Cocrystallization of the anticancer drug 5-fluorouracil and coformers urea, thiourea or pyrazinamide using supercritical CO₂ as an antisolvent (SAS) and as a solvent (CSS)

Highlights

• Co-crystals of the anticancer drug 5-fluorouracil (5-Fu) were prepared.

• 5-Fu and coformers urea, thiourea or pyrazinamide were processed in supercritical CO₂.

• 5-Fu-urea and 5-Fu-thiourea were crystallized via Supercritical Antisolvent (SAS).

• SAS conditions for the production of pure 5-Fu-urea co-crystals were identified.

• Cocrystallization with a Supercritical Solvent (CSS) required methanol as cosolvent.

Abstract

Co-crystals of 5-fluorouracil (5-Fu) and the coformers urea, thiourea and pyrazinamide (PZA) were attempted for the first time through the supercritical antisolvent (SAS) and the cocrystallization with supercritical solvent (CSS) techniques. SAS operational conditions were temperature (313 K), pressure (7.0–15.0 MPa) and 5-Fu concentration in methanol (5 and 2.5 mg/mL). Coformer concentration was always in the desired stoichiometric ratio. Co-crystals were characterized using powder X-ray diffraction (PXRD), differential scanning calorimetry (DSC), infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). Pure 5-Fu-urea cocrystals were obtained via SAS at 313 K and 8.0 MPa using a 5 mg/mL 5-Fu solution. All other SAS conditions studied led to 5-Fu homocrystal impurities. For comparison purposes 5-Fu, urea and thiourea were also processed by SAS. CSS produced a mixture of co-crystals and homocrystals only when supercritical CO₂ was modified with methanol. Advantages and disadvantages of the two supercritical cocrystallization techniques are discussed.

Introduction

5-Fluorouracil (5-Fu) is a well-known drug used in chemotherapy for the treatment of cancer. 5-Fu is a class III drug according to the biopharmaceutics classification system (BCS), i.e., high solubility and low permeability. There are two distinctive 5-Fu polymorphs known; polymorph I [1] (triclinic) and polymorph II [2] (monoclinic). Due to its antimetabolite action, 5-Fu is one of the most commonly used drugs to treat a wide range of cancers, including head and neck cancer [3], some skin cancers [4], and breast cancer [5]. It is though in colorectal cancer where 5-Fu has demonstrated a higher impact with increased overall and disease-free survival of patients undergoing chemotherapy [6]. Nevertheless, response rates to 5-Fu based chemotherapy are relatively low (10–15 %) in advanced colorectal cancer [6], and although they improve in combined chemotherapies (40–50 % [7]) there is still a need to overcome its main drawbacks such as tumours developing resistance, lack of selectivity, toxicity and effectiveness (more than 80 % of the administered 5-Fu catabolizes in the liver limiting its bioavailability in normal and tumour cells).

The main research paths that have been taken to improve the performance of 5-Fu are polychemeotherapy [8], identification of the pathways that are involved in tumour cell response to 5-Fu [9], development of 5-Fu derivatives [10], development of enhanced drug delivery systems [11] and enhancement of bioavailability and physicochemical properties of 5-Fu through cocrystalization [[12], [13], [14], [15], [16], [17], [18], [19], [20]]. The latter path is the one chosen in this work.

Supercritical fluid technology has been mainly employed in the 5-Fu micronization and the development of novel, enhanced delivery systems for 5-Fu. The techniques and achievements that have been accomplished in this field with the aid of supercritical fluids are outlined next.

Supercritical CO₂ has been used by several authors to impregnate 5-Fu to polymers. First reported studies are those of Guney and Akgerman [21]. These authors achieved a controlled release of the drug by supercritical impregnation of 5-Fu in poly(Lactide-coGlycolide) (PLGA). Cabezas et al. [22,23] used supercritical CO₂ to foam a polymeric support of poly(D,L-lactide) and PLGA, and simultaneously impregnate it with 5-Fu. A similar technique was used by Salerno et al. [24,25] to impregnate polycaprolactone achieving a 5-Fu progressive release over six days. Hao et al. [26] used supercritical carbon dioxide to entrap 5-Fu into a mesocellular silica foam modified with thermal and pH functional groups.

Many authors have used CO₂ as an antisolvent to produce enhanced 5-Fu formulations. The solution enhanced dispersion by supercritical fluids (SEDS) technique was employed by Chen et al. [27] to prepare microparticles of 5-Fu and poly (L-lactide) (PLLA). These authors also used the SEDS technology to prepare 5-Fu-SiO₂-PLLA microcapsules [28]. A “reverse emulsion-SEDS” technique was employed by Zhang et al. [29] to prepare nanoparticles of 5-Fu-PLLA-polyethylene glycol. The gas antisolvent (GAS) and supercritical antisolvent (SAS) techniques have also been explored. Kalantarian et al. [30] produced partly spherical 5-Fu nanoparticles using the SAS technique. The same authors used the GAS and SAS techniques to produce coprecipitates of 5-Fu and PLGA [31] accomplishing long term release profiles with partial burst effect. The SAS technique combined with the supercritical impregnation was used by Zhan et al. [32] to generate 5-Fu loaded microparticles of PLLA. The GAS technique was used by Esfandiari and Ghoreishi [33,34] to micronize 5-Fu. Our research group has also used the SAS technique to micronize 5-Fu and to prepare a 5-Fu-PLLA composite [11]. The produced composite presented a controlled drug delivery with a much slower release rate compared to the pure drug.

Although several supercritical techniques have been employed in the production of enhanced 5-Fu formulations, none of them has been used to prepare 5-Fu co-crystals. Cocrystals generate great interest in the pharmaceutical industry as they present a path to improve the overall bioavailability and performance of an active pharmaceutical ingredient (API) [35]. A pharmaceutical co-crystal is formed by an API and a suitable coformer or another API. Both co-crystal components form a supramolecular crystal structure through a non-covalent bond. By choosing the right coformer the properties of the API may be tuned improving the drug dissolution rate, its thermal stability, hygroscopicity, mechanical and even organoleptic properties [36]. Several authors have obtained different co-crystals of 5-Fu by conventional methods [[12], [13], [14], [15], [16], [17], [18], [19], [20],37]

The use of conventional methods in the co-crystal preparation presents several drawbacks such as scaling-up difficulties or the presence of homocrystals in the final product and often require post purification steps to eliminate solvents. The SAS precipitation has been shown to overcome the difficulties associated to these methods [[38], [39], [40]]. In this study, the feasibility of the SAS technique in producing co-crystals of 5-Fu with the coformers urea, thiourea, or PZA was investigated. The production of these co-crystals was also attempted through cocrystallization with supercritical solvent (CSS) in order to compare both supercritical techniques. In the SAS technique CO₂ plays the role of the antisolvent while in the CSS technique CO₂ plays the role of the solvent. Padrela et al. [41] and Ribas et al. [42] have successfully prepared co-crystals via CSS. The molecular structures of the polymorphs of 5-Fu [1,2], urea [43], thiourea [44] and PZA [45] involved in this study and those of the 5-Fu-urea [18] and 5-Fu-thiourea [18] co-crystals are given in Fig. 1.

Amides such as PZA are often used as coformers of pharmaceutical co-crystals [46]. PZA is an antituberculosis drug that has been used as a coformer with the anti-inflammatory drug diflunisal [47]. The 5-Fu-PZA co-crystal is a combination drug with great potential in a dual therapy. Urea and thiourea are also considered good coformers as they can easily form molecular complexes in the solid state. The NH₂ hydrogen atoms act as donors in hydrogen bonding while the lone pairs on the carbonyl oxygen atom act as acceptors. Furthermore, the pharmacological activity of urea and thiourea derivatives as antimicrobial and anticancer drugs and the anticancer potential of 5-Fu-urea and 5-Fu-thiourea co-crystals have been explored [18,48].