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1 EFFECT OF SOLVENT TYPE AND CONCENTRATION ON MORPHOLOGY OF LEVOFLOXACIN AND MOXIFLOXACIN MICROPARTICLES OBTAINED BY SUPERCRITICAL ANTISOLVENT PRECIPITATION Vorobei A. M. 1,2 *, Pokrovskiy O. I. 2, Parenago O. O. 1,2, Lunin V. V. 1,2 1 Department of Chemistry, Moscow State University, Moscow, , Russia 2 Kurnakov Institute of General and Inorganic Chemistry of the Russian Academy of Sciences, Moscow, , Russia vorobei@supercritical.ru The influence of solvent and concentration in the solution on morphology of fluoroquinolone particles obtained by SAS was investigated. Solvents used can be divided into two main groups depending on morphology of particles obtained. The use of chlorohydrocarbons leads to the formation of plate-like particles. Formation of parallelepipeds is observed in the case of solvents of the second group. (DMF, DMSO, methanol etc.) It was found that dependencies of average size on concentration of fluoroquinolones in solution are nonmonotonic. INTRODUCTION Supercritical fluid approaches can be used as alternative methods for controlled-release drugs preparation. Due to a number of advantages such as high speed of the transition from a solution to a solid phase and good controllability of the process, they provide an opportunity to avoid difficulties typical for conventional methods. Most of pharmaceutical substances are insoluble in SC-CO 2. Therefore, supercritical antisolvent (SAS) method is especially relevant for this task. However, there are many parameters in SAS process such as temperature, pressure, flow rates of both organic solution and SC-CO 2 etc., which can influence particle morphology drastically. Consequently, it is very useful to know how these parameters affect particles obtained. The effect of hydrodynamic parameters [1, 2], pressure and temperature [3-6] is sufficiently studied. In our opinion, effect of solvent type and concentration of micronized substance in the solution is investigated in a lesser degree. Particularly, most works devoted to this problem use only 2-3 solvents, frequently structurally close ones. Therefore, it is impossible to consider specific features of different solvents. Moreover, typically the range of concentration of API in solutions sprayed in SAS within one research is not broad. Hence, there are different examples when increase of concentration in the solution leads to increase [7], decrease [8] and negligible changes [9] of average particle size. In this work we focused on influence of these two parameters, type of solvent used and concentration of micronized substance in the solution. Two antituberculous fluoroquinolone drugs were chosen as objects of our research: levofloxacin and moxifloxacin. In our research we try to use maximal number of solvents for each fluoroquinolone. Furthermore, to understand the effect of concentration on size and morphology particles precipitated the concentration was varied in wide range.

2 MATERIALS AND METHODS The laboratory apparatus used for SAS process is represented schematically in fig. 1. Fig. 1. Schematic representation of the SAS experimental apparatus. 1- СО 2 cylinder; 2- cooler; 3- flowmeter; 4 - СО 2 pump; 5 heat exchanger; 6 polymer solution; 7 solution pump; 8 precipitator; 9 automatic back pressure regulator; 10 separator; 11 manual back pressure regulator; 12 valve The procedure of supercritical antisolvent precipitation using this apparatus was described in details elsewhere [10]. Briefly, micronization was carried out as follows. Solutions of fluoroquinolones were pre-prepared in different organic solvents. Solution pump was filled with pure solvent. Upon reaching operating parameters of pressure, temperature and CO 2 flow rate fluoroquinolone solution was sprayed by a high-pressure pump through a narrow nozzle into a high-pressure vessel in which a constant flow of supercritical CO 2 was sustained by another high-pressure pump. A back pressure regulator placed after the vessel maintains defined pressure. A solution is sprayed and rapidly mixes with the antisolvent which causes precipitation. To wash precipitated fluoroquinolone particles constant CO 2 flow was maintained for some time after solution spraying. Fluoroquinolone solution was sprayed into supercritical antisolvent at temperature 40 о С, solution flow rate was 1 ml/min, СО 2 flow rate was 50 ml/min, CO 2 pressure was 150 bar, nozzle diameter was 100 microns in every experiment. Fluoroquinolone concentration in the solution was varied from 6.25 to 50 g/l. Dimethylformamide (DMF), dimethyl sulfoxide (DMSO), methanol, hexafluoroisopropanol (HFIP) and acetic acid were used to dissolve moxifloxacin. Ethyl acetate, acetone, acetonitrile, methanol, ethanol, dichloromethane, chloroform, 1,2-dichloroethane, acetic acid, propionic acid, DMF, DMSO and HFIP were used to dissolve levofloxacin. All organic solvents were supplied by JT Baker. Food grade carbon dioxide was obtained from LindeGasRus (Russia). Electron microscopy was performed using scanning electron microscope (SEM) «LEO 1450» (Carl Zeiss, Germany). The sample was put on a carbon conductive bilateral adhesive tape pasted on copper-zinc sample holder. Samples were covered by layer of gold with 2.5 nm thickness using magnetron sputtering method. This procedure was performed using Quorum Q150R ES in vacuum. Accelerating voltage (from 0.5 to 30 kv) as well as working distance

3 (8-25 mm) were tuned depending on the structure and composition of the sample. Size assessment of micronized particles was performed using IP3 software. RESULTS SEM images of initial particles of levofloxacin and moxifloxacin are presented in fig. 2a and 2b respectively. Moxifloxacin particles are parallelepipeds elongated along one of the axes with an average size of 8 microns. Throughout this paper by average particle size we mean the particle average length in its longest dimension. Levofloxacin particles are different irregular fragments with average size 60 micrones. They are often elongated. a b Fig. 2. Initial levofloxacin (a) and moxifloxacin (b) particles Firstly, the effect of different solvents on size and morphology of fluoroquinolone particles obtained by SAS micronization was investigated. SEM images of levofloxacin micronized by SAS using different solvents are shown in fig. 3. Size and morphology of levofloxacin particles are summarized in Table 1. Levofloxacin concentration in the solution is 6.25 g/l for all samples. Particle size is µm depending on solvent used. All micronized particles are significantly smaller than initial levofloxacin particles. Size of particles obtained using chloroform, acetic acid and ethyl acetate is minimal, while size of crystalls precipitated using DMF is the largest. Particle morphology also depends on chosen solvent. Analysis of SEM images allows dividing all tested solvents into two main groups depending on morphology of particles obtained. The first group includes chlorohydrocarbons, namely chloroform, dichloromethane and 1,2-dichloroethane. The use of these solvent leads to thin plate-like particles, often with jagged edge. Typical morphology is demonstrated in fig. 3c. The second group includes DMF, DMSO, methanol, ethanol and acetonitrile. Particles obtained using these solvents are parallelepipeds elongated along one of axes. Typical examples of such morphology are shown in fig. 3a and 3b. Aspect ratio of such parallelepipeds is different for different solvents. The similarity of particle morphology is observed inside the second group, despite signifficant difference of structures and properties of solvents. Micronization using ethyl acetate, acetic and propionic acids leads to intermediate morphology. For example, there are both parallelepipeds and plates in a sample obtained from acetic acid (fig. 3d).

4 Table. 1. Morphology of levofloxacin particles obtained by SAS using different solvents Solvent Average particle diameter, µm Morphology DMF 31±2 elongated parallelepipeds methanol 18±0.8 elongated parallelepipeds DMSO 6.7±0.7 elongated parallelepipeds ethanol 5.7±0.4 elongated parallelepipeds acetonitrile 4.8±0.4 elongated parallelepipeds dichloromethane 3.7±0.1 plates 1,2-dichloroethane 2.9±0.2 plates chloroform 1.4±0.1 plates propionic acid 3.0±0.2 elongated parallelepipeds/plates acetic acid 1.7±0.1 elongated parallelepipeds/plates ethyl acetate 1.6±0.1 elongated parallelepipeds/plates a b c d Fig. 3. SEM images of levofloxacin particles precipitated using methanol (a), DMF (b), chloroform (c) and acetic acid (d) SEM images of moxifloxacin obtained by SAS using different solvents are presented in fig. 4. Average size and morphology of moxifloxacin particles are summarized in table 2. Moxifloxacin concentration in the solution is 6.25 g/l for all samples.

5 Table 2. Morphology of moxifloxacin particles obtained by SAS using different solvents Solvent Average particle diameter, µm Morphology HFIP 8,1±0,6 elongated parallelepipeds DMF 2,0±0,1 elongated parallelepipeds DMSO 1,3±0,1 elongated parallelepipeds methanol 0,7±0,1 predominantly elongated parallelepipeds acetic acid 0,58±0,03 polygonal plates a b Fig. 4. SEM images of moxifloxacin particles precipitated using HFIP (a) and acetic acid (b) Average size of particles strongly depends on solvent as in the case of levofloxacin. It varies from 0.6 µm for acetic acid to 8.1 µm for HFIP. All micronized particles are significantly smaller than initial levofloxacin particles excepting those obtained using HFIP. Minimal size of particles was observed when acetic acid and methanol were used as solvents. Particles precipitated using the most part of solvents (HFIP, DMF, DMSO, methanol) are parallelepipeds with different aspect ratios. Typical example of this morphology is presented in fig. 4a. The morphology changes dramatically when acetic acid is used for micronization. Particles obtained using this solvent are precipitated in the form of plates (fig.4b). It is interesting to note that this division of solvents is similar to results obtained for levofloxacin micronization. Unfortunately, moxifloxacin is insoluble in most chlorohydrocarbons (chloroform, dichloromethane etc.). Therefore, it is impossible to test other solvents apart from acetic acid which gave plate-like morphology for levofloxacin. To study the effect of concentration on particle average diameter several solvents from each group were chosen. The dependence of average particle size on concentration of levofloxacin for different solvents is demonstrated in fig. 5.

6 Fig. 5. Dependences of average particle diameter on concentration of levofloxacin in solution In our opinion, there are two types of curves in fig. 5. Curves which were obtained for DMF, DMSO and methanol can be combined into one group, while those of acetic acid and chloroform are fundamentally different. Specific features of first type curves are mostly expressed for DMF curve. Firstly, the dependence of average size on concentration of levofloxacin in the solution is nonmonotonic, it passes through minimum. Moreover, this dependence can be divided into three different areas. Drastic fall of average particle size from 30 to 8 µm takes place at concentration increase from 6.25 to 12.5 g/l. Particle size decrease becomes significantly less pronounced in the second interval, namely from 8 to 4 µm. This trend changes with further increase of levofloxacin concentration, in the third interval an increase in concentration causes an increase in particle size.. In our opinion, this dependence takes place because the degree of supersaturation is small during mixing СО 2 and solution in the case of dilute solution (first interval). Therefore, growth rate is considerably higher than nucleation rate. As solutions get more concentrated the degree of supersaturation is sufficient for active nucleation. Thus, crystal growth occurs in a larger number of crystallization centers, particle nucleation rate becomes comparable to particle growth rate and this fact determines smaller particle size. Further increase of concentration leads to a situation when concentration is sufficient for significant growth of all nucleation centers as well as for crystal intergrowth which leads to an increase of particle size in the last interval. We expect that similar dependences should theoretically be observed for methanol and DMSO. Unfortunately, it is impossible to obtain particles using DMSO at low concentration, because levofloxacin dissolves in СО 2 - DMSO mixture. Consequently, it is possible to find only the second area of particle size decrease for DMSO. Moreover, it is impossible to work at high concentration because required solubility of levofloxacin in initial solution cannot be achieved due to its limited solubility in those solvent.

7 The concentration curves for acetic acid and chloroform are radically different from the previous ones. The minimum takes place but particle size changes still less depending on concentration. a b с Fig.6. SEM images of levofloxacin particles precipitated using DMF. Levofloxacin concentration in the solution is 6.25 (a), 37.5 (b) and 50 (c) g/l It is important to mention that concentration of levofloxacin influence particle morphology significantly. Different morphology types of particles depending on concentration are demonstrated in fig. 6 for DMF concentration dependence. Elongated parallelepiped particles are precipitated at low concentration (fig. 6a). Increase of levofloxacin concentration in the solution leads to change of morphology and aspect ratio of particles decrease. There are plate particles predominantly at minimum of concentration curve (fig. 6b). Further concentration increase leads back to parallelepipeds (fig. 6c). Fundamentally different situation is in the case of chloroform and acetic acid. The morphology changes significantly less compared to DMF. Plates are precipitated using chloroform and acetic acid at low concentration, while there are some elongated parallelepiped particles at high concentration of levofloxacin in the solution. The dependences of moxifloxacin average particle size on concentration for DMF, DMSO and acetic acid are presented in fig. 7. Fig. 7. The dependance of average particle diameter on concentration of moxifloxacin in the solution

8 Despite obvious difference, all concentration curves are nonmonotonic, as in the case of levofloxacin. The dependence of average particle size on moxifloxacin concentration for DMF is quite similar to that of levofloxacin. Drastic fall of average particle size from 5.8 to 0.7 µm takes place at concentration increase from 1 to 12.5 g/l. However, the second interval of particle size decrease is represented in a lesser extent compared to levofloxacin. Concentration curve obtained for acetic acid and DMSO have other features. Particle size decreases significantly less than in the case of DMF. CONCLUSIONS Morphology of fluoroquinolone particles obtained by SAS strongly depends on chosen solvent. All solvents used can be divided into two main groups depending on morphology of particles obtained. The first group includes chlorohydrocarbons. The use of these solvents leads to the formation of plate-like particles. DMF, DMSO, methanol, ethanol and acetonitrile can be combined in second group. Parallelepipeds are precipitated using solvents of the second group. Dependencies of average size on concentration of fluoroquinolone in the solution are nonmonotonic. Three intervals can be distinguished on concentration curves: an interval of steep size drop, and interval of shallow size decrease and an interval of size growth with concentration increase. Concentration also influences particle morphology. ACKNOWLEDGEMENTS This work was financially supported by Russian Scientific Foundation, grant N REFERENCES [1] PETIT-GAS, T. et. al., The Journal of Supercritical Fluids, Vol. 51, 2009, p. 248 [2] MARTIN, A. COCERO M. J., The Journal of Supercritical Fluids, Vol. 32, 2004, p. 203 [3] MIGUEL, F., The Journal of Supercritical Fluids, Vol. 36, 2006, p. 225 [4] KROBER, H., The Journal of Supercritical Fluids, Vol. 22, 2002, p. 229 [5] REVERCHON, E., Powder Technology, Vol. 106, 1999, p. 23 [6] DE MARCO, I., REVERCHON, E., Powder Technology, Vol. 58, 2011, p. 295 [7] KIM., M.-S., Powder Technology, Vol. 177, 2007, p. 64 [8] REVERCHON, E. et. al., Powder Technology, Vol. 114, 2001, p. 17 [9] TAVARES CARDOSO M. A., The Journal of Supercritical Fluids, Vol. 44, 2008, p. 238 [10] VOROBEI, A.M. et. al., Russian Journal of Physical Chemistry B, Vol. 9, Issue , p. 1103