Structural Characterization of Co-Crystals of Chlordiazepoxide

pharmaceutics

Article Structural Characterization of Co-Crystals of Chlordiazepoxide with p-Aminobenzoic Acid and Lorazepam with Nicotinamide by DSC, X-ray Diﬀraction, FTIR and Raman Spectroscopy

Patrycja Garbacz 1 , Dominik Paukszta 2 , Artur Sikorski 3 and Marek Wesolowski 1,*

1 Department of Analytical Chemistry, Faculty of Pharmacy, Medical University of Gdansk, Gen. J. Hallera 107, 80-416 Gdansk, Poland; [email protected] 2 Institute of Chemical Technology and Engineering, Poznan University of Technology, Berdychowo 4, 60-695 Poznan, Poland; [email protected] 3 Department of Physical Chemistry, Faculty of Chemistry, University of Gdansk, Wita Stwosza 63, 80-308 Gdansk, Poland; [email protected] * Correspondence: [email protected]; Tel.: +48-58-349-10-96

Received: 18 June 2020; Accepted: 7 July 2020; Published: 9 July 2020

Abstract: The low water solubility of benzodiazepines seriously affects their bioavailability and, in consequence, their biological activity. Since co-crystallization has been found to be a promising way to modify undesirable properties in active pharmaceutical ingredients, the objective of this study was to prepare co-crystals of two benzodiazepines, chlordiazepoxide and lorazepam. Using different co-crystallization procedures, slurry evaporation and liquid-assisted grinding, co-crystals of chlordiazepoxide with p-aminobenzoic acid and lorazepam with nicotinamide were prepared for the first time. Confirmation that co-crystals were obtained was achieved through a comparison of the data acquired for both co-crystals using differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), Fourier-transform infrared (FTIR) and Raman spectroscopy, with comparisons acquired for the physical mixtures of both benzodiazepines and coformers. The compatibility of PXRD patterns of both benzodiazepines co-crystals with those contained in the base Powder Diffraction File (PDF-4+) suggests that new crystal structures were indeed created under the co-crystallization procedure. Single-crystal X-ray diffraction revealed that a chlordiazepoxide co-crystal with p-aminobenzoic acid and a lorazepam co-crystal with nicotinamide crystallized in the monoclinic P21/n and P21/c space group, respectively, with one molecule of benzodiazepine and one of coformer in the asymmetric unit. FTIR and Raman spectroscopy corroborated that benzodiazepine and coformer are linked by a hydrogen bond without proton exchange. Furthermore, a DSC study revealed that single endothermic DSC peaks assigned to the melting of co-crystals differ slightly depending on the co-crystallization procedures and solvent used, as well as differing from those of starting components.

Keywords: chlordiazepoxide; lorazepam; co-crystals; differential scanning calorimetry (DSC); powder X-ray diffraction (PXRD); single-crystal x-ray diffraction (SCXRD); Fourier-transform infrared (FTIR); Raman spectroscopy

1. Introduction The co-crystallization of active pharmaceutical ingredients (APIs) with coformers oﬀers a unique way to improve key physicochemical properties without changing the chemical nature and pharmacological activity of APIs [1,2]. Solubility, dissolution rate, bioavailability [3–8], melting point [9], stability [10–12], tabletability [13], taste [6], and other properties can undergo change during

Pharmaceutics 2020, 12, 648; doi:10.3390/pharmaceutics12070648 www.mdpi.com/journal/pharmaceutics Pharmaceutics 2020, 12, 648 2 of 17 co-crystallization. Orally disintegrating tablets consisting of gabapentin co-crystals (antiepileptic and analgesic agent) with saccharin (sweetener) have shown a number of benefits in comparison to the same formulation with a physical mixture of starting components [6]. Gabapentin co-crystals displayed improved solubility, particle size distribution and bioavailability. Co-crystals can also enhance patients’ compliance due to enhanced formulation taste. Increased solubility and the rapid release of API was also observed for atorvastatin calcium co-crystals with nicotinamide and citric acid [14]. It has recently been proven that co-crystallization of two APIs can improve the clinical efficiency and safety of treatment [15]. The co-crystallization of tramadol with celecoxib changed the pharmacokinetic profiles of both APIs, lowered the dose of tramadol (improved the safety of treatment) and accelerated the onset of celecoxib-mediated analgesia. These co-crystals are currently undergoing a third phase of clinical trials. Some co-crystals are already employed in medical practice [1,15,16]. The US Food and Drug Administration (FDA) has approved sacubitril co-crystals with valsartan (EntrestoTM), a multidrug used to lower the risk of heart failure, ertugliflozin co-crystals with L-pyroglutamic acid (SteglatroTM) for the treatment of type 2 diabetes, and aripiprazole co-crystals with fumaric acid (Abilify®) for the treatment of schizophrenia [1]. Ipragliflozin co-crystals with L-proline in the treatment of type 2 diabetes were recently approved in Japan [15]. Escitalopram (antidepressant), a salt now considered as a co-crystal/salt system consisting of two escitalopram anions, oxalate dianion, neutral oxalic acid and water in one crystal unit (Lexapro®) was approved by the FDA in 2009 [15,17]. Valproic acid, approved by the FDA for epilepsy treatment (Depakote®), constitutes a special case of co-crystals. A liquid at ambient temperature, it is co-crystalized with solid sodium valproate, which increases its stability [15]. Furthermore, numerous patents on co-crystals have been registered and some co-crystals are currently the subject of clinical trials [1]. Co-crystals can be prepared using solid-state and solution-based methods [18]. Among the solid-state methods, neat-grinding (without the addition of solvent) and liquid-assisted grinding (with the addition of small amounts of solvent) can be specified. Grinding can be performed using a mortar or ball mill. A small amount of solvent is added to accelerate co-crystallization. These methods are environmentally benign, relatively short in duration and temperature invariable [19]. Solution-based methods, in turn, consist of crystallization due to evaporation, slurry formation or cooling. The methods differ in the amount of solvent used and evaporation rate. Co-crystallization by evaporation is a commonly used method for co-crystal preparation, especially to obtain a single crystal for co-crystal structure identification [18]. In this method, API and coformer are completely dissolved in solvent and the solution is left to evaporate slowly at ambient temperature. Slurry co-crystallization requires smaller quantities of solvent since the substances are not fully dissolved. The evaporation rate is controlled by the cooling method only. Recently, advanced methods for co-crystal preparation have been developed, for example, using hot melt extrusion [20], supercritical fluid [21,22], or ultrasound [23]. Since co-crystallization usually improves the physicochemical properties of APIs, the most challenging issue to overcome is looking for new co-crystals of those APIs which have been used in medical practice for a long time and whose properties still need to be improved. The 1,4-benzodiazepine derivatives such as chlordiazepoxide and lorazepam may serve as an example. Both APIs have anxiolytic, anticonvulsant, muscle relaxant and hypnotic action [24–27]. Chlordiazepoxide is applied to alleviate alcohol withdrawal, lorazepam in turn, is useful for treatment of status epilepticus, insomnia, anxiety and pre-anesthetic medication. To date, many studies have been conducted to improve the solubility and stability of these APIs. For example, lorazepam was incorporated into a cyclodextrin cavity [24], included in poly D, L-lactide-co-glycolic acid nanoparticles [28] or coated with vinyl polymer [29]. For this reason, the aim of this study was to examine newly obtained benzodiazepine co-crystals, chlordiazepoxide with p-aminobenzoic acid and lorazepam with nicotinamide, prepared for the first time using two different co-crystallization methods, liquid-assisted grinding and slurry evaporation from solvents. The chemical structures of benzodiazepines and coformers used in this study are shown Pharmaceutics 2020, 12, 648 3 of 17

Pharmaceutics 2020, 12 3 of 18 in Figure1. p-Aminobenzoic acid is included on the list of substances called Everything Added to Foods in the United States (EAFUS), while nicotinamide belongs to the substances Generally Recognized as Everything Added to Foods in the United States (EAFUS), while nicotinamide belongs to the Safe (GRAS) [21,30]. p-Aminobenzoic acid and nicotinamide belong to vitamin B group. To the best of substances Generally Recognized as Safe (GRAS) [21,30]. p-Aminobenzoic acid and nicotinamide our knowledge, there are no data reporting on pharmacological interactions between the coformers belong to vitamin B group. To the best of our knowledge, there are no data reporting on and benzodiazepines used in this study. pharmacological interactions between the coformers and benzodiazepines used in this study. The thermal properties of co-crystals, their crystal structures and spectroscopic characteristics were The thermal properties of co-crystals, their crystal structures and spectroscopic characteristics investigated using differential scanning calorimetry (DSC), powder and single-crystal X-ray diffraction were investigated using differential scanning calorimetry (DSC), powder and single-crystal X-ray (PXRD and SCXRD), Fourier-transform infrared (FTIR) and Raman spectroscopy. A comparison of diffraction (PXRD and SCXRD), Fourier-transform infrared (FTIR) and Raman spectroscopy. A the data acquired for both co-crystals using DSC, PXRD, SCXRD, FTIR and Raman techniques with comparison of the data acquired for both co-crystals using DSC, PXRD, SCXRD, FTIR and Raman those obtained for physical mixtures of chlordiazepoxide with p-aminobenzoic acid and lorazepam techniques with those obtained for physical mixtures of chlordiazepoxide with p-aminobenzoic acid with nicotinamide permits the confirmation of co-crystal formation and an assessment of their and lorazepam with nicotinamide permits the confirmation of co-crystal formation and an basic properties. assessment of their basic properties.

Figure 1. Chemical structures of: (a) chlordiazepoxide, (b) p-aminobenzoic acid, (c) lorazepam, Figure 1. Chemical structures of: (a) chlordiazepoxide, (b) p-aminobenzoic acid, (c) lorazepam, and and (d) nicotinamide. (d) nicotinamide. 2. Experimental 2. Experimental 2.1. Materials 2.1.Chlordiazepoxide Materials and lorazepam were provided by Polfa Tarchomin (Warsaw, Poland). p-AminobenzoicChlordiazepoxide acid and and nicotinamide lorazepam werewere bought provided from by Sigma Polfa AldrichTarchomin (St. Louis,(Warsaw, MO, Poland). USA). Sincep-Aminobenzoic the purity of materialsacid and was nicotinamide above 99%, were both wereboug usedht from without Sigma further Aldrich puriﬁcation. (St. Louis, Methanol MO, USA). and ethylSince acetate the purity (pure of for materials analysis) was were above obtained 99%, from both POCHwere used (Gliwice, without Poland) further and purification. acetonitrile fromMethanol J. T. Bakerand ethyl (Phillipsburg, acetate (pure NJ, USA). for analysis) were obtained from POCH (Gliwice, Poland) and acetonitrile from J. T. Baker (Phillipsburg, NJ, USA). Sample Preparation SampleBinary Preparation physical mixtures of chlordiazepoxide with p-aminobenzoic acid and lorazepam with nicotinamideBinary werephysical prepared mixtures at 1:1 of molar chlordiazepoxide ratios. Components with p accurately-aminobenzoic weighed acidusing and lorazepam Mettler Toledo with XA105nicotinamide Dual Range were balance prepared (Schwerzenbach, at 1:1 molar Switzerland)ratios. Components were transferred accurately into weighed micro tubes using (Sarstedt, Mettler Nümbrecht,Toledo XA105 Germany) Dual Range and mixed balance using (Schwerzenbach, a laboratory stirrer Switzerland) (Kamush, were Gdansk, transferred Poland) into for micro 15 min tubes at 20(Sarstedt, rpm. Nümbrecht, Germany) and mixed using a laboratory stirrer (Kamush, Gdansk, Poland) for 15 minBenzodiazepines at 20 rpm. co-crystals were prepared by two co-crystallization methods, slurry evaporation and liquid-assistedBenzodiazepines grinding co-crystals using binary were physical prepared mixtures by two as starting co-crystallization materials. methods, slurry evaporationTo obtain and co-crystals liquid-assisted by slurry grinding evaporation, using binary a 100 µphysicalL of solvent, mixtures acetonitrile as starting and materials. ethyl acetate, and methanolTo obtain and co-crystals ethyl acetate, by slurry was addedevaporation, into a mixture a 100 µL of of chlordiazepoxide solvent, acetonitrile with andp-aminobenzoic ethyl acetate, acidand and methanol lorazepam and withethyl nicotinamide.acetate, was added The samples into a mixture were mixed of chlordiazepoxide for 30 min using with a laboratory p-aminobenzoic stirrer acid and lorazepam with nicotinamide. The samples were mixed for 30 min using a laboratory stirrer at 20 rpm and left in sealed micro tubes for 24 h. After this time the micro tubes were unsealed and left for free evaporation.

Pharmaceutics 2020, 12, 648 4 of 17 at 20 rpm and left in sealed micro tubes for 24 h. After this time the micro tubes were unsealed and left for free evaporation. To obtain co-crystals by liquid-assisted grinding co-crystallization, benzodiazepine mixtures were ground in micro tubes with two agate grinding balls 5 mm in diameter (Eqiumed, Cracow, Poland) using a laboratory stirrer for 30 min at 20 rpm. Before grinding, a small amount of a solvent (ﬁve drops) was added. Acetonitrile and ethyl acetate were used for mixture of chlordiazepoxide and p-minobenzoic acid, methanol and ethyl acetate for lorazepam mixture with nicotinamide. The ground samples were unsealed and left for evaporation.

2.2. Methods

2.2.1. Differential Scanning Calorimetry (DSC) The DSC curves of the samples examined were acquired by a heat-flux Mettler Toledo DSC device, model 822e (Schwerzenbach, Switzerland), coupled with STARe software, ver. 15.00. The experiments were run in dynamic nitrogen atmosphere at a flux rate 70 mL/min and at 5 ◦C/min heating rate over a range of 25–300 ◦C. About 4 mg of samples were weighed in flat-bottomed pans, which then were closed with perforated lids.

2.2.2. Powder X-ray Diffraction (PXRD) Powdered samples were measured using modernized and computer controlled horizontal TUR M-62 device (VEB TUR, Dresden, Germany). A PXRD technique with CuKα radiation at 1.5418 Å was used to collect X-ray diffraction patterns. Instrumental parameters were as follows: 2θ angle range 5–50◦, counting time 3 s per step, counting step (2θ) 0.04◦. Measurements were performed with nickel filtering. The diffraction patterns of starting components, physical mixtures and co-crystals were obtained using standard software. Identification of the compounds tested was carried out using the XRAYAN program (software for material identification by PXRD technique) [31]. This program is connected to the database Powder Diffraction File (PDF-4+) ICDD (International Centre for Diffraction Data, Newtown Square, PA, USA).

2.2.3. Single-Crystal X-ray Diffraction (SCXRD) Single crystals for SCXRD investigations were prepared by the solvent evaporation co-crystallization method using physical mixtures of benzodiazepines with coformers at a 1:1 molar ratio. Accurately weighed components were placed in a beaker consisting of 10–20 mL of solvent (acetonitrile, methanol or ethyl acetate) and mixed for 15 min using a magnetic stirrer. Afterwards the solution was filtered through paper filters (Bionovo, Legnica, Poland) and left for slow evaporation of solvent. Crystals suitable for SCXRD study were obtained using acetonitrile for chlordiazepoxide co-crystals with p-aminobenzoic acid, and acetonitrile and ethyl acetate for lorazepam co-crystals with nicotinamide. The SCXRD experiments for single-crystals of chlordiazepoxide co-crystals with p-aminobenzoic acid and lorazepam co-crystals with nicotinamide were recorded using Oxford Diffraction Gemini R ULTRA Ruby CCD diffractometer (T = 295(2) K and λMo = 0.71073 Å) (Table1). Software packages used were as follows: data collections—CrysAlis CCD [32]; cell refinement, data reduction and multi-scan absorption corrections—CrysAlis RED [32]; crystal structure solving and refining—SHELX [32,33]; calculations of intermolecular interactions—PLATON [34]; preparing of molecular graphics—ORTEPII [35], PLUTO-78 [36] and Mercury [37]. The chlorobenzene ring (C14–C19 and Cl20 atoms) in lorazepam co-crystals with nicotinamide has orientation disorders with refined site-occupancy factors of the disordered parts 0.897 and 0.103 (the disordered benzene rings were refined as rigid ideal hexagons with C–C = 1.39 Å). The C-bound hydrogen atoms were refined isotropically with d(C–H) = 0.93–0.96 Å and Uiso (H) = 1.2 Ueq (C), whereas O/N-bound hydrogen atoms were refined isotropically with Uiso (H) = 1.5 Ueq (O/N). Pharmaceutics 2020, 12, 648 5 of 17

Full crystallographic details of title compound have been deposited in the Cambridge Crystallographic Data Center (deposition No. CCDC 2007857 and CCDC 2007858).

Table 1. Crystal data and structure reﬁnement parameters for chlordiazepoxide co-crystals with p-aminobenzoic acid and lorazepam co-crystals with nicotinamide.

Chlordiazepoxide Co-Crystals Lorazepam Co-Crystals with Compound with p-Aminobenzoic Acid Nicotinamide

Chemical formula C24H21N4O3Cl C21H16N4O3Cl2 FW/g mol 1 436.89 443.28 · − T/K 295(2) 295(2) λMo/Å 0.71073 0.71073 Crystal system Monoclinic monoclinic Space group P21/n P21/c a/Å 7.5291(4) 14.8848(8) b/Å 20.8841(9) 11.3696(5) c/Å 13.6102(5) 12.6114(6) α/◦ 90 90 β/◦ 99.061(4) 109.380(6) γ/◦ 90 90 V/Å3 2113.3(2) 2013.4(2) Z 4 4 3 ρcalc/g cm− 1.373 1.462 · 1 µ/mm− 0.214 0.354 F(000) 912 912 θ range/◦ 3.47–25.00 3.58–25.00 Completeness θ/% 99.7 99.9 Reflections collected 14735 13021 Reflections unique 3704 [Rint = 0.0246] 3532 [Rint = 0.0229] Data/restraints/parameters 3704/0/293 3532/12/312 Goodness of fit on F2 1.108 1.101 Final R1 value (I > 2σ(I)) 0.0441 0.0445 Final wR2 value (I > 2σ(I)) 0.0984 0.0955 Final R1 value (all data) 0.0507 0.0516 Final wR2 value (all data) 0.1013 0.0991 CCDC number 2007857 2007858

2.2.4. Fourier-Transform Infrared (FTIR) FTIR analyses of the co-crystals under study were conducted using a Thermo Fischer Scientiﬁc FTIR spectrometer, model Nicolet 380 (Madison, WI, USA) coupled with a DTGS KBr detector and OMNIC software. A hydraulic press (Specac, Orpington, UK) was used to prepare samples containing 1 mg of sample and 100 mg of KBr (Merck, Darmstadt, Germany). FTIR spectra were collected over a 1 1 spectral range of 4000–400 cm− , with 4 cm− resolution at ambient temperature. The background spectrum was checked prior to each measurement.

2.2.5. Raman Spectroscopy A Thermo Fisher Scientiﬁc DXR Smart Raman spectrometer (Madison, WI, USA) was used to record Raman spectra. The device was equipped with OMNIC software, a 15-mW DXR 780 nm laser with a slit width of 25 µm, CCD detector and Raleigh ﬁlter. The Raman spectra were collected over a 1 1 range of 3413–99 cm− with a resolution of 2 cm− . Exposure time was 1 s (twice).

3. Results and Discussion The aim of this study was to prepare and examine newly obtained benzodiazepine co-crystals, viz. chlordiazepoxide with p-aminobenzoic acid and lorazepam with nicotinamide, prepared by different methods (slurry evaporation and liquid-assisted grinding) with selected solvents. The thermal Pharmaceutics 2020, 12, 648 6 of 17 profile and crystal structure of the co-crystals in question were studied using DSC, PXRD and SCXRD techniques, while the hydrogen bonding formation between APIs and coformers was assessed using spectroscopic techniques, i.e., FTIR and Raman. Co-crystal formation was fully verified by comparing their thermal, diffractometric and spectroscopic data with those for physical mixtures and starting components.

3.1. DSC The DSC curves for chlordiazepoxide co-crystals with p-aminobenzoic acid prepared by different methods and solvents revealed a single endothermic peak followed by an exothermic one (Table2). The endothermic effect is associated with the melting of co-crystal and differs slightly, depending on the co-crystallization procedure and solvent used. For the slurry evaporation method, the peaks were at 219.6 ◦C (Figure2a) and 221.7 ◦C for the sample prepared using acetonitrile and ethyl acetate, respectively. The liquid-assisted grinding procedure, in turn, leads to peaks at slightly higher temperatures, i.e., 220.4 ◦C for acetonitrile and 223.1 ◦C for ethyl acetate. This indicates that the melting point of co-crystals differs from those of starting components, 242.3 ◦C for chlordiazepoxide and 187.2 ◦C for p-aminobenzoic acid. A mixture of the two components heated at higher rate (10 ◦C/min) displays an endothermic peak at 180.1 ◦C due to eutectic melting followed by a co-crystal recrystallization at 185.1 ◦C (Figure2b). An exothermic e ffect immediately after the endothermic is characteristic of physical mixtures whose components co-crystallize under heating to form co-crystals [38,39]. Thus, the DSC method confirms chlordiazepoxide co-crystallization with p-aminobenzoic acid. It is noteworthy that melted co-crystals undergo decomposition, as proved by the second, exothermic, DSC peak. Thermogravimetric examination confirms that chlordiazepoxide decomposes immediately after melting with ~60% mass loss at ~300 ◦C (data not published).

Table 2. Thermal data acquired from diﬀerential scanning calorimetry (DSC) curves for benzodiazepines co-crystals prepared using diﬀerent co-crystallization methods and solvents, and for physical mixtures of benzodiazepines and coformers.

Co-Crystals Physical Mixtures Components Slurry Evaporation Liquid-Assisted Grinding

Ton (Tp) ◦C ∆H (J/g) Ton (Tp) ◦C ∆H (J/g) Ton (Tp) ◦C ∆H (J/g) Acetonitrile 217.9 (219.6) 56.19 endo 207.0 (220.4) 61.83 endo 180.1 (182.0) 13.52 endo Chlordiazepoxide and 220.7 (223.3) 258.9 exo 222.4 (224.2) 300.2 exo 183.6 (185.1) 9.59 exo p-aminobenzoic acid ethyl acetate 219.5 (223.4) 64.74 endo 225.3 (231.1) 304.5 exo 220.5 (221.7) 60.89 endo 221.9 (223.1) 94.91 endo 222.4 (225.4) 291.4 exo 224.8 (227.1) 338.5 exo Methanol 176.7 (177.5) 260.3 endo 174.9 (176.5) 257.4 endo 126.0 (127.3) 187.3 (204.0) 61.09 exo 187.8 (203.7) 47.91 exo 28.51 endo Lorazepam and 129.3 (130.1) 10.48 exo nicotinamide ethyl acetate 168.2 (171.5) 198.8 endo 126.4 (127.2) 0.44 endo 183.3 (197.3) 32.80 exo 177.3 (178.9) 205.8 endo 175.3 (176.7) 266.5 endo 190.5 (206.3) 29.26 exo 187.6 (204.2) 52.94 exo

Melting of lorazepam co-crystals with nicotinamide also depends on the preparation method and solvent used but regardless of co-crystallization procedure, DSC curves indicated endothermic peaks above 177 ◦C (Table2). Co-crystals prepared by slurry evaporation revealed higher melting points, at 177.5 ◦C and 178.9 ◦C for co-crystallization from methanol and ethyl acetate, respectively. The co-crystals obtained by liquid-assisted grinding melted at ~176 ◦C (Figure2c). An additional, slight endothermic DSC peak at 127.2 ◦C was found for co-crystals prepared by this procedure with Pharmaceutics 2020, 12, 648 7 of 17

ethyl acetate as a solvent. This is probably attributable to the melting of a slight quantity of coformer (Tp for nicotinamide = 128.3 ◦C). The melting point of lorazepam co-crystals coincides with that of lorazepam (Tp = 177.0 ◦C). The DSC curve of mixture for both components indicated three peaks (Figure2d), the first was endothermic at 127.3 ◦C, followed by an exothermic one at 130.1 ◦C. The last, an endothermic effect, can be assigned to the melting of co-crystals at 171.5 ◦C. Like chlordiazepoxide co-crystals, an exothermic effect immediately after the endothermic one confirms co-crystallization, whereas melted lorazepam co-crystals undergo decomposition, as proved by the last exothermic Pharmaceutics 2020, 12 7 of 18 DSC peak.

Figure 2. DSC curves of: (a) chlordiazepoxide co-crystals with p-aminobenzoic acid prepared by slurry Figure 2. DSC curves of: (a) chlordiazepoxide co-crystals with p-aminobenzoic acid prepared by evaporation method with ethyl acetate as a solvent, (b) physical mixture of chlordiazepoxide with slurry evaporation method with ethyl acetate as a solvent, (b) physical mixture of chlordiazepoxide p-aminobenzoic acid, (c) lorazepam co-crystals with nicotinamide prepared by liquid-assisted grinding with p-aminobenzoic acid, (c) lorazepam co-crystals with nicotinamide prepared by liquid-assisted method using methanol as a solvent, and (d) physical mixture of lorazepam with nicotinamide. grinding method using methanol as a solvent, and (d) physical mixture of lorazepam with 3.2. PXRDnicotinamide.

3.2. PXRDPXRD patterns confirmed that both the benzodiazepines and coformers used in this study were in crystalline form. The compliance of the diffraction pattern for chlordiazepoxide mixture with p-aminobenzoicPXRD patterns acid confirmed into those containedthat both the in the benzodia powderzepines base corroborated and coformers that used only crystallinein this study forms were of inbenzodiazepine crystalline form. and The coformer compliance can befound of the in diffraction the mixture. pattern Hence, for the chlordiazepoxide diffraction pattern mixture of the mixture with pis-aminobenzoic the sum of the acid diff ractioninto those maxima contained for chlordiazepoxide in the powder andbasep-aminobenzoic corroborated that acid, only which crystalline suggests formsthat there of benzodiazepine was no interaction and coformer between ingredientscan be found after in the simple mixture. mixing Hence, by 15 the min diffraction using a laboratorypattern of thestirrer mixture at 20 rpmis the (Figure sum of3a). the The diffra mixturection maxima displayed for sharp chlordiazepoxide di ffraction peaks and atp-aminobenzoic 2θ of 11.45◦, 13.88 acid,◦ , which14.77◦ ,suggests 15.33◦, 17.22 that◦ andthere 21.90 was◦. no A comparison interaction ofbetween these data ingredients with those after for chlordiazepoxide simple mixing by co-crystals 15 min usingwith pa-aminobenzoic laboratory stirrer acid at prepared20 rpm (Figure by slurry 3a). evaporationThe mixture methoddisplayed (Figure sharp3 diffractionb) and liquid-assisted peaks at 2θ ofgrinding 11.45°, method13.88°, (Figure14.77°, 3c)15.33°, revealed 17.22° that and di ff raction21.90°. patternsA comparison of co-crystals of these and data the physicalwith those mixture for chlordiazepoxideare disparate. The co-crystals most intensive with newp-aminobenzoic diffraction peaks acid wereprepared observed by slurry in the evaporation pattern of co-crystals method (Figure 3b) and liquid-assisted grinding method (Figure 3c) revealed that diffraction patterns of prepared by liquid-assisted grinding method at 10.69◦, 12.60◦, 14.28◦, 16.88◦, 17.40◦, 17.92◦, 19.27◦, co-crystals and the physical mixture are disparate. The most intensive new diffraction peaks were 21.18◦, 23.04◦, 24.19◦, 25.37◦, 27.16◦ and 29.34◦. Moreover, intensive diffraction peaks characteristic of observed in the pattern of co-crystals prepared by liquid-assisted grinding method at 10.69°, 12.60°, the mixture at 11.45◦, 15.33◦, 17.22◦ and 21.90◦ disappeared or reduced significantly in intensity in the 14.28°,co-crystals’ 16.88°, di ff17.40°,raction pattern.17.92°, 19.27°, The implication 21.18°, 23.0 is that4°, 24.19°, a new crystal25.37°, phase27.16° was and created. 29.34°. Moreover, intensiveThe compliancediffraction ofpeaks the di characteristicffraction pattern of forthe chlordiazepoxide mixture at 11.45°, co-crystals 15.33°, with 17.22°p-aminobenzoic and 21.90° disappearedacid prepared or by reduced slurry evaporation significantly method in intensity with those in contained the co-crystals’ in the base diffraction PDF-4+ didpattern. not display The implicationdiffraction peaksis that characteristic a new crystal of phase benzodiazepine was created. and coformer (Figure S1, Supplementary Materials). SinceThe the compliance co-crystals’ diofff theraction diffraction pattern pattern did not for match chlordiazepoxide those for components co-crystals alone, with it may p-aminobenzoic be concluded acidthat aprepared new crystal by structureslurry evaporation is created inmethod the co-crystallization with those contained process. in the base PDF-4+ did not display diffraction peaks characteristic of benzodiazepine and coformer (Figure S1, Supplementary Materials). Since the co-crystals’ diffraction pattern did not match those for components alone, it may be concluded that a new crystal structure is created in the co-crystallization process.

PharmaceuticsPharmaceutics2020 2020,,12 12, 648 88 ofof 1718 Pharmaceutics 2020, 12 8 of 18

Figure 3. Diffraction patterns of: (a) chlordiazepoxide mixture with p-aminobenzoic acid prepared at Figurea 1:1 molar 3. DiffractionDi ffratioraction by patternsa patterns laboratory of: of: ( a (stirrer,)a )chlordiazepoxide chlordiazepoxide (b) chlordiazepoxide mixture mixture with co-crystals with p-aminobenzoicp-aminobenzoic with p-aminobenzoic acid acid prepared prepared acid at ataprepared 1:1 a 1:1 molar molar by ratio slurry ratio by by evaporationa alaboratory laboratory method,stirrer, stirrer, ( b (andb) )chlordiazepoxide chlordiazepoxide (c) chlordiazepoxide co-crystals co-crystals co-crystals with with withp-aminobenzoic p-aminobenzoic acid preparedacid prepared byby slurryslurry by liquid-a evaporation evaporationssisted method, method,grinding and andmethod. (c) ( chlordiazepoxidec) chlordiazepoxide co-crystals co-crystals with withp-aminobenzoic p-aminobenzoic acid preparedacid prepared by liquid-assisted by liquid-assisted grinding grinding method. method. The compliance of the diffraction pattern of the lorazepam mixture with nicotinamide with thoseThe contained compliance in the of base the PDF-4+ didiffractionffraction enabled pattern the recognizable of the lorazepamlorazepam identification mixture of with both nicotinamide ingredients in with the thosemixture contained based on in thetheir base intensive PDF-4PDF-4++ enableddiffraction the peaksrecognizable at 6.63°, identification identification 14.73°, 17.43°, of both 17.78°, ingredients 19.94°, 20.51°, in the mixture24.48°, 25.04°, basedbased on25.78°on their their and intensive intensive 27.26° di ff(Figurediffractionraction S2, peaks peaksSupplementary at 6.63 at ◦6.63°,, 14.73 ◦14.73°,Materials)., 17.43 ◦17.43°,, 17.78 On◦ ,17.78°, 19.94the other◦ ,19.94°, 20.51 hand,◦ ,20.51°, 24.48 the◦, 25.0424.48°,diffraction◦, 25.7825.04°, pattern◦ and 25.78° 27.26 of and lorazepam◦ (Figure 27.26° S2, (Figureco-crystals Supplementary S2, Supplementarywith nicotinamide Materials). Materials). On prepared the other On by hand,the the other theliquid-assisted dihand,ffraction the patterndiffractiongrinding of and lorazepampattern slurry of evaporation co-crystalslorazepam withmethodsco-crystals nicotinamide differ with significantly nicotinamide prepared from by theprepared that liquid-assisted of lorazepam by the liquid-assisted grindingmixture with and slurrygrindingnicotinamide evaporation and slurry(Figure methods evaporation 4). Co-crystals differ methods significantly prepared differ by fromsignificantly the thatslurry of lorazepammethod from that have mixtureof lorazepamnew diffraction with nicotinamidemixture peaks with at (Figurenicotinamide9.98°, 13.63°,4). Co-crystals (Figure14.07°, 15.56°,4). prepared Co-crystals 19.88°, by the23.81°, prepared slurry 24.75° methodby thande slurry26.47°, have newmethod indicative di ff haveraction of new a peaksnew diffraction crystalline at 9.98 ◦peaks, 13.63phase at◦, 14.079.98°,being◦ ,13.63°,created 15.56◦ ,14.07°, 19.88during◦ ,15.56°, 23.81the co-crystallization◦ ,19.88°, 24.75◦ 23.81°,and 26.47 24.75° process.◦, indicative and A 26.47°, comparison of aindicative new crystallineof co-crystalsof a new phase crystallineprepared being created byphase the duringbeingliquid-assisted created the co-crystallization during grinding the and co-crystallization slurry process. evaporation A comparison process. methods A of comparison co-crystals with those prepared ofcontained co-crystals by in the theprepared liquid-assisted bases showed by the grindingliquid-assistedthat the anddiffraction slurry grinding evaporation maxima and slurryof methodslorazepam evaporation with and those methods nicotinamide contained with in thosedid the basesnot contained overlap showed in with thatthe bases thethose di ffshowed ractionfor the maximathatco-crystals. the ofdiffraction lorazepam Thus, themaxima and PXRD nicotinamide of studylorazepam didconfirms notand overlap nithatcotinamide witha new those didcrystal for not the overlapphase co-crystals. is with obtained Thus, those the forduring PXRD the studyco-crystals.co-crystallization. confirms Thus, that the a new PXRD crystal study phase confirms is obtained that during a new co-crystallization. crystal phase is obtained during co-crystallization.

Figure 4. Diﬀraction patterns of: (a) lorazepam mixture with nicotinamide prepared at a 1:1 molar ratio Figure 4. Diffraction patterns of: (a) lorazepam mixture with nicotinamide prepared at a 1:1 molar by a laboratory stirrer, (b) lorazepam co-crystals with nicotinamide prepared by liquid-assisted grinding Figureratio by 4. a Diffractionlaboratory stirrer,patterns (b of:) lorazepam (a) lorazepam co-crystals mixture with with nicotinamide nicotinamide prepared prepared by liquid-assistedat a 1:1 molar method, and (c) lorazepam co-crystals with nicotinamide prepared by slurry evaporation method. ratiogrinding by a method,laboratory and stirrer, (c) lorazepam (b) lorazepam co-crystals co-crystals with withnicotinami nicotinamidede prepared prepared by slurry by liquid-assisted evaporation grindingmethod. method, and (c) lorazepam co-crystals with nicotinamide prepared by slurry evaporation method.

Pharmaceutics 2020, 12, 648 9 of 17 Pharmaceutics 2020, 12 9 of 18 Differences in the peak positions between patterns of co-crystals obtained by different methods Differences in the peak positions between patterns of co-crystals obtained by different methods were slight, proving that, irrespective of preparation methods and solvents used, the same co-crystal were slight, proving that, irrespective of preparation methods and solvents used, the same co-crystal structure can be obtained. Moreover, the same diffraction maxima at 2θ values for the lorazepam structure can be obtained. Moreover, the same diffraction maxima at 2θ values for the lorazepam mixture with nicotinamide and both components excludes co-crystallization after lorazepam is mixture with nicotinamide and both components excludes co-crystallization after lorazepam is mixed with nicotinamide. It follows that the simple mixing of both components did not lead to mixed with nicotinamide. It follows that the simple mixing of both components did not lead to co-crystal formation. co-crystal formation. 3.3. SCXRD 3.3. SCXRD SCXRD measurements show that the chlordiazepoxide co-crystal with p-aminobenzoic acid and SCXRD measurements show that the chlordiazepoxide co-crystal with p-aminobenzoic acid and the lorazepam co-crystal with nicotinamide crystallized in the monoclinic P21/n and P21/c space group, the lorazepam co-crystal with nicotinamide crystallized in the monoclinic P21/n and P21/c space respectively, with one molecule of benzodiazepine and one molecule of coformer in the asymmetric group, respectively, with one molecule of benzodiazepine and one molecule of coformer in the unit (Table1). In the crystal structure of chlordiazepoxide co-crystals with p-aminobenzoic acid, asymmetric unit (Table 1). In the crystal structure of chlordiazepoxide co-crystals with the geometric parameters, i.e., bond lengths and angles, are typical for those observed in crystals p-aminobenzoic acid, the geometric parameters, i.e., bond lengths and angles, are typical for those containing the chlordiazepoxide molecule [40,41]. In the crystal of chlordiazepoxide compounds observed in crystals containing the chlordiazepoxide molecule [40,41]. In the crystal of with p-aminobenzoic acid, the C–O bond lengths [1.217(2)–1.334(2) Å] in the COOH group of the chlordiazepoxide compounds with p-aminobenzoic acid, the C–O bond lengths [1.217(2)–1.334(2) Å] p-aminobenzoic acid molecule show that no proton transfer has occurred but that a co-crystal is formed, in the COOH group of the p-aminobenzoic acid molecule show that no proton transfer has occurred where the chlordiazepoxide and p-aminobenzoic acid molecules are linked via N12–H12 O30 and but that a co-crystal is formed, where the chlordiazepoxide and p-aminobenzoic acid molecules··· are O29–H29 O14 hydrogen bonds to form heterodimer (Table3, Figure5). The neighboring heterodimers linked via··· N12–H12···O30 and O29–H29···O14 hydrogen bonds to form heterodimer (Table 3, Figure are connected through a N31–H31A N1 hydrogen bond to produce chains along the [0 0 1] direction 5). The neighboring heterodimers··· are connected through a N31–H31A···N1 hydrogen bond to (Table3, Figure6a). Adjacent chains are connected by a N31–H31B O14 hydrogen bond to create produce chains along the [0 0 1] direction (Table 3, Figure 6a). Adjacent··· chains are connected by a layers along the a-axis (Table3, Figure6b). These layers are, in turn, linked via a weak C20–H20 Cl21 N31–H31B···O14 hydrogen bond to create layers along the a-axis (Table 3, Figure 6b). These ···layers hydrogen bond to form a 3D framework. are, in turn, linked via a weak C20–H20···Cl21 hydrogen bond to form a 3D framework. Table 3. Hydrogen bonding interactions in the crystal structure of chlordiazepoxide co-crystals with Table 3. Hydrogen bonding interactions in the crystal structure of chlordiazepoxide co-crystals with p-aminobenzoic acid. p-aminobenzoic acid.

D–H A d(D–H) (Å) d(H A) (Å) d(D A) (Å)

FigureFigure 5.5. Molecular structure of chlordiazepoxide co-crystals with pp-aminobenzoic-aminobenzoic acid,acid, showingshowing thethe atom-labelingatom-labeling schemescheme (displacement(displacement ellipsoidsellipsoids areare drawndrawn atat aa 25%25% probabilityprobability levellevel andand HH atomsatoms areare shownshown asas smallsmall spheresspheres ofof arbitraryarbitrary radius;radius; hydrogenhydrogen bondsbonds areare representedrepresented by by dashed dashed lines). lines).

Pharmaceutics 2020, 12, 648 10 of 17 Pharmaceutics 2020, 12 10 of 18

Figure 6.6.( a()a Chains) Chains formed formed by chlordiazepoxideby chlordiazepoxide and p -aminobenzoicand p-aminobenzoic acid molecules acid molecules in the co-crystals. in the (co-crystals.b) Crystal packing(b) Crystal of chlordiazepoxide packing of chlordiazepoxide co-crystals with co-crystalsp-aminobenzoic with p-aminobenzoic acid viewed along acid the vieweda-axis (hydrogenalong the a-axis bonds (hydrogen are represented bonds byare dashedrepresented lines; by symmetry dashed lines; codes: symmetry (i) x, y, 1 codes:+ z; (ii) (i) x,1 +y, x,1+z;1 (ii)y, − 2 2 − −1½+x,+ z; (iii)½−y, 2 ½+z;x, 1(iii)y, 2−x,z. 1−y, −z. 2 − − − In thethe crystalcrystal structurestructure of of lorazepam lorazepam co-crystals co-crystal withs with nicotinamide, nicotinamide, the the geometric geometric parameters parameters are similarare similar to those to those observed observed in the in crystals the crystals containing contai thening lorazepam the lorazepam molecule molecule (Table 4(Table, Figure 4,7 )[Figure42,43 7)]. In[42,43]. the crystal In the of crystal lorazepam of lorazepam compounded compounded with nicotinamide, with nicotinami the C–Ode, bondthe C–O length bond [1.395(2) length Å][1.395(2) in the hydroxylÅ] in the group,hydroxyl as well group, as N–C as bondwell lengthsas N–C [1.351(2)bond lengths and 1.409(2) [1.351(2) Å], involvingand 1.409(2) the endocyclicÅ], involving N-atom the inendocyclic the lorazepam N-atom molecule, in the showlorazepam that proton molecule, transfer show is absent that andproton that transfer a co-crystal is absent is formed. and Onthat this a occasion,co-crystalthe is formed. lorazepam On and this nicotinamide occasion, the molecules lorazepam in theandco-crystal nicotinamide are connected molecules by in O13–H13 the co-crystalO30 ··· andare N29–H29Aconnected byO12 O13–H13···O30 hydrogen bonds and to formN29–H29A·· a heterodimer·O12 hydrogen (Table4, Figure bonds7). to Adjacent form a heterodimers heterodimer ··· are(Table linked 4, Figure via N1–H1 7). AdjacentN22, N29–H29B heterodimersO13 ar ande linked C25–H25 via N1–H1···N22,N4 hydrogen N29–H29B···O13 bonds to create tapes and alongC25– ··· ··· ··· theH25···N4 [0 1 0] hydrogen direction (Tablebonds4 ,to Figure create8a). tapes The along neighboring the [0 tapes1 0] direction are connected (Table by 4, πFigureπ interactions 8a). The ··· (withneighboring centroid tapescentroid are connected distances by 3.568(3) π···π interactions Å) Å to form (with a 3D centroid···centroid framework (Figure distances8b). 3.568(3) Å) ··· Å to form a 3D framework (Figure 8b). Table 4. Hydrogen bonding interactions in the crystal structure of lorazepam co-crystals with nicotinamide. Table 4. Hydrogen bonding interactions in the crystal structure of lorazepam co-crystals with D–H A d(D–H) (Å) d(H A) (Å) d(D A) (Å)

Pharmaceutics 2020, 12, 648 11 of 17 Pharmaceutics 2020, 12 11 of 18

Pharmaceutics 2020, 12 11 of 18

FigureFigureFigure 7. 7.Molecular Molecular7. Molecular structure structure structure of of of lorazepam lorazepamlorazepam co-crystalsco-crystals co-crystals with with with nicotinamide, nicotinamide, nicotinamide, showing showing showing the theatom-labeling the atom-labeling atom-labeling schemeschemescheme (displacement (displacement (displacement ellipsoids ellipsoids ellipsoids are are drawn drawndrawn at aat at25% a a25% 25% probability probability probability level level level and and Hand H atoms atoms H atoms are are shown shown are shown as as small as spheressmallsmall spheres of spheres arbitrary of of arbitrary radius; arbitrary hydrogen radius;radius; bondshydrogen are represented bondbonds sare are represented byrepresented dashed by lines; bydashed disordereddashed lines; lines; disordered chlorobenzene disordered ringchlorobenzenechlorobenzene (C14–C19 and ring ring Cl20 (C14–C19 (C14–C19 atoms) and wasand Cl20 omitted atoms)atoms) for was clarity).was omitted omitted for for clarity). clarity).

FigureFigure 8. (a 8.) Tapes(a) Tapes formed formed by by lorazepam lorazepam and and nicotinamidenicotinamide molecules molecules in inthe the co-crystals. co-crystals. (b) Crystal (b) Crystal b packingpacking of lorazepamof lorazepam co-crystals co-crystals with with nicotinamidenicotinamide viewed viewed along along the the b-axis-axis (hydrogen (hydrogen bonds bonds are are representedrepresented by dashedby dashed lines, lines, whereas whereasπ π···interactionsπ interactions by by dotted dotted lines; lines; disordered disordered chlorobenzene chlorobenzene ring ··· was omittedring was foromitted clarity; for symmetryclarity; symmetry codes: codes: (i) 1 (i)x, 1−1x,+ ½+y,y, 1 ½−z. Figure 8. (a) Tapes formed by lorazepam and− nicotinamide2 2 − molecules in the co-crystals. (b) Crystal packing of lorazepam co-crystals with nicotinamide viewed along the b-axis (hydrogen bonds are

represented by dashed lines, whereas π···π interactions by dotted lines; disordered chlorobenzene ring was omitted for clarity; symmetry codes: (i) 1−x, ½+y, ½−z.

Pharmaceutics 2020, 12 12 of 18

3.4. FTIR and Raman Spectroscopy The FTIR data compiled in Table 5 indicate discrepancies between the spectra of chlordiazepoxide co-crystals and the physical mixture. It is significant that the spectra of co-crystals prepared by various procedures showed no significant divergence, nor did the spectrum of physical mixture reveal major changes in band position compared with starting components. The spectra of benzodiazepine and coformer are consistent with the literature data [30,44]. It therefore follows that chlordiazepoxide and p-aminobenzoic acid do not interact after mixing. Characteristic bands at 3460, 3363, 3196 and 3057 cm–1 due to stretching vibrations of the amine group in the physical mixture were shifted to lower or higher values at 3443, 3322, 3216 and 3122 cm–1 in the spectra of co-crystals, suggesting a hydrogen bonding formation between the amine group of chlordiazepoxide and p-aminobenzoic acid. Pharmaceutics 2020, 12, 648 12 of 17 Table 5. Characteristic FTIR and Raman bands of chlordiazepoxide co-crystals with p-aminobenzoic acid, prepared using different co-crystallization methods and solvents. 3.4. FTIR and Raman Spectroscopy Vibrations Co-Crystallization Methods The FTIR data compiled in Table5 indicate discrepancies between the spectra–NH2 str as of–NH chlordiazepoxide2str s –NH C=Ostr N=C–NC2H5/ϭNH2 C–OHstr 3216.9 w co-crystals and the physical mixture. It is significant that the spectra ofFTIR co-crystals 3443.2 m prepared3322.7 s by various1675.5 s 1621.7 vs - 3122.1 w procedures showed no significant divergence,Slurry evaporation nor did and the acetonitrile spectrum of physical mixture reveal major Raman - - - 1672.6 m 1619.5 sh vw 1284.7 s changes in band position compared with starting components. The spectra of benzodiazepine and 3216.0 w FTIR 3443.3 m 3321.9 s 1675.5 s 1621.7 vs - coformer are consistent with the literatureLiquid-assisted data[ grinding30,44]. and It acetonitrile therefore follows that chlordiazepoxide3121.7 w and p-aminobenzoic acid do not interact after mixing. CharacteristicRaman bands - at 3460,- 3363,- 31961671.4 and m 1618.5 sh vw 1283.4 s 1 3217.0 w 3057 cm− due to stretching vibrations of the amine group in the physicalFTIR 3443.7 mixture m 3323.1 were s shifted1676.4 to s 1621.8 vs - Slurry evaporation and ethyl acetate 3122.2 w lower or higher values at 3443, 3322, 3216 and 3122 cm 1 in the spectra of co-crystals, suggesting a − Raman - - - 1672.3 m 1619.4 sh vw 1284.5 s hydrogen bonding formation between the amine group of chlordiazepoxide and p-aminobenzoic3216.1 w acid. FTIR 3443.6 m 3322.2 s 1675.2 s 1621.9 vs - Liquid-assisted grinding and ethyl acetate 3122.3 w Table 5. Characteristic FTIR and Raman bands of chlordiazepoxide co-crystalsRaman with- p-aminobenzoic- - 1671.7 m - 1284.0 s 3196.3 m acid, prepared using different co-crystallization methods and solvents. FTIR 3460.5 m 3363.0 m - 1625.3 vs 1286.8 s Physical mixture 3057.2 m Vibrations Raman - - - - - 1287.2 s Co-Crystallization Methods –NH2 str as S–NHtretching2str s vibration–NH (Tr), in- Cplane=Ostr bendingN= C–NCvibration2H5 (/ϭ)NH, symmetric2 C–OH (s)str, asymmetric (as), very strong (vs), strong (s),3216.9 medium w (m), weak (w). Slurry evaporation FTIR 3443.2 m 3322.7 s 1675.5 s 1621.7 vs - 3122.1 w and acetonitrile The formation of hydrogen bonding can also be confirmed by consecutive changes in the Raman - - - 1672.6 m 1619.5 sh vw 1284.7 s co-crystal spectra. Two new bands at ~2490 and 2934 cm–1 were identified, which were over the Liquid-assisted 3216.0 w FTIR 3443.3spectral m 3321.9 range s of 2300–3000 cm1675.5–1 assigned s to hydrogen 1621.7 vs bonding formation - [30]. A new band at 1675 grinding and 3121.7 w –1 –1 acetonitrile Raman -cm was -also created, - probably 1671.4 due m to the shift 1618.5 of sh a vwband at 1665.6 1283.4 cm s , attributed to the stretching vibrations of the C=O group of p-aminobenzoic acid. However, a strong band at ~1283 cm–1 assigned 3217.0 w Slurry evaporation FTIR 3443.7 m 3323.1 s 1676.4 s 1621.8 vs - to the C–OH stretching3122.2 wvibration of coformer disappeared, which confirms the participation of this and ethyl acetate Raman -group in the - formation - of hydrogen 1672.3 m bonding 1619.4 with sh chlordiazepoxide. vw 1284.5 s Furthermore, the band at 1625 cm–1 in the physical mixture, due to bending vibrations in the plane of the amine group of coformer Liquid-assisted 3216.1 w FTIR 3443.6 m 3322.2 s 1675.2 s 1621.9 vs - –1 grinding and ethyl and the methylamine3122.3 group w of chlordiazepoxide, is shifted to 1621 cm in the co-crystal spectra. In acetate Raman -addition, -bands observed - at 1170, 1671.7 1150 m and 1134 cm -–1 in the spectra 1284.0 of s physical mixture are shifted to –1 lower values at ~1161,3196.3 1146 m and 1115 cm in the co-crystal spectra. Hence, the FTIR study confirms FTIR 3460.5 m 3363.0 m - 1625.3 vs 1286.8 s Physical mixture the participation 3057.2 of the m carboxyl and amine groups of coformer and the methylamine group of Raman -chlordiazepoxide - in the - creation of - hydrogen bond -ing. 1287.2 s Stretching vibration (Tr), in-plane bending vibrationAs was ( the), symmetric case with (s), chlordiazepoxide asymmetric (as), very co-crystals, strong (vs), discrepancies strong (s), were observed between the medium (m), weak (w). FTIR spectra of lorazepam co-crystals and the physical mixture (Figure 9, Table 6). Characteristic bands at 3459 and 3363 cm–1, assigned to stretching vibrations of amine and hydroxyl groups of The formation of hydrogen bondinglorazepam can also and be asymmetric confirmed stretching by consecutive of the amide changes group in of the nicotinamide co-crystal [24,28,29,45], were shifted to lower values1 at ~3418 and 3313 cm–1 in the co-crystal spectra, indicating the formation of hydrogen spectra. Two new bands at ~2490 and 2934 cm− were identified, which were over the spectral range of 1 bonding between benzodiazepine and coformer. The participation1 of C=O stretching vibrations in 2300–3000 cm− assigned to hydrogen bonding formation [30]. A new band at 1675 cm− was also 1 created, probably due to the shift of a band at 1665.6 cm− , attributed to the stretching vibrations 1 of the C=O group of p-aminobenzoic acid. However, a strong band at ~1283 cm− assigned to the C–OH stretching vibration of coformer disappeared, which confirms the participation of this group in 1 the formation of hydrogen bonding with chlordiazepoxide. Furthermore, the band at 1625 cm− in the physical mixture, due to bending vibrations in the plane of the amine group of coformer and the 1 methylamine group of chlordiazepoxide, is shifted to 1621 cm− in the co-crystal spectra. In addition, 1 bands observed at 1170, 1150 and 1134 cm− in the spectra of physical mixture are shifted to lower values 1 at ~1161, 1146 and 1115 cm− in the co-crystal spectra. Hence, the FTIR study confirms the participation of the carboxyl and amine groups of coformer and the methylamine group of chlordiazepoxide in the creation of hydrogen bonding. As was the case with chlordiazepoxide co-crystals, discrepancies were observed between the FTIR spectra of lorazepam co-crystals and the physical mixture (Figure9, Table6). Characteristic bands at 1 3459 and 3363 cm− , assigned to stretching vibrations of amine and hydroxyl groups of lorazepam and asymmetric stretching of the amide group of nicotinamide [24,28,29,45], were shifted to lower 1 values at ~3418 and 3313 cm− in the co-crystal spectra, indicating the formation of hydrogen bonding between benzodiazepine and coformer. The participation of C=O stretching vibrations in hydrogen 1 bonding is confirmed by the shift in physical mixture bands at 1703 and 1686 cm− to lower values at Pharmaceutics 2020, 12 13 of 18 hydrogen bonding is confirmed by the shift in physical mixture bands at 1703 and 1686 cm–1 to lower valuesPharmaceutics at 16952020 and, 12, 6481662 cm–1. Moreover, distinctive bands at 1143 and 1133 cm–1 assigned to 13–OH of 17 stretching vibrations of lorazepam are invaluable for co-crystal identification. The shift of the band assigned to –NH2 deformation1 vibrations of nicotinamide from 1618 cm1 –1 in the physical mixture 1695 and 1662 cm− . Moreover, distinctive bands at 1143 and 1133 cm− assigned to –OH stretching –1 spectrumvibrations to of 1602 lorazepam cm in are the invaluable co-crystal for spectra co-crystal confirms identification. the participation The shift of of the bandamide assigned group in to hydrogen bonding. 1 –NH2 deformation vibrations of nicotinamide from 1618 cm− in the physical mixture spectrum to 1 1602 cm− in the co-crystal spectra confirms the participation of the amide group in hydrogen bonding. Table 6. Characteristic FTIR and Raman bands of lorazepam co-crystals with nicotinamide prepared usingTable different 6. Characteristic co-crystallization FTIR and methods Raman bands and solvents. of lorazepam co-crystals with nicotinamide prepared using different co-crystallization methods and solvents. Vibrations

Co-Crystallization Methods –NHstr/– –NHstr/–OH C=Ostr VibrationsC=Ostr –NH2 def C–N(amide) str –OHstr OH/–NH2 as str Co-Crystallization Methods 3312.9 –NHw str/ 1143.6 s FTIR 3418.0–NHstr s /–OH 1696.1 vsC =Ostr 1662.9C vs= Ostr 1602.4–NH s2 def C–N 1381.8(amide) s str –OHstr Slurry 3203.5–OH w/–NH 2 as str 1133.2 s evaporation and 3312.9 w 1169.91143.6 vs s ethyl acetate RamanFTIR 3418.0 s 1689.4 w1696.1 vs 1662.9 vs1592.5 1602.4 vs s 1383.8 1381.8 m s Slurry evaporation - - 3203.5 w - 1133.01133.2 m s and ethyl acetate 1169.9 vs Raman -3313.0 w - 1689.4 w - 1592.5 vs 1383.8 m 1143.8 s FTIR 3418.4 s 1696.5 vs 1662.2 vs 1602.7 s 1381.7 vs 1133.0 m Liquid-assisted 3203.3 w 1133.5 s grinding and 3313.0 w 1143.8 s Liquid-assisted FTIR 3418.4 s 1696.5 vs 1662.2 vs 1602.7 s 1381.7 vs 1168.5 vs ethyl acetate Raman - - 3203.3 w1692.6 m - 1592.8 vs 1383.5 m 1133.5 s grinding and ethyl 1133.6 m 1168.5 vs acetate Raman - - 1692.6 m - 1592.8 vs 1383.5 m 1143.7 m FTIR 3418.0 s 3206.1 w 1693.9 s 1662.7 s 1602.6 m 1382.2 m 1133.6 m Slurry 1133.1 m evaporation and 1143.7 m FTIR 3418.0 s 3206.1 w 1693.9 s 1662.7 s 1602.6 m 1382.2 m 1168.6 vs methanolSlurry evaporationRaman - - 1692.5 m - 1593.0 vs 1383.3 m 1133.1 m and methanol 1133.41168.6 vss Raman - - 1692.5 m - 1593.0 vs 1383.3 m 3313.1 w 1143.71133.4 s Liquid-assisted FTIR 3418.0 s 1695.3 vs 1662.8 vs 1602.8 s 1382.2 s 3204.23313.1 w w3 1133.41143.7 s s grindingLiquid-assisted and FTIR 3418.0 s 1695.3 vs 1662.8 vs 1602.8 s 1382.2 s 204.2 w 1168.51133.4 s methanolgrinding and Raman 1692.1 m 1662.6 w 1592.8 vs 1383.2 m - - 1133.71168.5 w s methanol Raman - - 1692.1 m 1662.6 w 1592.8 vs 1383.2 m 1686.7 vs 1162.01133.7 ws FTIR 3459.1 m 3363.4 m 1703.5 sh 1618.3 s 1387.9 m 1671.21686.7 sh vs 1130.11162.0 m s FTIR 3459.1 m 3363.4 m 1703.5 sh 1618.3 s 1387.9 m Physical mixture 1671.2 sh 1130.1 m Physical mixture 1619.0 sh Raman - - - 1674.3 m 1619.0 sh - 1167.9 m Raman - - - 1674.3 m 1599.8 vs - 1167.9 m 1599.8 vs str—stretching vibrations, def—deformation vibrations, vs—very strong, s—strong, m—medium, str—stretching vibrations, def—deformation vibrations, vs—very strong, s—strong, m—medium, w—weak,w—weak, sh—shoulder.sh—shoulder.

Figure 9. FTIR spectra of: (a) lorazepam physical mixture with nicotinamide, (b) lorazepam co-crystals Figure 9. FTIR spectra of: (a) lorazepam physical mixture with nicotinamide, (b) lorazepam with nicotinamide prepared by slurry evaporation method and ethyl acetate as a solvent, and (c) co-crystals with nicotinamide prepared by slurry evaporation method and ethyl acetate as a solvent, lorazepam co-crystals with nicotinamide prepared by liquid-assisted grinding method with methanol and (c) lorazepam co-crystals with nicotinamide prepared by liquid-assisted grinding method with as a solvent. methanol as a solvent. Overall, the FTIR study conﬁrms that the amide group of nicotinamide and hydroxyl and carbonyl groups of lorazepam participate in the formation of hydrogen bonding. Additionally, changes in Pharmaceutics 2020, 12 14 of 18

PharmaceuticsOverall,2020 the, 12 ,FTIR 648 study confirms that the amide group of nicotinamide and hydroxyl14 and of 17 carbonyl groups of lorazepam participate in the formation of hydrogen bonding. Additionally, changesthe band in positions the band of positions –NH of of lorazepam –NH of lorazepam imply hydrogen imply bondinghydrogen by bonding the N1 by ring the of N1 diazepine. ring of diazepine.As lorazepam As lorazepam is known to is form known dimers to form in the dimers solid in state the [ 24solid], the state dimer [24], could the dimer be created could between be created the betweenN1 ring ofthe diazepine N1 ring fromof diazepine one molecule from andone anmolecu oxygenle and atom an of oxygen the carbonyl atom groupof the fromcarbonyl the secondgroup frommolecule the second of lorazepam. molecule Moreover, of lorazepam. it is possible Moreover, to create it is possible a hydrogen to create bond a between hydrogen the bond carbonyl between and thehydroxyl carbonyl groups and ofhydroxyl the diazepine groups ring of the in twodiazepine adjacent ring molecules in two adjacent of lorazepam. molecules The of FTIR lorazepam. study shows The FTIRthat lorazepam study shows can that create lorazepam hydrogen can bonds create with hydr nicotinamideogen bonds in with the samenicotinamide manner asin dimersthe same are manner formed asin dimers the solid are state. formed in the solid state. The co-crystalsco-crystals preparedprepared by by di ffdifferenterent methods methods show show no significant no significant changes changes in their in band their spectral band spectralpositions. positions. The spectrum The spectrum of the physical of the mixture physical was, mixture in turn, was, a simple in turn, overlap a simple of the benzodiazepineoverlap of the benzodiazepineand coformer spectra and andcoformer does notspectra reveal and major does changes not reveal to band major position changes when to compared band position with starting when comparedcomponents. with This starting excludes components. the interaction This excludes of components the interaction after mixing. of components after mixing. Raman spectra displayed merely minor changes in band intensity and position (up to 2 cm–11) for Raman spectra displayed merely minor changes in band intensity and position (up to 2 cm− ) for thethe co-crystalsco-crystals of of both both chlordiazepoxide chlordiazepoxide and and lorazepam lorazepam prepared prepared using diusingfferent different methods methods and solvents. and solvents.Moreover, Moreover, the Raman spectrathe Raman of physical spectra mixtures of physical of chlordiazepoxide mixtures of with chlordiazepoxidep-aminobenzoic with acid pand-aminobenzoic lorazepamwith acidnicotinamide and lorazepam do notwith reflect nicotinamide any distinctive do not changes reflect inany band distinctive position orchanges intensity in bandcompared position with or the intensity starting compared components; with hence, the starti it canng be components; asserted that nohence, co-crystallization it can be asserted occurs that when no co-crystallizationbenzodiazepines withoccurs coformers when benzodiazepines are mixed. However, with comparingcoformers are the mixed. Raman However, spectra of co-crystalscomparing with the Raman spectra of co-crystals with those of physical mixtures revealed new unique bands1 at ~1672, those of physical mixtures revealed new unique bands at ~1672, 1552, 1255 and 1162 cm− , and at ~1692, 1552, 1255 and 11621 cm–1, and at ~1692, 1506 and 1383 cm–1, for chlordiazepoxide and lorazepam 1506 and 1383 cm− , for chlordiazepoxide and lorazepam co-crystals, respectively. These changes in co-crystals,Raman spectra respectively. and those These specified changes below in revealRaman that spectra new and structures those specified are created, below thereby reveal confirming that new structuresco-crystal formation.are created, thereby confirming co-crystal formation. Raman spectra of of chlordiazepoxide co-crystals co-crystals with with pp-aminobenzoic-aminobenzoic acid acid prepared prepared by by different different methods and and of the physical mixture of both comp componentsonents are shown in Figure 1010.. Raman bands at 1181.0 and 1133.0 cm–1 found1 in the chlordiazepoxide physical mixture spectrum disappeared in the 1181.0 and 1133.0 cm− found in the chlordiazepoxide physical mixture spectrum disappeared in co-crystal spectra, while another at 1515.9 cm–1 is shifted1 to 1521 cm–1. Moreover,1 the band at 1287cm–1 the co-crystal spectra, while another at 1515.9 cm− is shifted to 1521 cm− . Moreover, the band at in the mixture1 spectrum assigned to the hydroxyl stretching of p-aminobenzoic acid [46] changes 1287cm− in the mixture spectrum assigned to the hydroxyl stretching of p-aminobenzoic acid [46] position slightly to ~1284 cm–1 and a1 new strong band at 1255 cm–1, near1 the hydroxyl band, is changes position slightly to ~1284 cm− and a new strong band at 1255 cm− , near the hydroxyl band, createdis created in inthe the co-crystal co-crystal spectra. spectra.

Figure 10. Raman spectra of: (a) chlordiazepoxide and p-aminobenzoic acid physical mixture, Figure 10. Raman spectra of: (a) chlordiazepoxide and p-aminobenzoic acid physical mixture, (b) (b) chlordiazepoxide co-crystals with p-aminobenzoic acid prepared by grinding assisted acetonitrile, chlordiazepoxide co-crystals with p-aminobenzoic acid prepared by grinding assisted acetonitrile, and (c) chlordiazepoxide co-crystals with p-aminobenzoic acid co-crystals prepared by slurry and (c) chlordiazepoxide co-crystals with p-aminobenzoic acid co-crystals prepared by slurry evaporation method and ethyl acetate as a solvent. evaporation method and ethyl acetate as a solvent. 1 In the lorazepam co-crystal spectra, a band at 1674.3 cm− assigned to the C=O stretching vibration 1 of nicotinamide [47] is absent. The intensity of the band at 1167.9 cm− increased in relation to that in

Pharmaceutics 2020, 12, 648 15 of 17

1 the mixture spectrum. Moreover, additional weak peaks at ~1196, 1132, 722 and 696 cm− appeared, conﬁrming the interaction of lorazepam with nicotinamide due to co-crystallization.

4. Conclusions The outcomes of this research reveal that two new co-crystals, i.e., chlordiazepoxide with p-aminobenzoic acid and lorazepam with nicotinamide, were prepared using liquid-assisted grinding and slurry evaporation procedures. Both co-crystallization methods could be used for further preparation of benzodiazepine co-crystals, since they are environmentally benign procedures. However, in the case of lorazepam co-crystals with nicotinamide, prepared by a liquid-assisted grinding procedure with ethyl acetate as a solvent, a DSC study revealed a minute amount of unreacted starting component; hence, the usage of ethyl acetate in this procedure should be reconsidered. Crystal structures, spectroscopic characteristics and thermal profiles of both benzodiazepines co-crystals were described using powder X-ray diffraction (PXRD), single-crystal X-ray diffraction (SCXRD), Fourier-transform infrared (FTIR), Raman spectroscopy, and differential scanning calorimetry (DSC). The study revealed that benzodiazepine co-crystals are crystalline materials, where the compliance of co-crystal patterns with those contained in the base PDF-4+ show that new structures were obtained. The chlordiazepoxide co-crystal with p-aminobenzoic acid and the lorazepam co-crystal with nicotinamide crystallized in the monoclinic P21/n and P21/c space group at a 1:1 molar ratio. New, unique bands and the shifting of band positions observed in the Raman spectra of co-crystals verify that new structures were obtained after the co-crystallization process.

Supplementary Materials: The following are available online at http://www.mdpi.com/1999-4923/12/7/648/s1, Figure S1. Identification cards of chlordiazepoxide and p-aminobenzoic acid (from the database PDF-4+ ICDD) and the diffraction pattern of chlordiazepoxide co-crystals with p-aminobenzoic acid prepared by slurry evaporation method, Figure S2. Identification cards of lorazepam and nicotinamide (from the database PDF-4+ ICDD) and the diffraction pattern of lorazepam mixture with nicotinamide. Author Contributions: Conceptualization, P.G. and M.W.; methodology, P.G.; formal analysis, P.G., D.P. and A.S.; investigation, P.G., D.P. and A.S.; data curation, P.G., D.P. and A.S.; writing—original draft preparation, P.G., D.P., A.S. and M.W.; writing—review and editing, P.G. and M.W.; supervision, M.W. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the European Union through the European Social Fund under the Operational Programme Knowledge Education Development 2014–2020, project POWR.03.02.00-00-I026/17-00, and the Ministry of Science and Higher Education, Poland, grant nos. 02-0015/07/505 and 0912/SBAD/0903. Conflicts of Interest: The authors declare no conflict of interest.

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