a similar adsorption behavior (Fig. 15).

All these results can be better understood if the mathematical definition of the retention time is taken into account. In fact, as already highlighted in previous studies, t N it is the contribution of many interactions sites i and the interaction energy between the probe and the sites i [START_REF] Cares-Pacheco | Physicochemical characterization of D-mannitol polymorphs: the challenging surface energy determination by inverse gas chromatography in the infinite dilution region[END_REF]. So, if the more active sites are not numerous enough its influence can be attenuated by the presence of less actives ones, moreover, the influence of less actives sites will be more important when increasing the injection size. The study of surface heterogeneity at higher surface coverage can give a more general idea of this point. Thus, for a-CM samples, it seems that the more active sites created by this process are not numerous enough because the cryogenic milling of a pure samples seems to only generate surface defects, which ones seem to be less active than the most energetically active face of the a crystal.

Surface energy analysis by IGC at Henry's domain highlighted the influence of particles habits and size over solid's surface behavior. The combined set IGC and DVS validated this statement. Nevertheless, it is quite difficult to separate both effects because most processes affect at the same time solid's habits and size, even particles separation processes, such as sieving, are related to particles morphologies.

From a critical point of view, the study of surface energies in Henry's domain by IGC-ID, does not provide a global idea of the solid behavior. Indeed, SD and CM samples presented quite small linear adsorption region. Thus, only 1% of the surface can be "cover" to be in Henry's domain (u s < 0.01). So the question is, how far are we of the solid's "real" behavior; are we able to actually distinguish crystalline facets influence (surface chemistry), are those important? It seems necessary to have another technique of surface analysis such as DVS, or even molecular modeling. It will be also interesting to study surface heterogeneity by IGC using an equipment that allows a higher surface coverage and to use other mathematical models such as the energy distribution function.

Conclusions

Powder behavior depends directly of its interfacial interactions, thus of the physical and chemical characteristics of the particles of which it is composed. Nevertheless, it is difficult to link crystalline structure, habits, particles agglomeration state, size distribution and surface energy. Classically, surface energy increments after physical and mechanical treatment such as grinding, are attributed to changes in particles size (surface area), morphology (habits), solid state structure (polymorphism or amorphization) and/or to the exposure of more energetically active planes. In this work we studied the influence of particles habits and size distribution over the solid surface energy. To achieve this goal, two batches of pure a and b mannitol, generated by successive crystallization protocols, were cryogenic milled and/or spray dried.

For the b form, three batches of different size and shape were studied. The first one, obtained by antisolvent precipitation, is composed by well defined sticks with smooth surfaces, the second one b-SD, is composed by well-defined spheres while the batch b-CM is formed by agglomerations of quite irregular particles. It is quite difficult to separate the effect of the size from the effect of particles habits because the two processes used, CM and SD, affected at the same time solid's habits and size. Moreover, even particles separation processes to study particles size, such as sieving, are related to particles morphologies.

IGC-ID studies highlighted surface energy differences between these three batches. Surface heterogeneity analyses, in Henry's domain (0.0004 < u s < 0.01), showed a quite heterogeneous and more energetically active b-CM (61.9-52.4 mJ m À2 ) and b-SD For the a form, two batches of different size and a quite conserved needle-like morphology were studied. The first batch was generated by seeding and fast cooling while the second one, a-CM, by the cryogenic milling of the first sample. Surface energy analysis at really low surface coverage, u s < 0.005, showed a more energetically active a-CM sample.

These results allow us to conclude that particles habits appear to be a major factor in the solid surface adsorption behavior of Dmannitol polymorphs. Moreover, it seems that there is a "threshold" above which the influence of more active sites, here attributed to surface irregularities, is attenuated and different batches of a same polymorph tend towards similar adsorption behavior. This threshold depends directly of the value of the more active sites but also of their number. If the more active sites are not numerous enough, or not sufficiently higher than the most energetic active face, its influence will be misled by the techniques which provides a mean value of the solid surface energy.

IGC-ID provides key information in terms of surface energy and appears to be a powerful tool to monitoring surface energy evolution as function of the generation process or after mechanical or thermal treatment. Even if the solid surface anisotropy is weak and maybe undetectable by standard analytical methods, it can make the solid more energetically active and thus more reactive. IGC-ID is able to detect weak surface energy variation, which may explain the behavior differences between batches, of a same polymorph, over time but also in atmospheres more or less moist.

  Nomenclature a s (m 2 /g) Specific surface area of the solid d (n,0.5) (mm) Number median diameter D [y,0.5] (mm) Median volume diameter Dg ads (J/mol) Molar free energy variation for an isothermal adsorption of probe molecules m (g) Sample mass n (mol) Desorbed mole number n ads (mmol/g) Adsorbed mole number per gram of solid n m (mol) Monolayer capacity or number of adsorbed moles corresponding to a monolayer P (Pa) Vapor pressure or partial pressure P sat /m 2 or N/m) Liquid surface energy (or surface tension) g d s (J/m 2 or N/m) Dispersive component of solid surface energy g s (J/m 2 or N/m) Total surface energy of a solid g sp s (J/m 2 or N/m) Specific component of solid surface energy u s (-) Surface coverage

  2.2.1. CrystallizationPure b and a forms were generated by antisolvent precipitation (acetone) and by seeding and fast cooling respectively as described previously in[START_REF] Cares-Pacheco | Physicochemical characterization of D-mannitol polymorphs: the challenging surface energy determination by inverse gas chromatography in the infinite dilution region[END_REF].

  .1. D-mannitol depart samples 3.1.1. Powder crystallinity and purity XPRD patterns of the commercial form P160C and the pure forms b and a mannitol are shown in Fig. 1. As it is depicted from the figure, a mannitol has characteristic reflection peaks at 2u positions of 13.7 , 17.3 and 19.9 and 21.4 while for b mannitol are located at 14.7 , 18.8 , 23.6 and 29.5 . No differences in the characteristic reflection peaks between the commercial powder

Fig. 1 .

 1 Fig. 1. X-ray patterns of depart samples, a and b mannitol and the commercial form Pearlitol 160C.

3. 2 .

 2 Spray dried samples (SD) Two samples were generated by spray drying two different b mannitol batches. The first one correspond to a pure b mannitol obtained by antisolvent crystallization and the second batch to the commercial powder Pearlitol 160C. The abbreviations b-SD and P160C-SD are used in this manuscript to differentiate them.

Fig. 3 .Fig. 4 .

 34 Fig. 3. SEM micrographs of (a) commercial powder Pearlitol160C, (b) pure b mannitol and (c) pure a mannitol.

Fig. 5 .

 5 Fig. 5. SEM micrographs of pure b mannitol sample after spray drying.

Fig. 6 .

 6 Fig. 6. SEM micrographs of commercial powder P.160C after spray drying.

Fig. 7 .Fig. 8 .

 78 Fig. 7. Adsorption isotherms of b-SD and P160C-SD obtained by DVS using n-nonane as vapour probe.

  Fig. 10. SEM micrographs of a mannitol cryomilled samples.

Fig. 11 .Fig. 12 .

 1112 Fig. 11. SEM micrographs of b mannitol cryomilled samples.

Fig. 13 .

 13 Fig.13. Surface heterogeneity analysis of a and b mannitol samples, before and after cryogenic milling (CM).

Fig. 14 .

 14 Fig. 14. Henry's domain determination by analysing the adsorption isotherms: example of b-SD/C 9 .

Fig. 15 .

 15 Fig. 15. Surface energy analysis by DVS (dotted line) and IGC of different pure b mannitol batches: b generated by antisolvent precipitation, after spray drying b-SD and after cryogenic milling b-CM.

(

  57.2-51.7 mJ m À2 ) samples, and a quite homogeneous and less active b form ($40 mJ m À2 ). Surface energy analysis at low surface coverage, u s < 0.01, allowed us to differentiate the SD samples of the CM samples. The solid size reduction, due to CM and SD, generates an increase of the specific surface area of the b pure powder, allowing DVS analysis of the b-CM (47 mJ m À2 ) and b-SD (40 mJ m À2 ) samples.

Table 1

 1 An overview of the influence of particles size reduction over the surface properties of pharmaceutical ingredients by IGC.

	Pharmaceutical solid	g d s	Reference
	Acetaminophen	"	Trowbridge et al. (1998) Heng et al. (2006)
	Cefditoren pivoxil DL-propanolol hydrochloride Felodipine	# " "	Ohta and Buckton (2004) York et al. (1998) Chamarthy and Pinal (2008) Chamarthy and Pinal (2008)
	Griseofulvine	"	Feng et al. (2008) Otte and Carvajal (2011)
	Ibipinabant Indomethacin	# " "	Otte et al. (2012) Gamble et al. (2012) Planinsek et al. (2010) Lim et al. (2013)
			Ahfat et al. (2000)
			Feeley et al. (2002)
	Lactose	"	Newell and Buckton (2004) Thielmann et al. (2007) Shariare et al. (2011)
			Brum and Burnett (2011)
			Jones et al. (2012)
	Mannitol Salbutamol sulfate	"	Ho et al. (2012) Ticehurst et al. (1994)
		"	Feeley et al. (1998)
	Salmeterol Xinofoate	"	Tong et al. (2001, 2006) Das et al. (2009)
	Sucrose	"	Surana et al. (2003) Hasegawa et al. (2009) Luner et al. (2012)
	Succinic acid	"	Luner et al. (2012)

Table 2

 2 Summary of physical solid-state characterization obtained by DSC.

	P160C	b-form	a-form

Fig. 2. DSC profile of the commercial powder Pearlitol 160C, heating rate of 5 C/min.

Table 3

 3 Specific surface areas and dispersive surface energies of different D-mannitol samples. The "X" represents the analysis that can not be done (technique limits) while "-" represents the ones that have not being done.

		a s (m 2 g À1 )			g d s (mJ m À2 )
	D-mannitol	ASAP-Ar IGC-C 9			IGC
				DVS	u s = 0.04% u s = 0.1% u s = 1% u s = 8%
	P160C	0.4	0.4	X	70.7	69.1	58.0	44.9
	a	8.4	8.5	51	74.9	75.0	72.1	45.5
	b	2.8	0.4	X	40.9	40.9	39.4	38.2
	P160C-SD	3.4	4.0	40	62.6	62.3	55.4	X
	b-SD	2.9	4.4	41	57.2	57.3	51.7	X
	a-CM	-	11.9	-	85.6	81.7	70.7	X
	b-CM	3.6	3.3	47	61.9	62.0	52.4	X

P. 160C -SD β -SD

Acknowledgment

In the memory of Elisabeth Rodier .


Jacqueline Resende De Azevedo

Jean-Jacques Letourneau

Fabienne Espitalier

Maria Ineŝ Reú

Solubility of a New Cardioactive Prototype Drug in Ionic Liquids

Solubility in different solvents is an intrinsic material characteristic for a defined molecule. The solubility of a new cardioactive prototype drug, 3,4-methylenedioxybenzoyl-2-thienylhydrazone (LASSBio-294), was determined in seven different imidazolium based ionic liquids (ILs). The ionic liquids selected were 1-ethyl-3-methylimidazolium methyl phosphonate; 1-ethyl-3-methylimidazolium ethyl phosphonate; 1-ethyl-3-methylimidazolium acetate; 1,3-dimethylimidazolium methyl phosphonate; 1-butyl-3-methylimidazolium acetate; 1-butyl-3-methylimidazolium tetrafluoroborate; and 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide. In addition, solubility data of LASSBio-294 in water and in mixtures of water and ILs were also measured. For all solvents, the concentration of drug in saturated solutions was determined by high performance liquid chromatography (HPLC). Solid-liquid equilibrium measurements were performed in a range of temperatures from 288.15 K to 308.15 K, at atmospheric pressure. From the solubility data it can be concluded that the drug showed sufficient solubility in five of the tested ILs so that these compounds can be suitable for further crystallization studies. The aim of this work is also to discuss and understand the solubility of LASSBio-294 in these alternative solvents. The results indicate that the solubility of the drug is dependent on IL structural variations (cation-anion combinations).

■ INTRODUCTION 3,4-Methylenedioxybenzoyl-2-thienylhydrazone, LASSBio-294, a new bioactive of N-acylhydrazone class, was synthesized in an attempt to develop new therapies which simplify the treatment of cardiovascular diseases. [START_REF] Lima | Synthesis and analgesic activity of novel N-acylarylhydrazones and isosters, derived from natural safrole[END_REF][START_REF] Barreiro | Estrateǵias de simplificacaõ molecular no planejamento racional de faŕmacos: a descoberta de novo agente cardioativo (Molecular simplification strategies in rational drug design to discover new cardioactive agent)[END_REF] Biological studies have characterized the LASSBio-294 as a molecule able to increase the contraction of the muscles of the heart and promote vasodilation through a mechanism different from that displayed by cardiac glycosides and β-adrenergic agonist. [START_REF] Gonzalez-Serratos | A novel thienylhydrazone, (2-thienylidene)3,4methylenedioxybenzoylhydrazine, increases inotropism and decreases fatigue of skeletal muscle[END_REF][START_REF] Sudo | The new compound, LASSBio-294, increases the contractility of intact and saponin-skinned cardiac muscle from Wistar rats[END_REF][START_REF] Silva | Cyclic GMP-dependent vasodilatory properties of LASSBio-294 in rat aorta[END_REF] These promising therapeutic effects strongly indicate that LASSBio-294 is a novel drug candidate, effective and safer for the treatment of cardiac failure [START_REF] Gonzalez-Serratos | A novel thienylhydrazone, (2-thienylidene)3,4methylenedioxybenzoylhydrazine, increases inotropism and decreases fatigue of skeletal muscle[END_REF] and for prevention of myocardial infarction induced by cardiac dysfunction. [START_REF] Costa | A compound with inotropic and lusitropic activity, decreases cardiac remodeling and improves Ca +2 influx into sarcoplasmic reticulum after myocardial infarction[END_REF][START_REF] Sudo | Thienylhydrazon with digitalis-like properties (positive inotropic effects)[END_REF] However, as a poorly water-soluble drug, it exhibits a low solubility and dissolution rate in the gastrointestinal tract, which limits its effective absorption and bioavailability after oral administration.

Drug properties such as crystal morphology, crystallinity, particle size distribution, and dissolution kinetics can be modified in different ways including recrystallization. Among the recrystallization techniques, antisolvent crystallization has been successfully used to improve dissolution kinetics, as shown by a number of reports in the literature. [START_REF] Zhang | Micronization of atorvastatin calcium by anti-solvent precipitation process[END_REF][START_REF] Muller | State of the art of nanocrystalsSpecial features, production, nanotoxicology aspects and intracellular delivery[END_REF][START_REF] Ali | Hydrocortisone nanosuspensions for ophthalmic delivery: A comparative study between microfluidic nanoprecipitation and wet milling[END_REF][START_REF] Vicosa | An innovative anti-solvent precipitation as a promising technique to prepare ultrafine rifampicin particles[END_REF] Antisolvent crystallization represents a class of process, which is characterized by the mixing between a solution and an antisolvent to produce solid particles. This mixing generates high supersaturation that subsequently induces nucleation, simultaneous growth, and possibly agglomeration of crystals.

Recrystallization works only when the proper solvent is used. The purpose of this work is to identify alternative solvents for the antisolvent crystallization of LASSBio-294 in the presence of water as antisolvent. The alternative solvents investigated here are the ionic liquids (ILs).

Unlike conventional solvents, ILs are entirely composed of ions. ILs are organic salts, usually liquid at room temperature, which are composed of a relatively large asymmetric organic cation (e.g., alkylpyridinium, dialkyl imidazolium ions) and an inorganic or organic anion (e.g., halide, hexafluophosphate, tetrafluoroborate, and ions based on fluorinated amides). These materials are commercially available, and the number of reported ionic liquids continues to increase. They are emerging as novel replacements for volatile organic compounds traditionally used as industrial solvents, owing to their negligible vapor pressures that reduce the risk of air pollution. [START_REF] Lourenco | Solubility advantage of pyrazine-2-carboxamide: Application of alternative solvents on the way to the future pharmaceutical development[END_REF] Furthermore, ILs present the prospective of "designer solvents" since their physicochemical properties can be finely tuned by an appropriate choice of the cation and/or the anion, and there is an enormous range of potential combinations of cation and anion that could be synthesized to produce an IL. [START_REF] Smith | Solubilities of Pharmaceutical Compounds in Ionic Liquids[END_REF] As a result, these solvents can be designed for a particular application or to present a particular set of intrinsic properties such as acidicity, basicity, hydrophilicity/ hydrophobicity and water miscibility, [START_REF] Huddleston | Characterization and comparison of hydrophilic and hydrophobic room temperature ionic liquids incorporating the imidazolium cation[END_REF] as well as toxicity. [START_REF] Azevedo | Imidazolium ionic liquids as solvents of pharmaceuticals: Influence on HSA binding and partition coefficient of nimesulide[END_REF] The combination of these excellent properties thus allows for designing green processes based on ILs. [START_REF] Freire | An overview of the mutual solubilities of waterimidazolium-based ionic liquids systems[END_REF][START_REF] Alevizou | Prediction of phase equilibrium in mixtures containing ionic liquids using UNIFAC[END_REF] The possibility of engineering the properties of ILs by manipulating the anion-cation combination, in association with their solvent properties and in some cases water-miscibility, makes them promising as potential pharmaceutical solvents in different situations. For instance, these combinations can be used in liquid-liquid extraction, [START_REF] Manic | Solubility of erythromycin in ionic liquids[END_REF] and to influence crystallization habits such as demonstrated in the case of pyrazine-2carboxamide, [START_REF] Lourenco | Solubility advantage of pyrazine-2-carboxamide: Application of alternative solvents on the way to the future pharmaceutical development[END_REF][START_REF] Melo | Ammonium ionic liquids as green solvents for drugs[END_REF] isoniazid [START_REF] Melo | Ammonium ionic liquids as green solvents for drugs[END_REF][START_REF] Forte | A favourable solubility of isoniazid, an antitubercular antibiotic drug, in alternative solvents[END_REF] and paracetamol, [START_REF] Smith | Solubilities of Pharmaceutical Compounds in Ionic Liquids[END_REF] as well as to dissolve poorly water-soluble drugs. Researches in this latter field have been focused on the ability of ILs to solubilize class II BCS compounds such as ibuprofen, [START_REF] Smith | Solubilities of Pharmaceutical Compounds in Ionic Liquids[END_REF] albendazole, and danazol. [START_REF] Mizuuchi | Room temperature ionic liquids and their mixtures: Potential pharmaceutical solvents[END_REF] Furthermore, our group has reported promising results for the antisolvent crystallization of rifampicin using an imidazolium ionic liquid as solvent and phosphate buffer as antisolvent. [START_REF] Vicosa | An innovative anti-solvent precipitation as a promising technique to prepare ultrafine rifampicin particles[END_REF] The ultrafine particles obtained were amorphous with enhanced solubility and faster dissolution rate.

To understand and control such antisolvent crystallization processes, solubility data of the solute in the IL-water mixtures are essential. In the present work a systematic study on the solubility of LASSBio-294 in imidazolium ionic liquids at different temperatures (288.15 K to 308.15 K) is reported. In particular, seven ILs were used: 1-ethyl-3-methylimidazolium methyl phosphonate; 1-ethyl-3-methylimidazolium ethyl phosphonate; 1-ethyl-3-methylimidazolium acetate; 1,3-dimethylimidazolium methyl phosphonate; 1-butyl-3-methylimidazolium acetate; 1-butyl-3-methylimidazolium tetrafluoroborate; and 1butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide. The solubility data were also analyzed in terms of possible solute-solvent interactions in the solvation process.

As with all solvents, solubility needs to be evaluated for LASSBio-294 in the ILs and in the antisolvent/IL mixtures chosen for recrystallization. The solubility of LASSBio-294 was then measured in water (antisolvent) and in mixtures of water and ILs at different temperatures (288.15 K to 308.15 K) to set up optimal operating conditions in the crystallization process.

■ EXPERIMENTAL SECTION Materials. The LASSBio-294, C 13 H 10 N 2 O 3 S, was obtained from Cristaĺia Ltd.a (Itapira, SP, Brazil). Its structural formula is shown in Figure 1. It is in the form of a yellowish solid with a molecular weight of 274.3 g•mol -1 and 99.96 % purity.

All ionic liquids were purchased from Solvionic (Toulouse, France) and their full names, abbreviations, chemical structures, and purity are given in Table 1. Acetronitrile (HPLC grade) was purchased from Scharlau SL (Spain). Water was distilled and purified using Milli-Q Water Purification System (Purelab Classic DI MK2, Elga, UK). All other chemicals were of an analytical grade.

Solubility Measurement Procedure. The solubility of LASSBio-294 in different ILs, in water, and in mixtures of water and ILs at different temperatures (288.15 K, 298.15 K and 308.15 K) was determined by the following procedure: stirred solutions containing an excess of solid were kept in a bath at a controlled temperature (measured using a calibrated thermocouple with an accuracy of 0.5 K) and atmospheric pressure for 72 h. In fact, from preliminary experiments, saturation was found to be reached after 24 h, and consequently all samples could be left for at least 24 h to ensure that the saturation solubility was reached (left for 72 h in this work). The suspensions were then centrifuged for 15 min at 4000 rpm. An aliquot of the supernatant was adequately diluted in a mixture of 60 % acetonitrile and 40 % water (w/w). The solutions were filtered (Acrodisc GHP, pore size 0.2 μm, Milipore, Bedford, MA, UK) and analyzed by high performance liquid chromatographic (HPLC) to assess the amounts of solute dissolved. The system consisted of an Agilent chromatograph (model 1100 series) equipped with a UV-vis detector. A Xterra RP18 column 5 μm (150 mm by 3.9 mm) was maintained at room temperature and equilibrated with the analytical mobile phase before injection. The mobile phase consisted of 50 % acetonitrile and 50 % water. The flow rate was 0.8 μm 3 •min -1 , the elution was monitored at 318 nm, and the injection volume was 0.02 cm 3 .

■ RESULTS AND DISCUSSION

This study concerns the solubility of a new molecule, LASSBio-294 in ILs as alternative solvents. As solubility depends on the structure and solid-state properties of the molecule, some properties of the original solid sample of LASSBio-294 were initially determined: the melting point (T m ) and the enthalpy of fusion (ΔH m ) by differential scanning calorimetry (DSC), the identification of the solid state by X-ray diffraction (XRD), and its morphology by scanning electron microscopy (SEM). The methodology used and the experimental data obtained are given in the Supporting Information. The original LASSBio-294 has a crystalline solid structure characterized by a T m of 479.06 K and a ΔH m = 122.9 kJ•kg -1 . This value of T m is close to that previously given in the literature for LASSBio-294 (478.15 K). [START_REF] Silva | Synthesis and vasodilatory activity of new Nacylhydrazone derivatives, designed as LASSBio-294 analogues[END_REF] The same crystalline form (Supporting Information, Figure S2) was identified in previous works. [START_REF] Kummerle | Studies towards the identification of putative bioactive conformation of potent vasodilator arylidene N-acylhydrazone derivatives[END_REF][START_REF] Costa | Structure re-determination of LASSBio-294a cardioactive compound of the N-acylhydrazone classusing Xray powder diffraction data[END_REF] The original crystals present an elongated shape (Supporting Information, Figure S3).

LASSBio-294 Solubility in Ionic Liquids. The solubility of LASSBio-294 (mass fraction) in the chosen ILs at 298.15 ± 0.5 K and at atmospheric pressure is shown in Table 2. Data represent averages from two or three independent experiments. The uncertainties were calculated by the experimental standard deviation of the means. Depending on the molecular structure of the drug substance, one could expect the solubility to change with the nature of the solvent. In fact, Table 2 shows that five imidazolium-based family cation ILs tested in this study have been found capable of dissolving LASSBio-294.

The success of the dissolution of a drug depends on the ability of the solvent to interact with the drug to form interactions more stable than interactions solute-solute and solvent-solvent. The properties of ILs are based on the anion-cation combination. Although imidazolium-based ILs are commonly used, their microscopic nature is not well understood. [START_REF] Freire | An overview of the mutual solubilities of waterimidazolium-based ionic liquids systems[END_REF] However, it is known that they can act both as hydrogen-bond acceptors (anion) and hydrogen-bond donors (cation):

• The electronic structure of imidazolium cation contains delocalized 3-center-4-electron configuration across the N 1 -C 2 -N 3 moiety, a double bond between C 4 and C 5 on the opposite side of the ring, and a weak delocalization in the central region. charged owing to the electron deficit in the CN bond, whereas C 4 and C 5 are practically neutral. [START_REF] Gonfa | Unique structure and solute-solvent interaction in imidazolium based ionic liquids: A review[END_REF] • The aromatic structure of imidazolium cation, hydrophilic domain, offers the possibilities of formation of electrostatic interaction and π-stacking with drugs.

• The hydrogen atom of C 2 can serve as a site for formation of a hydrogen bond. The hydrophobic domain, alkyl chain, allows the formation of van der Waals interactions. In addition, the anion can have the ability to delocalize its charge and act as an acceptor of hydrogen bond and/or Lewis base (Scheme 1). [START_REF] Gonfa | Unique structure and solute-solvent interaction in imidazolium based ionic liquids: A review[END_REF][START_REF] Olivier-Bourbigou | Ionic liquids and catalysis: Recent progress from knowledge to applications[END_REF] The levels of solubility observed for LASSBio-294 in

[emim][CH 3 O(H)PO 2 ], [emim][CH 3 CH 2 O(H)PO 2 ],
[emim][CH 3 OO], and [dmim][CH 3 O(H)PO 2 ] could be due to solute-solvent interactions. Interactions such as hydrogen bonds, van der Waals forces, and π-π interactions between solute-solvent have been suggested as responsible for the solubilization of hydrophobic drugs in ILs. [START_REF] Lourenco | Solubility advantage of pyrazine-2-carboxamide: Application of alternative solvents on the way to the future pharmaceutical development[END_REF][START_REF] Smith | Solubilities of Pharmaceutical Compounds in Ionic Liquids[END_REF][START_REF] Melo | Ammonium ionic liquids as green solvents for drugs[END_REF][START_REF] Mizuuchi | Room temperature ionic liquids and their mixtures: Potential pharmaceutical solvents[END_REF][START_REF] Holbrey | Liquid clathrate formation in ionic liquid-aromatic mixtures[END_REF] On the basis of the analysis of the LASSBio-294 structure (Scheme 1), it is hypothesized here that the same types of interactions may exist between the LASSBio-294 and the imidazolium-based ILs tested in this study.

Differences in the LASSBio-294 solubility in the seven ILs tested have been observed. To explain these findings, a more detailed analysis of each IL structure is performed, considering favorable and unfavorable interactions between the drug molecular structure and the anions and cations of ILs.

Table 1. ILs Used in This Study

*

The purity is provided by the supplier (determined from Karl Fisher titration, IR spectra, and Ionic Chromatography).

[emim][CH 3 O(H)PO 2 ] 254 ± 2 b [emim][CH 3 CH 2 O(H)PO 2 ] 474 ± 3 [emim][CH 3 OO] 457 ± 5 b [dmim][CH 3 O(H)PO 2 ] 367 ± 2 b [bmim][CH 3 OO] 473 ± 6 b [bmim][NTf 2 ] insoluble [bmim][BF 4 ]
insoluble a These values correspond to the mean solubility of two independent experiments. The uncertainty given in the table represents the standard deviation of the mean. b Three independent experiments. The reported repeatabilities are assumed to be equal the standard uncertainty for the measured solubilities. Standard uncertainties u is u(T) = 0.5 K.

Anion Influence. The contribution of the anion to solubilize the drug is demonstrated by comparing the results obtained with ILs-derived [bmim] and [emim] cations. The combination [bmim] cation-[CH 3 OO] anion could dissolve the LASSBio-294, contrarily to the combinations [bmim] cation-[BF 4 ] anion and [bmim] cation-[NTf 2 ] anion (Scheme 2).

First, the solubility of the LASSBio-294 in ILs derived with a CH 3 OO -anion can be associated with the good capacity of this anion to form a hydrogen bond and delocalize charges between the oxygen atoms. [START_REF] Pinkert | Reflections on the solubility of cellulose[END_REF] In turn, although the formation of a hydrogen bond between NTf 2 -anion and the drug (N-H group) has been described as the mechanism responsible for the ability of this anion to dissolve drugs such as isoniazid and pyrazinecarboxamide, [START_REF] Lourenco | Solubility advantage of pyrazine-2-carboxamide: Application of alternative solvents on the way to the future pharmaceutical development[END_REF][START_REF] Melo | Ammonium ionic liquids as green solvents for drugs[END_REF][START_REF] Forte | A favourable solubility of isoniazid, an antitubercular antibiotic drug, in alternative solvents[END_REF] the presence of the thienyl group of LASSBio-294 probably helps prevent solubilization of this drug. Furthermore, the greater structural complexity of LASSBio-294 in relation to isoniazid and pyrazinecarboxamide as well as the bulky substituents of the NTf 2 -anion probably impedes such interactions with LASSBio-294. [START_REF] Pinkert | Reflections on the solubility of cellulose[END_REF] Finally, the low capability of the BF 4

-anion to dissolve LASSBio-294 may be related to its tetrahedral shape that may hinder interactions with this drug. [START_REF] Pinkert | Reflections on the solubility of cellulose[END_REF] The discussion relating the effect of the anion on the solvency power toward LASSBio-294 of imidazolium-based ILs can be extended to another type of cation, 1-ethyl-3-methylimidazolium -, and CH 3 OO - shows that the differences may be related to the ability to form hydrogen bonds and also to the ability to delocalize the charges between the oxygen atoms (Scheme 3). [START_REF] Pinkert | Reflections on the solubility of cellulose[END_REF] Cation Influence. The effect of the cation is observed when the solubility data obtained with ILs-derived [emim] and [bmim] cations complexed with CH 3 OO -anion and [emim] and [dmim] cations complexed with the CH 3 O(H)PO 2 -anion are compared. In the first case, the greater hydrophobicity of the [bmim] cation contributes to interactions with the hydrophobic molecule leading to a higher solubility as described by Muzuuchi and co-workers. [START_REF] Mizuuchi | Room temperature ionic liquids and their mixtures: Potential pharmaceutical solvents[END_REF] In the second case, the influence of hydrophobicity is not observed. The solubility of LASSBio-294 was higher with [dmim] cation. This suggests that the solubility depends not only on the contribution of each ion separately but also on a combined cation-anion effect. A combined cation-anion effect in a dissolution process has already been described with cellulose, [START_REF] Zhang | 1-Allyl-3-methylimidazolium chloride room temperature ionic liquid: A new and powerful nonderivatizing solvent for cellulose[END_REF][START_REF] Feng | Research progress on dissolution and functional modification of cellulose in ionic liquids[END_REF][START_REF] Kosan | Solution states of cellulose in selected direct dissolution agents[END_REF][START_REF] Zhang | NMR spectroscopic studies of cellobiose solvation in EmimAc aimed to understand the dissolution mechanism of cellulose in ionic liquids[END_REF] although other scientific works have attributed most of the dissolution ability to the anion. [START_REF] Zhu | Dissolution of cellulose with ionic liquids and its application: A minireview[END_REF][START_REF] Remsing | Mechanism of cellulose dissolution in the ionic liquid 1-n-butyl-3methylimidazolium chloride: A 13 C and 35/37 Cl NMR relaxation study on model systems[END_REF][START_REF] Remsing | Solvation of carbohydrates in N,Ndialkylimidazolium ionic liquids: A multinuclear NMR spectroscopy study[END_REF][START_REF] Brandt | The effect of the ionic liquid anion in the pretreatment of pine wood chips[END_REF] Effect of Temperature on LASSBio-294 Solubility in Water and in [emim] Cation-Based ILs. The solubility of LASSBio-294 in water was measured as a function of temperature between 288.15 K and 308.15 K. The mass fraction at the equilibrium is lower than 5•10 -6 and the accuracy and the precision of the analytical method at this concentration level (1• 10 -6 ) did not permit acquisition of the evolution of the solubility values as a function of temperature.

On the basis of the solubility values of LASSBio-294 in pure ILs at 298.15 ± 0.5 K (Table 2), three ILs were chosen to evaluate the influence of temperature on the drug solubility. The LASSBio-294 solubility was determined in the three ILs at temperatures ranging from 288.15 K to 308.15 K. The results are shown in Table 2 and Table 3. It can be noted that the solubility in these pure ILs increased with temperature in the studied range. However, the results for [emim][CH 3 CH 2 O(H)PO 2 ] at 288.15 K and 298.15 K show that the solubility is rather constant, which is not the case for the other ILs. This behavior may be due to a problem of the solubility measurement at 288.15 K with this IL. Indeed, this is more viscous (0.14 Pa•s) than the other ones (<0.11 Pa•s), and the separation liquid/solid is more difficult, inducing probably an overestimation of the solubility. At 308.15

T/K [emim] [CH 3 O(H)PO 2 ] [emim] [CH 3 CH 2 O(H)PO 2 ] [emim] [CH 3 OO] 288.15 233 ± 2 475 ± 1 b 437 ± 8 308.15 302 ± 1 > 508 ± 8 c > 641 ± 1 c
a These values correspond to the mean solubility of two independent experiments done at each temperature. The uncertainty given in the table represents the standard deviation of the mean. The reported repeatabilities are assumed to be equal the standard uncertainty for the measured solubilities. Standard uncertainties u is u(T) = 0.5 K. 4. They revealed that the solubility of LASSBio-294 decreased sharply when water was added to the drug-IL solutions.

These data indicate that the antisolvent crystallization method is an interesting way to recrystallize the LASSBio-294. The knowledge of the solid-liquid equilibrium in these systems will be used for determining the operating conditions for a crystallization procedure leading to a good solid performance.

Effect of the

■ CONCLUSIONS

This research is focused on ILs and their application as alternative solvents for LASSBio-294, a new cardioactive prototype and poorly water-soluble drug. The use of ILs as alternative solvents for this type of drug can open new possibilities for LASSBio-294 reprocessing because not only ILs essentially do not evaporate, and so they cannot lead to fugitive emissions, but also several of them acted as good solvents for this drug as shown in this study.

The dissolution properties of ILs depend on both the cation and the anion. Systematic studies about the cation and the anion influence on solubility are currently very scarce and are needed. Overall the results presented here suggest that a design strategy to solubilize a poorly water-soluble drug such as LASSBio-294 can be used for the selection of a specific IL, based on a proper knowledge of the drug molecule structure and of the IL chemistry. The structure of an IL could thus be tuned to maximize its interaction with a targeted drug, and then to favor the drug solubility.

Finally, the sufficient solubility of LASSBio-294 in ILs as alternative solvents obtained in this work can open new perspectives in pharmaceutical processing through recrystallization (antisolvent crystallization process). The residual content of ionic liquid in a solid after crystallization can for instance be removed by washing with water.

Figure 1 .

 1 Figure 1. Structural formula of LASSBio-294.

Scheme 1 .-, and BF 4 -)

 14 Scheme 1. Schematic Drawing of Hypothetical Interactions between the Imidazolium-Based Ionic Liquids and LASSBio-294. Scheme adapted with permission from ref 26. Copyright 2010 Elsevier.

  [emim], associated with different anions: CH 3 O(H)PO 2 -, CH 3 CH 2 O(H)PO 2 -, and CH 3 OO -. When the [emim] cation is combined with the CH 3 O(H)-PO 2 -and CH 3 CH 2 O(H)PO 2 -anions, the solubility can be linked to the alkyl chain length of the CH 3 CH 2 O(H)PO 2 -anion (Scheme 3). The length of this chain (the higher hydrophobicity) induces a greater solubility of the LASSBio-294 in the ILs. A comparison of the solubility data obtained with ILsderived CH 3 O(H)PO 2 -, CH 3 CH 2 O(H)PO 2

  They are [emim][CH 3 O(H)PO 2 ], [emim][CH 3 CH 2 O(H)-PO 2 ], and [emim][CH 3 OO] ILs.

Scheme 3 .

 3 Scheme 3. Hypothetical Relationship between the Anion Combined to 1-Ethyl-3-methylimidazolium [emim] and the Solvency Power of the IL toward LASSBio-294. Anions: CH 3 O(H)PO 2 -, CH 3 CH 2 O(H)PO 2 -, and CH 3 OO -

■

  ASSOCIATED CONTENT * S Supporting Information Experimental section describing the determination of T m and ΔH m , XRD diffractogram, and SEM images of the original solid

Table 2 .

 2 Solubility of LASSBio-294 in Various ILs at 298.15 ± 0.5 K and 0.1 MPa

	ionic liquids	solubility

a (mass fraction•10 3 )

  b Three independent experiments. c Equilibrium not reached. K, the equilibrium solubility data in [emim][CH 3 CH 2 O(H)-PO 2 ] and [emim][CH 3 OO] could not be determined because the drug-IL mixture became a gel. LASSBio-294 Solubility in Mixtures of Water and ILs Consisting of [emim] Cation Associated with Three Different Anions, [CH 3 O(H)PO 2 ], [CH 3 CH 2 O(H)PO 2 ], and [CH 3 OO]. The first step to design a drug recrystallization process when using an antisolvent crystallization method is to define the antisolvent/solvent mass proportion to obtain high yields of solid product. In this part of the study, the solubility of LASSBio-294 in mixtures of water/[emim][CH 3 O(H)PO 2 ], water/[emim]-[CH 3 CH 2 O(H)PO 2 ], and water/[emim][CH 3 OO] was determined at 298.15 ± 0.5 K. The experimental data obtained are shown in Table

  Temperature on the Solubility of LASSBio-294 in Mixtures of Water and ILs Consisting of [emim] Cation Associated with Three Different Anions, CH 3 O-(H)PO 2 -, CH 3 CH 2 O(H)PO 2 -, and CH 3 OO -. The solubility of LASSBio-294 (mass fraction) in mixtures of water/[emim]-[CH 3 O(H)PO 2 ], water/[emim][CH 3 CH 2 O(H)PO 2 ], and water/[emim][CH 3 OO] for water/IL mass ratios of 0.05 and 1.0 at different temperatures is shown in Table 5. The results confirm that the solubility of LASSBio-294 in mixtures of water with ILs generally increases with temperature. In the mixture water/[emim][CH 3 CH 2 O(H)PO 2 ], the solubility change does not follow the same trend as the other mixtures: the solubility does not increase as a function of temperature for the water/IL mass ratio of 1.0 by contrast with the ratio of 0.05. This unexpected result may be due to experimental uncertainties, as already found for pure [emim][CH 3 CH 2 O(H)PO 2 ].

Table 4 .

 4 Solubility of LASSBio-294 in Mixtures of Water and ILs at 298.15 ± 0.5 K and 0.1 MPa These values correspond to the mean solubility of two independent experiments done at each mass ratio. The uncertainty given in the table represents the standard deviation of the mean. The reported repeatabilities are assumed to be equal to the standard uncertainty for the measured solubilities. Standard uncertainties u is u(T) = 0.5 K. (-) Not measured.

		solubility a (mass fraction•10 3 )
	mass ratio water/IL	[emim] [CH 3 O(H)PO 2 ]	[emim] [CH 3 CH 2 O(H)PO 2 ]	[emim] [CH 3 OO]
	9.00	0.002 ± 0.001	-	-
	4.00	0.004 ± 0.001	-	-
	2.30	0.012 ± 0.001	-	-
	1.50	0.021 ± 0.004	-	-
	1.00	0.211 ± 0.004	0.243 ± 0.011	0.254 ± 0.014
	0.67	0.307 ± 0.031	0.553 ± 0.001	-
	0.43	1.240 ± 0.004	1.323 ± 0.012	-
	0.25	5.664 ± 0.054	8.661 ± 0.348	-
	0.18	10.701 ± 0.241	-	-
	0.11	33 ± 7	105 ± 1	-
	0.05	85 ± 1 b	315 ± 5	258 ± 6
	0.02	160 ± 7	-	-

a b Three independent experiments.

Table 5 .

 5 Solubility of LASSBio-294 in Mixtures of Water and ILs at Different Temperatures and 0.1 MPa solubility a (mass fraction•10 3 ) These values correspond to the mean solubility of two independent experiments done at each mass ratio and temperature. The uncertainty given in the table represents the standard deviation of the mean. The reported repeatabilities are assumed to be equal to the standard uncertainty for the measured solubilities. Standard uncertainties u is u(T) = 0.5 K. b Three independent experiments. sample of LASSBio-294, whose solubility in different ILs was the subject of this work. The experimental results are given in FiguresS1, S2, and S3. This material is available free of charge via the Internet at http://pubs.acs.org.

	T/K	mass ratio water/IL	[emim] [CH 3 O(H)PO 2 ]	[emim] [CH 3 CH 2 O(H)PO 2 ]	[emim] [CH 3 OO]
	288.15	1.00	0.172 ± 0.040	0.280 ± 0.080	0.226 ± 0.010
		0.05	62 ± 1	264 ± 1 b	217 ± 3
	298.15	1.00	0.211 ± 0. 004	0.243 ± 0.011	0.254 ± 0.014
		0.05	85 ± 1 b	314 ± 5	258 ± 6
	308.15	1.00	0.253 ± 0.030 b	0.377 ± 0.050	0.256 ± 0.010
		0.05	122 ± 1	318 ± 3	312 ± 1

a

Funding

This research was partially supported by CNPq (Brazilian National Research Council) Research Scholarship (Proc.200835/2010-6).

Notes

The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

The authors would like to thank Crystalia Ltda (Itapira, SP, Brazil) for providing the drug LASSBio-294.