The Effect of Cooling on the Degree of Crystallinity, Solid-State Properties

pharmaceutics

Article The Eﬀect of Cooling on the Degree of Crystallinity, Solid-State Properties, and Dissolution Rate of Multi-Component Hot-Melt Extruded Solid Dispersions

Dean Hurley 1,2 , Mark Davis 2 , Gavin M. Walker 2, John G. Lyons 1 and Clement L. Higginbotham 1,2,*

1 Materials Research Institute, Athlone Institute of Technology, Athlone N37 F6D7, Ireland; [email protected] (D.H.); [email protected] (J.G.L.) 2 Synthesis and Solid State Pharmaceutical Centre (SSPC), Bernal Institute, University of Limerick, Limerick V94 T9PX, Ireland; [email protected] (M.D.); [email protected] (G.M.W.) * Correspondence: [email protected]; Tel.: +353-(0)-90-6468050

Received: 30 December 2019; Accepted: 17 February 2020; Published: 1 March 2020

Abstract: The effect of cooling on the degree of crystallinity, solid-state and dissolution properties of multi-component hot-melt extruded solid dispersions [SD] is of great interest for the successful formulation of amorphous SDs and is an area that is unreported, especially in the context of improving the stability of these specific systems. The thermal solid-state properties, degree of crystallinity, drug–polymer interactions, solubility and physical stability over time were investigated. X-ray powder diffraction [XRPD] and hyper differential scanning calorimetry [DSC] confirmed that indomethacin [INM] was converted to the amorphous state; however, the addition of poloxamer 407 [P407] had a significant effect on the degree of crystallinity and the solubility of the SD formulations. Spectroscopy studies identified the mechanism of interaction and solubility studies, showing a higher dissolution rate compared to amorphous and pure INM in pH 1.2 with a kinetic solubility of 20.63 µg/mL and 34.7 µg/mL after 3 and 24 h. XRPD confirmed that INM remained amorphous after 5 months stability testing in solid solutions with Poly(vinylpyrrolidone-co-vinyl acetate) [PVP VA64] and Plasdone S-630 [PL-S630]. Although cooling had a significant effect on the degree of crystallinity and on solubility of INM, the cooling method used did not have any significant effect on the amorphous stability of INM over time.

Keywords: solid dispersion; cooling; glass transition; extrusion; solubility; amorphous; crystallization

1. Introduction Within both the pharmaceutical industry and academia, there is a strong interest in utilizing hot melt extrusion [HME] to prepare amorphous solid dispersions [SDs] which have the potential to significantly improve the kinetic solubility and thus, the bioavailability of BCS class II drugs which have poor aqueous solubility and high permeability [1]. The conversion of the active pharmaceutical ingredient [API] to the amorphous state increases dissolution rate and apparent solubility as the penetration of solvents is not prohibited by an endothermic barrier attributed to the disorder of crystalline lattices [2]. During dissolution the carrier traps the dispersed API in a high energy state, maintaining supersaturation in the medium over time and preventing crystallization [3]. Bravo et al. (2004) reported that HME has many significant benefits such as the elimination of solvents, good content uniformity due to the low drug loading used, dosage forms of a desired shape, including tablets, pellets and implants can be easily manufactured by this process; HME is

Pharmaceutics 2020, 12, 212; doi:10.3390/pharmaceutics12030212 www.mdpi.com/journal/pharmaceutics Pharmaceutics 2020, 12, 212 2 of 24 also a continuous process and can be scaled up [4] and finally, HME allows conversion of crystalline drugs to amorphous form or dispersion of API into very small particles, enhancing bioavailability and improving patient compliance. However, the application of high shear inherent to the process and high processing temperatures presents a significant challenge when dealing with APIs that are thermo-sensitive. However the inclusion of plasticizers as excipients can lower the processing temperatures associated with HME and can modify the physicochemical and dissolution properties of poorly water-soluble APIs [2,5]. Understanding the physicochemical properties of both the API and polymeric carriers is important when manufacturing SDs as it is the properties of the formulation that is being extruded that dictate the dissolution behaviour. Although the potential of amorphous SDs is enormous, the physicochemical factors of these systems need to be fully understood as they have a significant effect on the dissolution and stability of the amorphous drug. Various factors can affect the solid-state and dissolution properties of these systems and can result in undesirable behaviours, such as nucleation, phase separation and recrystallization during storage [6]. Previous studies have focused on how various extrusion process parameters affect the dissolution behaviour of indomethacin [INM]. For example, Liu et al. (2009) reported the effect of extrusion process parameters on the dissolution behaviour of INM in Eudragit EPO SDs. In this study, three process conditions—set mixer temperature, screw speed and residence time—were studied. The results show that the dissolution rate of INM increased with increasing screw rotating speed or the mixer set temperature. Further research was carried out using semi-crystalline polymers such as Poloxamer 407 [P407] to overcome the drawbacks associated with HME such as its high processing temperatures [5,7], as the processing temperatures can restrict the proccesing of thermally labile drugs [8]. However, the cooling method used during the extrusion process can also have a significant effect on the crystallization tendency and solubility of the API. Very little has been reported on how various cooling methods effect the degree of crystallinity, solid state properties and dissolution rate of multi-component amorphous SDs. INM has been used as a model drug in this study which is a BCS class II API (low solubility, high permeability). It is well reported in the literature that preparing an SD of INM significantly improved the amorphous stability and solubility of INM [5]. Poly(vinylpyrrolidone-co-vinyl acetate) [PVP VA64] and Plasdone-S630 [PL-S630] in terms of monographs, have the same chemical structure; however, they have different solid-state properties and solubilities depending on the manufacturing process used. PVP VA64 and PL-S630 are commonly used carriers for optimizing the solubility of INM, and the effect of these specific carriers will be examined in this study. This study is also a continuation of the work carried out by Hurley et al., (2019) [6]. P407 was chosen as it is reported in literature that P407 cannot only overcome the drawbacks associated with HME, but increase the solubility of INM significantly [9]. The 30% INM drug–polymer mixtures were used as it was reported that maximum solubility of INM reported in the literature was 30 µg/mL after 72 h using 30% INM [10]. The relationship between cooling and the physicochemical/dissolution properties of the drug and polymeric carriers is an area that is very important in the manufacture and development of SDs. It is reported in the literature that slow cooling enhances the physical stability of amorphous INM [11]; however, Meng et al. (2015) stated that the faster the cooling rate, the lower the crystallinity of the API [12]. Therefore, in this study, the effect of various cooling processes are studied. This study was, to our knowledge, the first to report how various cooling methods affect the dissolution behaviour of INM using a semi-crystalline surfactant and how it affects the degree of crystallinity of amorphous SDs. Since this study uses a semi-crystalline plasticizer to improve the solubility of INM, the degree of crystallinity of the semi-crystalline polymer in the SD formulations must be examined as it can affect the processing and physical properties of the API. Pharmaceutics 2020, 12, 212 3 of 24

2. Materials and Methods

2.1.Pharmaceutics Materials 2020, 12, x FOR PEER REVIEW 3 of 24 Crystalline INM (1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-3-indoleacetic acid), purchased from Crystalline INM (1-(4-Chlorobenzoyl)-5-methoxy-2-methyl-3-indoleacetic acid), 1purchased Tokyo Chemical Industry (TCI) (Oxford Science Park, Oxford, UK) (N.V.) (Mw 357.79 g/mol− , Tm 160.0 from Tokyo Chemical Industry (TCI) (Oxford Science Park, Oxford, UK) (N.V.) (Mw 357.79 g/mol−1, ◦C Tg 42 ◦C) was used as the model drug [5]. INM has an aqueous solubility of 1.5 µg/mL at pH 1.2 [13]. Tm 160.0 °C Tg 42 °C) was used as the model drug [5]. INM has an aqueous solubility of 1.5 µg/ml at pH P407, a hydrophilic non-ionic surfactant, (Mw 12,000, Tm 55 ◦C, Tg 67 ◦C) and PVP VA64 (Mw 45,000, 1.2 [13]. P407, a hydrophilic non-ionic surfactant, (Mw 12,000, Tm− 55 °C, Tg −67 °C) and PVP VA64 Tg 107 ◦C, Tm 140 ◦C) was purchased from BASF Europe GmbH (Burgbernheim, Germany) [5]. PL-S630 (Mw 45,000, Tg 107 °C, Tm 140 °C) was purchased from BASF Europe GmbH (Burgbernheim, (Mw 47,000, Tg 106 ◦C, Tm 140 ◦C) was received as a gift from Ashland Specialties Ltd, (UK) [6]. All otherGermany) chemicals [5]. PL and-S630 reagents (Mw were47,000, purchased Tg 106 °C, from Tm Sigma140 °C) Aldrich was received (Wicklow, as Ireland) a gift from and Ashland were of analyticalSpecialties reagent Ltd, (UK) grade. [6]. TheAll chemicalother chemicals structures and of reagents the materials were used purchased in this from study Sigma are shown Aldrich in Figure(Wicklow,1. Ireland) and were of analytical reagent grade. The chemical structures of the materials used in this study are shown in Figure 1.

Figure 1. Chemical structures of (a) BCS class II model drug indomethacin [INM], (b) Poly(vinylpyrrolidone-co-vinyl acetate) [PVP VA64], (c) Plasdone S-630 [PL-S630] and (d) Poloxamer Figure 1. Chemical structures of (a) BCS class II model drug indomethacin [INM], (b) 407 [P407] used in this study. Poly(vinylpyrrolidone-co-vinyl acetate) [PVP VA64], (c) Plasdone S-630 [PL-S630] and (d) Poloxamer 2.2. Preparation407 [P407] used of Physical in this study. Blends

2.2. PreparationSix diﬀerent of Physical combinations Blends of the four components were examined and 50 g of powder were used for each batch. Physical blends were mixed in a mortar and pestle for 5 min after weighing, in accordanceSix different with combinations the drug/carrier of the percentages four components outlined were in examined Table1. The and 30–70% 50 g of powder INM-P407 were binary used for each batch. Physical blends were mixed in a mortar and pestle for 5 min after weighing, in mixtures were prepared as controls. Amorphous INM [aINM] was prepared by heating up to 160 ◦C in aaccordance stainless steel with beaker the drug/carrier using a hotplate percentage and quenchs outlin coolinged in T usingable 1. ﬂuid The nitrogen 30%–70% [5 ].INM-P407 binary mixtures were prepared as controls. Amorphous INM [aINM] was prepared by heating up to 160 °C in a stainless steel Tablebeaker 1. usingDrug/ carriera hotplate percentages and quench of the HMEcooling processed using fluid formulations. nitrogen [5].

Batch identifier No Composition (% w/w) 2.3. Hot Melt Extrusion PL-S630 (% w/w) PVP VA64 (% w/w) P407 (% w/w) INM (% w/w) The compositionsSD1 (%w/w) of 32.5 each of the physical 32.5 mixtures shown 5in Table 1 were extruded 30 using a bench-top coSD2-rotating Prism 16 27.5 mm twin-screw extruder 27.5 (Thermo Fisher 15 Scientific, Waltham, 30 MA, USA) with a 2SD3 mm diameter die and - a length to diameter 65 [L/D] ratio 5of 15/1. The physical 30 mixtures were extrudedSD4 using screws containing - conveying 55elements and a screw 15 speed of 100 30 RPM [5]. The extruder containedSD5 three heating zones 65 which, from -feeding zone to the 5 die, had set points 30 of 140, 160 and 180 °C. TheSD6 physical blends 55 were fed into the -15:1 L/D extruder 15 using an automatic 30 feeder at a rate of 13 ± 1 g/min. The diameter of each of the extrudates was 2mm. With the addition of the 2.3.surfactant, Hot Melt the Extrusion temperature profile was reduced by 10 °C. As the extrudates exited the die, they were air cooled to 25 °C, ground in a mortar via liquid nitrogen, and passed through a 200 µm sieve to The compositions (%w/w) of each of the physical mixtures shown in Table1 were extruded using obtain uniform sized fractions for further studies [5]. All the samples were placed in a desiccator a bench-top co-rotating Prism 16 mm twin-screw extruder (Thermo Fisher Scientific, Waltham, MA, alongside phosphorus pentoxide to remove any residual moisture. USA) with a 2 mm diameter die and a length to diameter [L/D] ratio of 15/1. The physical mixtures were extruded using screws containing conveying elements and a screw speed of 100 RPM [5]. The Table 1. Drug/carrier percentages of the HME processed formulations. extruder contained three heating zones which, from feeding zone to the die, had set points of 140, 160 andBatch 180 ◦identifierC. The physical No blends were fed intoComposition the 15:1 L/D (% extruder w/w) using an automatic feeder at a rate of 13 1 g/min. ThePL diameter-S630 (% w of/w each) PVP of theVA64 extrudates (% w/w) wasP407 2mm. (% w/ Withw) INM the addition (% w/w) of the ±SD1 32.5 32.5 5 30 surfactant, the temperature profile was reduced by 10 ◦C. As the extrudates exited the die, they were SD2 27.5 27.5 15 30 SD3 - 65 5 30 SD4 - 55 15 30 SD5 65 - 5 30 SD6 55 - 15 30

Pharmaceutics 2020, 12, 212 4 of 24

air cooled to 25 ◦C, ground in a mortar via liquid nitrogen, and passed through a 200 µm sieve to obtain uniform sized fractions for further studies [5]. All the samples were placed in a desiccator alongside phosphorus pentoxide to remove any residual moisture.

2.4. Cooling Process To investigate the crystallization tendency of the API, a selected SD formulation was air cooled [AC] to 25 ◦C by an air pipe connector attached to the 2 mm die of the extruder, cooled to normal room temperature (25 ◦C) [NT], the same as air cooling except with no air pipe connector and quench cooled using liquid nitrogen, [Liq N2], to examine the eﬀect of slow and rapid cooling processes on the amorphous stability and solubility of INM. For the purpose of this study, only one SD formulation was investigated.

2.5. Calculation of Solubility Parameters via Hansen Solubility Parameters (δ) This theory was established by Hansen in 1967 when he divided the cohesion energy into 3 diﬀerent components: polar, physical and hydrogen bonding forces, which all contribute to the molecular structure of any compound. This speciﬁc idea was the key aspect of Hansen solubility parameters theory [HSPs]. HSPs have been applied in the pharmaceutical industry to provide a complete understanding of the various cohesive energies within APIs and therefore, permits the prediction of their behavior under various conditions and manufacturing processes in addition to how they behave within the human body. The HSPs were calculated from their chemical structures using the combined group contribution methods of Hoftyzer and Van Krevelen, (1976), [14], according to the following Equation (1): 2 2 2 2 δ = δ d + δ p + δ h (1) where 2 ΣF (ΣFpi ) ΣF δ = di , δ = 1/2, δ = hi 1/2 d V p V h V where i, δ, δd, δp, and δh, are the functional group within the molecule, the total solubility parameter, contribution from dispersion forces, polar interactions and hydrogen bonding, respectively. Fdi, Fpi, and Ehi are the molar attraction constant due to molar dispersion forces, molar polarization forces and hydrogen bonding energy, respectively, and V is the volume within the lattice site [15]. Hildebrand and Scott (1950) developed the drug–polymer interaction parameter, χ, which can be calculated via the HSPs [16]. This is expressed as follows:

Vo χ = (δ δ )2 (2) RT drug − polymer where Vo is the volume of the lattice site, R is the gas constant and T is the absolute temperature.

2.6. Attenuated Total Reﬂectance-Fourier Transform Infrared [ATR-FTIR] Spectroscopy ATR-FTIR spectra of all SD formulations were analyzed via a Spectrum One, Perkin Elmer apparatus equipped with an ATR sampling universal accessory. The spectra were collected within the 1 range of 4000–420cm− , using a 16 scan per sample sequence. The compression force used was 80N, which is ﬁxed and universal. The analysis of the ATR-FTIR spectra was performed using Perkin Elmer Spectrum software [5].

2.7. Raman Spectroscopy Raman spectra of all SD formulations were attained using a Renishaw invia Raman confocal microscope (Renishaw Instruments, Gloucestershire, UK) which was attached to a motorized stage. A 785 nm laser was used to provide Raman scattered light, the laser operated at 300 mW with a laser Pharmaceutics 2020, 12, 212 5 of 24

1 power of 100%. The Raman Spectra of each formulation were within the range of 100 and 3200 cm− , and with an acquisition time of 10 s. A 20 lens was used and had a spot size of 50 µm [6]. A 1200 l/mm × (633/780) beam path grating was used.

2.8. X-ray Powder Diffraction [XRPD] XRPD spectra of all SD formulations were attained using a Philips PANalytical Empyrean X’Pert MPD Pro using a sample spinner PW3064. Each SD formulation once ground were positioned on silica disks to provide a zero-background [5]. The diffraction pattern was within the range of 5◦ to 40◦ (2θ) with a 0.0167◦ step size. The total collection time was 29.845 s, using a sample rotation of 15 rpm via a PANalytical Data Collector, version 2.0. A Cu Kα (λ = 1.5418 Å) source was used, using an accelerating 1 voltage of 40 Kv and a 35 mA anode current. A 4 ” fixed divergence slit and a nickel filter, which was 0.020 mm in size, were used [5].

2.9. Hyper Diﬀerential Scanning Calorimetry [DSC] Hyper DSC was carried out on all SD formulations using a PerkinElmer DSC 8500 that was attached to a cooling accessory which was refrigerated (PerkinElmer, UK). The purge gas used was Helium at 30 mL/min. The DSC instrument was calibrated using a heating rate of 100 ◦C/min using high-purity indium in order to standardize the heat ﬂow signal and temperature [5]. Then, 2.5 mg samples were weighed and then placed in crimped DSC pans. Samples were ramped from 10 to − 180 ◦C at 100 ◦C/min. The DSC analysis was performed using a PerkinElmer pyris thermal analysis software, version 10.1 and any numerical values reported are the mean SD of three independently ± prepared samples [5]. The degree of crystallinity of P407 was calculated using Equation (3):

∆Hm χc = 100% (3) ◦ ∆H m (w) where χc is the degree of crystallinity, ∆Hm◦ the heat of fusion of the 100% crystalline materials, ∆Hm is the heat of fusion of the sample and w is the weight fraction. The value of the heat of fusion of 100% crystalline material ∆Hm◦ was calculated using DSC.

2.10. Preparation of pH Buﬀer 1.2 An amount of 2 g of sodium chloride [NaCl] was added to 500 mL of deionized water, and 8.3 mL of 0.1 M HCL was pipetted and diluted to 1 litre with deionized water.

2.11. Solubility Studies Solubility studies of the SD formulations were carried out in triplicate via the method reported by Higuchi and Connors in pH buffer 1.2 [17]. pH buffer 1.2 was prepared. A specific amount of SD formulation was added to 10 ml volumetric flasks. The suspensions were maintained at 37 ◦C for 24 h. This timeframe was reported in the literature to be an appropriate timeframe to achieve equilibrium [5]. Two millilitres were filtered and withdrawn through 25 mm Millex—Polytetrafluoroethylene [PTFE] syringe filters (Merck Millipore LTD, Ireland) LCR code. The syringe filters were 0.45 µm in size and hydrophilic in nature. Once diluted, the filtrates were examined for dissolved INM spectrophotometrically at 320 nm [5].

2.12. In-Vitro Dissolution Studies The dissolution rate and supersaturation parameter [SP] of INM from SDs and physical blends was tested under non-sink conditions using the United States Pharmacopeia [USP] dissolution testing apparatus 1 (basket technique) (Distek 50947, USA) with a paddle speed of 50 rpm. The non-sink dissolution test was performed using 900 mL of pH buﬀer 1.2 at a temperature of 37 0.5 C. ± ◦ Pharmaceutics 2020, 12, 212 6 of 24

A formulation (in powder form) which was equal to 100 mg of INM in physical blends and SD formulations was placed within the dissolution medium. At set time points, 5 mL aliquots were withdrawn and separated through a 25mm millex-LCR PTFE hydrophilic syringe channel (0.45 µm, Merck Millipore LTD, Ireland). At each time point, a similar volume of new medium was replaced as withdrawn. The INM concentration in each example was examined utilizing a UV-1280 UV–Vis spectrophotometer (Shimadzu, Japan) and a standard curve. Pure and amorphous INM were used as controls. The INM concentration dissolved for each formulation (n = 3) was plotted as a function of dissolution time information being expressed as the mean standard deviation of duplicate absorbance ± estimations. The ability of the polymeric carriers to prolong and maintain INM supersaturation in pH buﬀer 1.2 was qualiﬁed by using the SP as reported by Chen et al. (2015) [18].

2.13. Statistical Analysis Dissolution profiles of all the SD formulations using an analysis of variance [ANOVA] statistical test were compared. The area under the curve [AUC] was statistically examined to examine the effect of the amorphous form (GraphPad Prism version 5.03 for Windows, La Jolla, San Diego, CA, USA). Tukey’s Multiple Comparison post-hoc test was used to compare the means. A significance level of * p < 0.05 was used to signify the significance for all formulations [5].

2.14. Accelerated Amorphous Stability Studies

Accelerated stability studies were carried out under accelerated conditions (40 ◦C, 75% relative humidity) by placing SDs in open class vials which were stored inside a desiccator containing a saturated solution of sodium chloride to produce a relative humidity of 75%. The relative humidity inside the desiccator was checked constantly utilizing a thermohygrometer [5]. The amorphous state of the stored SDs was examined using hyper DSC and XRPD, as described previously.

3. Results and Discussion

3.1. Drug-polymer Miscibility Studies.

3.1.1. Pre-Formulation Studies Gaikwad et al. (2017) reported that within the pharmaceutical industry, HSPs are commonly used to determine the miscibility of an API with pharmaceutical excipients/carriers in SDs. It has also been reported that HSPs have been used within the pharmaceutical industry to determine the compatibility of pharmaceutical materials, and are therefore recommended in pre-formulation and in the development of SDs and tablets [19]. Maniruzzaman et al. (2015) used the Flory–Huggins (F–H) lattice-based theory to explain intermolecular interactions between polymer and API is limited as it does not take into consideration the various multiple interactions in drug-polymer systems [20]. Therefore, the HSPs developed by Van Krevelen and Hoftyzer group were used as a substitute for the Flory–Huggins theory to explore the nature of interactions that occur within drug–polymer systems and is mainly based upon the chemical structures of each of the pure components. For an intermolecular interaction to occur between an API and polymer, the change in the free energy of mixing must be negative according to the laws of thermodynamics of mixing. This speciﬁc change in the free energy of mixing is related to enthalpy and entropy contributions according to the following equation (Equation (4)):

∆G = ∆H T x ∆S (4) mix mix − mix where ∆Gmix, ∆Hmix, ∆Smix and T are the Gibbs free energy, enthalpy of mixing, entropy of mixing and absolute temperature, respectively. The free energy change is spontaneous as a result of the increase in entropy of mixing and is therefore negative [20]. However, the presence of repulsive Pharmaceutics 2020, 12, 212 7 of 24 and cohesive intermolecular and intramolecular forces (e.g., dispersion force, hydrogen bonding and dipole–dipole interaction) which are present within the amorphous SD, making the intermolecular interaction between drug and polymer more complex. The drug–polymer interaction factor was calculated via the Hildebrand-Scott method which is a hypothetical method used to calculate F–H interaction parameters [16]. It is well reported in the literature that unfavourable intermolecular interactions will result in phase separation and recrystallization if ∆δ > 7 MPa1/2, whilst favourable intermolecular interactions and a uniform phase will occur if ∆δ < 7 MPa1/2 [21]. This is also the case for drug–polymer interactions if the value for χ is close to zero, as shown in Pharmaceutics 2020, 12, x FOR PEER REVIEW 7 of 24 Table2. ∆δ and χ between INM and the polymers was less than 7 MPa1/2 and close to zero respectively, This is also the case for drug–polymer interactions if the value for χ is close to zero, as shown in thereforeTable 2. theyΔδ and χ are between likely INM and to the be polyme misciblers was less [16 than]. 7 MPa1/2 and close to zero respectively, therefore they are likely to be miscible [16].

TableTable 2. 2. Calculated solubility solubility parameters using parameters Hansen solubility using parameters Hansen (δ) and drug–polymer solubility parameters (δ) and drug–polymer interactioninteraction factor factor (χ). (χ). Compound δt (MPa1/2) Δδ (MPa1/2) χ INM 23.00 - - 1/2 1/2 PVPCompound VA64 26.40 a 3.40δt a(MPa 0.46 ) ∆δ (MPa ) χ PL-S630 26.40 3.40 0.46 P407INM 25.50b 2.50b 23.000.36 - - a and b are values obtained from a previously published report [5]. PVP VA64 26.40 a 3.40a 0.46 The molar attraction constant for INM was high (10.37 MPa1/2) and was similar to the values calculated for the polymers (PVP PL-S630VA64: 11.86 MPa1/2 and PL-S630: 26.40 11.86 MPa1/2) due to the 3.40 interaction 0.46 via hydrogen bonding. The molar attraction constant values are vital for intermolecular interactions between drug and polymer to occurP407 during the transformation25.50 to theb amorphous state. 2.50See Tablesb 3 0.36 to 6 for HSPs calculations used to predict drug–polymer miscibility and drug–polymer interaction factor (χ). a and b are values obtained from a previously published report [5]. Maniruzzaman et al. (2015) prepared amorphous solid dispersions using hot-melt extrusion using the BCS class II drug Verapamil HCL, which had a molar attraction constant of 6.95 MPa1/2 and reported that due to the high molar attraction constants of the APIs and polymeric carriers, they were able to contribute to hydrogen bonding [20]. As the HSPs are only theoretical, drug–polymer1 /2 miscibilityThe molar was further attraction examined using constant various characterization, for INM was thermal high analysis (10.37 and solubility MPa ) and was similar to the values calculatedtechniques for to determine the polymers if the drug–polymer (PVP mixtur VA64:es were 11.86 miscible. MPa In order1/2 forand a drug PL-S630: and polymer 11.86 MPa1/2) due to the interaction to be miscible it requires the drug to be successfully converted to the amorphous form and to interact via hydrogenwith the polymeric bonding. carrier. Conversion The molar to the amorphous attraction state constantwas determined values using XRPD are and vital for intermolecular interactions hyper DSC to not only determine if the drug is amorphous, but also to determine if only one glass betweentransition drug temperature and polymer(Tg) is present in to the occur SD formulations. during A single the transformationTg means successful integration to the amorphous state. See Tables3–6 for HSPsof the drug calculations into the polymer. used Interaction to predict between drug drug–polymer and polymer was examined miscibility using ATR-FTIR and drug–polymer interaction factor (χ). and Raman spectroscopy.

Table3.Table 3. CalculationCalculation of HSPs of and HSPs molar and volume molar for INM volume according forto the INM Hoftyzer-Van according Krevelen to the Hoftyzer-Van Krevelen method. method.

Group Frequency Fdi F2Pi Fhi Vm a Vm a (J1/2 cm 3/2 mol1 −/12) (J3/1/22 cm 3/2 mol−11) (J/mol)2 (cm1/23/mol)3 (/V2o) 1 Group Frequency Fdi (J cm mol − )F Pi (J cm mol− ) Fhi (J/mol) 3 –CH3 2 840 0 0 67 (cm /mol) (Vo) –CH–CH2– 21 270 8400 0 32.2 0 0 67 =CH–3 3 600 0 0 40.5 >C= 5 350 0 0 −27.5 –CH2– 1 270 0 0 32.2 Phenylene (o,m,p) 1 1270 12,100 0 52.4 =CH––Cl 31 450 600302,500 400 24 0 0 40.5 –O– 1 100 160,000 3000 3.8 >C= 5 350 0 0 27.5 –CO– 1 290 770 2000 10.8 − Phenylene–COOH (o,m,p) 11 530 1270176,400 10,000 12,10028.5 0 52.4 –N< 1 20 640,000 5000 −9 Ring closure–Cl 5 or more atoms 1 2 380 450 0 0 302,500 32 400 24 Conjugation in ring for each double bond 4 0 0 0 -8.8 –O– 1 100 160,000 3000 3.8

–CO– 1 290 770 2000 10.8 –COOH 1 530 176,400 10,000 28.5 –N< 1 20 640,000 5000 9 − Ring closure 5 or more 2 380 0 0 32 atoms Conjugation in ring for 4 0 0 0 -8.8 each double bond P 5100 1291770 27400 254.7 Pharmaceutics 2020 12 Pharmaceutics 2020, 12, x FOR, PEER, 212 REVIEW 8 of 24 8 of 24

∑ 5100 1291770 27400 254.7 Table 4. Calculation of HSPs and molar volume for PVP VA64/PL-S630 according to the Hoftyzer-Van PharmaceuticsTable 2020 , 4.12 ,Calculation x FOR PEER ofREVIEW HSPs and molar volume for PVP VA64/PL-S630 according to the Hoftyzer-8 of 24 VanKrevelen Krevelen method. method. ∑ 5100 1291770 27400 254.7

Table 4. Calculation of HSPs and molar volume for PVP VA64/PL-S630 according to the Hoftyzer- Van Krevelen method.

Group Frequency Fdi (J1/2 cm 3/2 mol −1) F2Pi (J1/2 cm 3/2 mol −1) Fhi (J/mol) Vm a (cm3/mol) (Vo) Vm a Group Frequency F (J1/2 3/2 1)F2 (J1/2 3/2 1) F (J/mol) –CH3 1 420 di 0 cm mol − 0 Pi cm 33.5 mol − hi (cm3/mol) (Vo) –CH2– 2 540 0 0 32.2 –CH 1 420 0 0 33.5 –CH– 3 2 160 0 0 −2.00 Group–CO– –CH Frequency2–1 Fdi (J1/2 cm 3/2 mol 290 2 −1 ) F2Pi (J1/2 cm 3/2 mol 770 −1 ) 540 Fhi (J/mol)2000 Vm a (cm3/mol) 010.8 (V o) 0 32.2 3 –N< 1 20.0 800 5000 -9.00 –CH –CH–1 420 2 0 160 0 33.5 0 0 2.00 –CH2–COO–– 2 1 540 390 0 490 0 7000 32.2 18.0 − –CH–Ring 5 or more–CO– 2 1 160 190 1 0 0 290 0 0 −2.00 770 16.0 2000 10.8 –CO– ∑ –N< 1 290 2010.01 770 1,473,000 20.0 2000 14,000 10.8 800 99.50 5000 -9.00 –N< 1 20.0 800 5000 -9.00 –COO– 1 390 490 7000 18.0 Table–COO– 5. Calculation1 of HSPs and 390 molar volume 490for P407 according 7000 to the Hoftyzer-Van 18.0 Krevelen Ringmethod. 5 or moreRing 5 or more 1 190 1 0 190 0 16.0 0 0 16.0 ∑ P 2010.0 1,473,000 14,000 99.50 2010.0 1,473,000 14,000 99.50

Table 5. Calculation of HSPs and molar volume for P407 according to the Hoftyzer-Van Krevelen method.Table5. Calculation of HSPs and molar volume for P407 according to the Hoftyzer-VanKrevelen method.

Group Frequency Fdi (J1/2 cm 3/2 mol−1) F2Pi (J1/2 cm 3/2 mol−1) Fhi (J/mol) Vm a (cm3/mol) (Vo) –CH3 1 420 0 0 33.5 –CH2– 5 2100 0 0 80.5 –OH 2 420 500,000 20,000 20 a Group –O–Frequency 2 Fdi (J1/2 cm 3/2 mol 200−1) F2Pi (J1/2 cm 3/2 320,000 mol−1) 1 /2 Fhi (J/mol)3/2 3000 1 Vm a (cm23/mol)1 7.6/2 (V o) 3/2 1 Vm Group Frequency Fdi (J cm mol− )F Pi (J cm mol− ) Fhi (J/mol) 3 –CH3 ∑ 1 420 2390 0 820,000 0 46,000 33.5 141.6 (cm /mol) (Vo)

–CH2– –CH5 3 2100 1 0 420 0 80.5 0 0 33.5 –OHTable 6. Calculated2 HSPs 420 of and drug–polymer 500,000 interaction 20,000 factor for drug 20 and polymers. –CH – 5 2100 0 0 80.5 –O– 2 2 200 320,000 3000 7.6 Identifier∑ –OH 2390 2Solubility 820,000 Parameters 420 46,000 141.6 500,000 20,000 20 δd (MPa1/2) δp (MPa1/2) δh (MPa1/2) δv (MPa1/2) (Vo) δtotal (MPa1/2) Δδt (MPa1/2) χ INM –O–20.02 4.46 210.37 20.51 200 23.00 320,000 - - 3000 7.6 Table 6. CalculatedP HSPs of and drug–polymer interaction factor for drug and polymers. PVP VA64 20.20 12.20 11.86 23.592390 26.40 820,000 3.40 0.46 46,000 141.6 IdentifierPL-S630 20.20 12.20 Solubility11.86 Parameters 23.59 26.40 3.40 0.46

P407 δd (MPa1/216.88) δp (MPa1/26.40) δh (MPa1/218.02) δv (MPa1/2) 18.05(Vo) δtotal (MPa 25.501/2) Δδt (MPa1/2 2.50) χ 0.36 INM Table20.02 6. Calculated4.46 10.37 HSPs of and 20.51 drug–polymer 23.00 interaction - factor - for drug and polymers. 3.1.2.PVP ATR-FTIRVA64 20.20 and Raman12.20 Studies 11.86 23.59 26.40 3.40 0.46 PL-S630 20.20 12.20 11.86 23.59 Solubility 26.40 Parameters 3.40 0.46 P407ATR-FTIR 16.88was used 6.40in conjunction18.02 with Rama 18.05 n studies 25.50 to elucidate 2.50 the mechanism0.36 of interaction between INM and eachδ ofd the polymersδp which is necessaryδh for drug–polymer1/2 miscibility.δtotal ∆δt Identiﬁer 1/2 1/2 1/2 δv (MPa ) (Vo) 1/2 1/2 χ 3.1.2.XRPD ATR-FTIR and andhyper Raman DSC Studies was used (MPa to confirm) (MPa the am) orphous(MPa nature) of INM in each of(MPa the SD) (MPa ) formulations and to determine Tg. PVP VA64 and PL-S630 showed two strong bands at 1731 cm−1 and ATR-FTIR was usedINM in conjunction20.02 with Rama 4.46n studies 10.37 to elucidate the 20.51 mechanism 23.00of - - 1672 cm−1 which correspond to the C=O stretch of the vinyl acetate and the C=O stretch of the amide interaction between PVPINMVA64 and each of the20.20 polymers 12.20which is necessary 11.86 for drug–polymer 23.59 miscibility. 26.40 3.40 0.46 carbonyl respectively. When INM was converted to the amorphous form via hot melt extrusion, the XRPD and hyper DSCPL-S630 was used to 20.20confirm the 12.20amorphous nature 11.86 of INM 23.59in each of the 26.40SD 3.40 0.46 amide carbonyl C=O of PVP VA64 and PL-S630 shifted to 1680 cm−1. This indicates that hydrogen formulations and to determine Tg. PVP VA64 and PL-S630 showed two strong bands at 1731 cm−1 and bonding had takenP407 place between16.88 the amide carbonyl 6.40 of the 18.02 polymer and the 18.05 free C=O bond 25.50 of the 2.50 0.36 1672 cm−1 which correspond to the C=O stretch of the vinyl acetate and the C=O stretch of the amide carboxylic acid of INM [5]. This was the case for all SD formulations, as shown in Figure 2a. carbonyl respectively. When INM was converted to the amorphous form via hot melt extrusion, the −1 amide carbonylManiruzzaman C=O of PVP VA64 et and al. PL-S630 (2015) shifted prepared to 1680 amorphouscm . This indicates solid that dispersions hydrogen using hot-melt extrusion bonding had taken place between the amide carbonyl of the polymer and the free C=O bond of the 1/2 carboxylic using acid the of INM BCS [5]. class This IIwas drug the case Verapamil for all SD formulations, HCL, which as shown hada in molar Figure 2a. attraction constant of 6.95 MPa and reported that due to the high molar attraction constants of the APIs and polymeric carriers, they were able to contribute to hydrogen bonding [20]. As the HSPs are only theoretical, drug–polymer miscibility was further examined using various characterization, thermal analysis and solubility techniques to determine if the drug–polymer mixtures were miscible. In order for a drug and polymer to be miscible it requires the drug to be successfully converted to the amorphous form and to interact with the polymeric carrier. Conversion to the amorphous state was determined using XRPD and hyper DSC to not only determine if the drug is amorphous, but also to determine if only one glass transition temperature (Tg) is present in the SD formulations. A single Tg means successful integration of the drug into the polymer. Interaction between drug and polymer was examined using ATR-FTIR and Raman spectroscopy. Pharmaceutics 2020, 12, 212 9 of 24

3.1.2. ATR-FTIR and Raman Studies ATR-FTIR was used in conjunction with Raman studies to elucidate the mechanism of interaction between INM and each of the polymers which is necessary for drug–polymer miscibility. XRPD and hyper DSC was used to conﬁrm the amorphous nature of INM in each of the SD formulations and 1 1 to determine Tg. PVP VA64 and PL-S630 showed two strong bands at 1731 cm− and 1672 cm− which correspond to the C=O stretch of the vinyl acetate and the C=O stretch of the amide carbonyl respectively. When INM was converted to the amorphous form via hot melt extrusion, the amide 1 carbonyl C=O of PVP VA64 and PL-S630 shifted to 1680 cm− . This indicates that hydrogen bonding had taken place between the amide carbonyl of the polymer and the free C=O bond of the carboxylic

acidPharmaceutics of INM [205].20 This, 12, x wasFOR PEER the case REVIEW forall SD formulations, as shown in Figure2a. 9 of 24 Pharmaceutics 2020, 12, x FOR PEER REVIEW 9 of 24

FigureFigure 2. 2.(a ()a ATR-FTIR) ATR-FTIR Spectra Spectra of of pure pure components components and and selected selected SD SD formulations formulations (30% (30% INM) INM) showing showing Figure 2. (a) ATR-FTIR Spectra of pure components and1 −1 selected SD1 −1 formulations (30% INM) showing a shifta shift in in the the amide amide carbonyl carbonyl C= C=OO from from 1672 1672 cm cm− to to 1680 1680 cm cm− . There. There is is no no shift shift in in the the vinyl vinyl acetate acetate −1 −1 a shiftcarbonylcarbonyl in the peak amidepeak as as a carbonyl resulta result of ofC=O its its weaker fromweaker 1672 hydrogen hydrogen cm to bond bond1680 potential. cmpotential.. There (b ()b ATR-FTIRis) ATRno shift-FTIR spectrain spectrathe vinyl of of binary acetatebinary SDs SDs carbonyl(controls)(controls) peak of of 10%as 10 a %result and and 30% of30% its INM-P407 INMweaker-P407 hydrogen drug–polymer drug–polymer bond mixtures.potential. mixtures. (b ) ATR-FTIR spectra of binary SDs (controls) of 10% and 30% INM-P407 drug–polymer mixtures. ThereThere was was no change no change in the in vinyl the acetate vinyl acetate C=O carbonyl C=O carbonyl due to weaker due to hydrogen weaker bondinghydrogen potential bonding ofpotential theThere vinyl was of acetate the no vinyl change carbonyl acetate in the as carbonyl reported vinyl acetate as byreported Yuan C=O etby carbonyl al. Yuan (2015) et due al as. to(2 shown015) weaker as in shown hydrogenFigure in2 bFigure [ bonding22]. 2b Any [22] . potentialP407Any peaks P407 of the withinpeaks vinyl within the acetate pure the carbonyl P407 pure sample P407 as samplereported were completelywere by Yuancompletely et absent al. (2absent in015) the as in ATR-FTIR shownthe ATR in- spectraFTIRFigure spectra of2b the[22] of SD. the AnyformulationsSD P407 formulations peaks as within shown as the shown in pure Figures inP407 Figure2a sample and 32.a This wereand indicates completelyFigure 3 that. This absent P407 indicates possiblyin the ATRtha hast- FTIR P407 no intermolecular spectra possibly of hasthe no SDinteraction intermolecular formulations with as INM interaction shown at the in molecularwith Figure INM 2 levelaat and the [9 molecular].Figure 3. level This [9]indicates. that P407 possibly has no intermolecular interaction with INM at the molecular level [9].

Figure 3. ATR-FTIR Spectra of aINM and Physical blend. Crystalline INM is still present in the FigureFigurephysical 3. 3. ATRATR-FTIR blend-FTIR as Spectraexpected. Spectra of of aINM aINM and and Physical Physical blend. blend. Crystalline Crystalline INM INM is still is present still present in the physicalin the physicalblend asblend expected. as expected. Pure INM contains two strong non-hydrogen bonding C=O bands at 1690 cm-1 (acid-acid dimer -1 C=OPure stretch) INM contains and 1714 two cm strong−1 (free nonC=O-hydrogen of carboxylic bonding acid) C=Orespectively bands at [23] 1690. The cm hydrogen (acid-acid-bonded dimer O– C=OH stretch) stretch and of the 1714 acid, cm −shown1 (free C=O in Figure of carboxylic 4, is overlaid acid) respectively on the C–H [23] sharp. The stretches, hydrogen as-bonded literature O– has H stretchreported of thatthe acid,the free shown carboxylic in Figure acid 4O, –isH overlaid stretch as on a resultthe C –ofH hydrogen sharp stretches, bonding as can literature exist as hasdimers reported[23]. It that is also the importantfree carboxylic to note acid that O –inH the stretch ATR as-FTIR a result reference of hydrogen spectrum bonding of aINM, can the exist two as C=Odimers bands [23]at. It 1714 is also cm important−1 (free C=O to ofnote carboxylic that in the acid) ATR and-FTIR 1690 reference cm−1 (acid spectrum-acid dimer of aINM, C=O thestretch) two C=Oshifted bands to 1707 −1 −1 at 1714cm− 1cm and (free1679 C=Ocm−1 ,of respectively carboxylic, acid)due to and transformation 1690 cm (acid to -itsacid amorphous dimer C=O form stretch) [5], as shifted shown to in 1707 Figure cm−12a, and therefore 1679 cm do−1, respectivelynot align with, due the to polymer transformation peaks. The to its physical amorphous blends form contained [5], as shown crystalline in Figure INM as 2a, thereforeexpected doas shownnot align in Figurewith the 3 [5]polymer. peaks. The physical blends contained crystalline INM as expected as shown in Figure 3 [5].

Pharmaceutics 2020, 12, 212 10 of 24

Pure INM contains two strong non-hydrogen bonding C=O bands at 1690 cm-1 (acid-acid dimer 1 C=O stretch) and 1714 cm− (free C=O of carboxylic acid) respectively [23]. The hydrogen-bonded O–H stretch of the acid, shown in Figure4, is overlaid on the C–H sharp stretches, as literature has reported that the free carboxylic acid O–H stretch as a result of hydrogen bonding can exist as dimers [23]. It is also important to note that in the ATR-FTIR reference spectrum of aINM, the two C=O bands at 1714 1 1 1 cm− (free C=O of carboxylic acid) and 1690 cm− (acid-acid dimer C=O stretch) shifted to 1707 cm− 1 and 1679 cm− , respectively, due to transformation to its amorphous form [5], as shown in Figure2a, therefore do not align with the polymer peaks. The physical blends contained crystalline INM as PharmaceuticsPharmaceuticsexpected 20 as 2020 shown20, 1,2 1, 2x, FORx FOR in PEER Figure PEER REVIEW REVIEW3[5]. 1010 of of 24 24

FigureFigureFigure 4. 4.ATR 4. ATRATR-FTIR-FTIR-FTIR Spectr Spectr Spectrumumum of of ofcrystalline crystalline crystalline INM. INM. INM.

Pharmaceutical drugs which are usually conjugated compounds have very strong Raman signals, PharmaceuticalPharmaceutical drugs drugs which which are are usually usually conjugated conjugated compounds compounds have have very very strong strong Raman Raman while pharmaceutical excipients tend to have Raman signals which are weak [8]. Like ATR-FTIR, Raman signals,signals, while while pharmaceutical pharmaceutical excipients excipients tend tend to to have have Raman Raman signals signals which which are are weak weak [8] [8]. Like. Like ATR ATR- - spectroscopy can identify potential mechanism of intermolecular interaction among the polymeric FTIRFTIR, Raman, Raman spectroscopy spectroscopy can can identify identify potential potential mechanism mechanism of of intermolecular intermolecular interaction interaction among among the the carriers and INM due to a shift in the PVP VA64 and PL-S630. polymericpolymeric carriers carriers and and IN INMM due due to to a ashift shift in in the the PVP PVP VA6 VA64 4and and PL PL-S630.-S630. Hydrogen bonding was the predicted mechanism of interaction due to polarity of INM and HydrogenHydrogen bonding bonding was was the the predicted predicted mechanism mechanism of of interaction interaction due due to to polarity polarity of of INM INM and and polymeric carriers as a result of the relatively high INM molar attraction constant calculated using the polymericpolymeric carriers carriers as as a aresult result of of the the relatively relatively high high INM INM molar molar attraction attraction constant constant calculated calculated using using 1 1 HSPs. The amide carbonyl (vC=O) peak at 1673 cm shifted−1 to 1680 cm in−1 all SD formulations due thethe HSPs. HSPs. The The amide amide carbonyl carbonyl (v (C=O)vC=O) peak peak at at 1673 1673 cm− cm−1 shifted shifted to to 1680 1680 cm− cm−1 in in all all SD SD formulations formulations to hydrogen bond interaction with the –OH carboxylic acid of INM, as shown in Figure5. The intensity duedue to to hydrogen hydrogen bond bond interaction interaction with with the the – OH–OH carboxylic carboxylic acid acid of of INM INM, as, as shown shown in in Figure Figure 5 .5 The. The -1 of the vinyl acetate carbonyl group is weak and appears at 1745 cm due to the-1 weaker hydrogen intensityintensity of of the the vinyl vinyl acetate acetate carbonyl carbonyl group group is is weak weak and and appears appears at at 1745 1745 cm cm-1 due due to to the the weaker weaker bonding potential as reported by Yuan et al. (2015) [22]. hydrogenhydrogen bonding bonding potential potential as as reported reported by by Yuan Yuan et et al al. (2015). (2015) [22] [22]. .

Figure 5. Raman spectra of crystalline INM, polymeric carriers and chosen SD formulations (30% Figure 5. Raman spectra of crystalline INM, polymeric carriers and chosen SD formulations (30% FigureINM), 5. which Raman shows spectra interaction of crystalline via hydrogen INM, polymeric bonding as carriers a result ofand the chosen shift in SD the formulations amide C=O carbonyl (30% INM),INM), which which shows shows interaction interaction via via hydrogen hydrogen bonding bonding as as a a result result of of the the 1 shift shift in in the the amide amide C=O C=O stretch of PL-S630 and PVP VA64. The low intense peak at 1732.00 cm− corresponds to the vinyl carbonyl stretch of PL-S630 and PVP VA64. The low intense peak at 1732.00 cm−1 −1 corresponds to the carbonylacetate stretch C=O carbonyl. of PL-S630 and PVP VA64. The low intense peak at 1732.00 cm corresponds to the vinylvinyl acetate acetate C=O C=O carbonyl. carbonyl. 1 The acid vC=O present at 1702 cm− (free C=O of carboxylic acid) of INM disappears in the RamanTheThe acid spectra acid v C=OvC=O of present the present quaternary at at 1702 1702 cm and cm−1 ternary−(1free (free C=O C=O SD of formulationsof carboxylic carboxylic acid) dueacid) of to of INM the INM low disappears disappears intensity in ofin the INM.the Raman Raman Based spectraspectraon the of Ramanof the the quaternary quaternary spectra in and Figureand ternary ternary5, the SD peaksSD formulations formulations identiﬁed due in due the to to Ramanthe the low low spectra intensity intensity of aINMof of INM. INM. were Based Based not on present on the the RamanRaman sp spectraectra in in F igureFigure 5 ,5 the, the peaks peaks identified identified in in the the Raman Raman spectra spectra of of aINM aINM were were not not present present in in thethe Raman Raman spectra spectra of of the the amorphous amorphous SD SD formulations. formulations. The The broad broad peak peak at at 1482 1482 cm cm−1 −1in in the the Raman Raman spectrumspectrum of of solid solid-state-state P407 P407 reflected reflected the the deformation deformation vibration vibration of of the the δCH δCH2 group.2 group. TheThe v (Ov(O–H)–H) of of P407 P407 was was present present at at 3200 3200 cm cm−1 −and1 and v (Cv(C-O-O-C)-C) was was present present at at 850 850 cm cm−1.− 1Careful. Careful analysisanalysis of of the the S DSD formulations formulations indicates indicates that that the the bands bands associated associated with with P 407P407 were were not not present present in in the the SDSD formulations formulations due due to to the the lack lack of of an an inter intermolecularmolecular interaction. interaction. Raman Raman spectroscopy spectroscopy similar similar to to ATR ATR- - FTIRFTIR spectroscopyspectroscopy ideidentifiedntified that that hydrogenhydrogen bonding bonding occurred occurred between between PVP PVP VA64/ VA64/PLPL-S630-S630 regardregardlessless of of the the cooling cooling method method used. used.

3.3.1.1.3.3 XRPD. XRPD and and Hyper Hyper DSC DSC Studies Studies XRPDXRPD was was used used to to examine examine the the amorphous amorphous nature nature of of the the drug drug within within the the SD SD formulations. formulations. XRPD XRPD waswas performed performed on on a aPANalytical PANalytical Empyrean Empyrean X -Xray-ray diffractometer diffractometer attached attached to to a acomputer computer running running

Pharmaceutics 2020, 12, 212 11 of 24

1 in the Raman spectra of the amorphous SD formulations. The broad peak at 1482 cm− in the Raman spectrum of solid-state P407 reflected the deformation vibration of the δCH2 group. 1 1 The v(O–H) of P407 was present at 3200 cm− and v(C-O-C) was present at 850 cm− . Careful analysis of the SD formulations indicates that the bands associated with P407 were not present in Pharmaceutics 2020, 12, x FOR PEER REVIEW 11 of 24 the SD formulations due to the lack of an intermolecular interaction. Raman spectroscopy similar to ATR-FTIRHigh Score spectroscopy Plus to collect identified and process that data. hydrogen The XRPD bonding of INM occurred shows multiple between Bragg PVP peaks VA64 similar/PL-S630 regardlessto the of literature the cooling values method reported used. for INM [8], indicating the crystalline nature of the drug, as shown in Figure 6a. Figure 6a also shows the XRPD diffractograms of P407, PL-S630 and PVP VA64. Figure 3.1.3. XRPD7 shows and the HyperXRPD diffractograms DSC Studies of the SD formulations used to investigate the effect of cooling. The XRPD diffractograms of PL-S630 and PVP VA64 showed that they were amorphous in XRPDnature was due used to slight to examine amorphous the halo amorphous raised above nature the ofbaseline. the drug The within presence the of SD two formulations. Bragg peaks of XRPD was performedP407 at 19 on and a PANalytical24° respectively Empyrean indicates X-ray that P407 diffractometer was not converted attached to to the a computeramorphous running form as High Score Plusreported tocollect by Hurley and et processal. (2018) data.[5]. The The drug XRPD peaks associated of INM shows with crystalline multiple INM Bragg were peaks completely similar to the literatureabsent in values the melt reported extrudates for with INM various [8], indicating drug–polymer the ratios, crystalline indicating nature that ofINM the was drug, successfully as shown in Figure6converteda. Figure to6 aits also amorphous shows theform XRPD as shown di ff inractograms Figure 7. Hy ofper P407, DSC PL-S630, like the XRPD and PVP diffractograms VA64. Figure, 7 showsalso the confirmed XRPD diff thatractograms INM was ofsuccessfully the SD formulations converted to its used amorphous to investigate form and the P407 eff ectremained of cooling. semi- crystalline.

FigureFigure 6. (a) XRPD6. (a) XRPD diﬀractograms diffractograms of of the the pure pure components components and and (b (b) )hyper hyper DSC DSC thermograms thermograms of pure of pure componentscomponents after after heating heating at 100 at 100◦C /°C/minmin to to 180 180◦ C.°C. INMINM is transformed transformed to tothe the amorphous amorphous form. form. (b) (b) HyperHyper DSC shows DSC shows that purethat pure P407 P407 displays displays an an endothermic endothermic peak at at 57.13 57.13 °C◦ dueC due to its to semi its semi-crystalline-crystalline Pharmaceutics 2020, 12, x FOR PEER REVIEW 12 of 24 nature.nature. Glass Glass transition transition temperature temperature [T g[T]g is] is indicated indicated by a a tick. tick.

Hyper DSC was performed to characterize the solid state of the SD formulations and to determine the Tg of the SD formulations. The DSC thermograms of the pure components and SD formulations are shown in Figures 6b, 8 and 9. The % crystallinity of P407 for each of the samples was also determined. The thermogram of crystalline INM showed an endothermic peak at 165.47 °C corresponding to the melting endotherm of the stable γ INM form. Thermograms of all the SD formulations characterized showed the complete disappearance of the melting endotherm, indicating that INM was completely converted to its amorphous state, SD formulations existed as a two-phase system due to the presence of the semi-crystalline P407. In the low-temperature region of the DSC scan, the glass transition temperature [Tg] of aINM was detected at 45.5 °C [2]. Pezzoli et al. (2019) also reported a Tg of 45.5 °C for aINM when processed using HME [2]. PVP VA64/PL-S630 were indeed amorphous with a Tg at 109 and 107 °C respectively, as expected, based on the XRPD results.

FigureFigure 7. XRPD 7. XRPD diff ractogramsdiffractograms of of SD SD formulations formulations and and XRPD XRPD diffractograms diffractograms of the of SD the formulation SD formulation used toused investigate to investigate the the eff ecteffect of of cooling. cooling. P407 P407 was still still present present in inits itscrystalline crystalline form form in all informulations all formulations (AC = Air cooled, NT = Normal room temperature and Liq N2 = Liquid Nitrogen). (AC = Air cooled, NT = Normal room temperature and Liq N2 = Liquid Nitrogen).

A separate Tg attributable to INM was not present in the hyper DSC thermograms of the SD formulations, suggesting a complete absence of a separated phase rich in amorphous INM and a good

level of mixing. P407 also displayed an endothermic peak at 58.16 °C due to its semi-crystalline structure with an enthalpy of fusion (ΔHm) of 117.1 J/g which corresponds to its crystalline fraction. Therefore, assuming that 100% semi-crystalline P407 has an enthalpy of fusion of 117.1 J/g, the degree of crystallinity of P407 within SD formulations was calculated. The P407 enthalpy of fusion (J/g) was calculated by dividing the area of the endothermic (melt) peak by the heating rate, which was 100°C/min. The % crystallinity of each of the SD formulations was calculated by dividing the P407 enthalpy of fusion (J/g) of each of the formulations by the theoretical enthalpy of fusion (J/g) of both the 5% and 15% P407 formulations. For SD1-AC to SD6-AC, the P407 enthalpy of fusion (J/g) in Table 7 was divided by 5.86 J/g for the 5% P407 formulations and 17.56 J/g for the 15% P407 formulations and multiplied by 100 to determine the % crystallinity in each of the formulations. For example, for SD1-AC, 0.593 J/g was divided by 5.86 J/g and multiplied by 100 to give 10.12 % P407 crystallinity. Table 7 displays an overview of the degree of crystallinity exhibited by the various extrudates and glass transition temperatures (experimental vs predicted) relative to the pure crystalline drug. After careful analysis of the hyper DSC thermograms of the SD formulations, the endotherm attributed to the melting of the P407 fraction was detected in all formulations at 52.63 °C, with a mean ΔHm of 0.862 J/g for the 5% P407 SD formulations and 6.68 J/g for the 15% P407 SD formulations. When calculated, ΔHm of 100% crystalline P407 in 5% SD formulations was 5.86 J/g which corresponds to 14.7% crystalline P407. Similarly, 15% SD formulations contain in theory 38.0% P407.

Pharmaceutics 2020, 12, x FOR PEER REVIEW 13 of 24

Table 7. Experimental Tg, Predicted Tg, P407 enthalpy of fusion (J/g) and % crystallinity of various SD formulations and selected SD formulations used to investigate the effect of cooling on the solubility of INM.

Identifier No Experimental Tg Predicted Tg (Couchman-Karasz) P407 Enthalpy of fusion (J/g) % Crystallinity Crystalline INM 45.56 - - - P407 −67.00 - 117.1 - SD1-AC 74.91 83.54 0.593 10.12 SD2-AC 67.42 84.70 9.551 54.39 SD3-AC 71.82 80.14 0.963 16.43 SD4-AC 60.54 78.26 5.697 32.44 SD5-AC 74.58 86.94 1.032 17.61 SD6-AC 67.42 84.71 4.799 27.33 SD2-NT 69.24 84.70 11.90 67.78 SD2-Liq N2 70.19 84.70 9.478 53.97

Pharmaceutics 2020, 12, 212 12 of 24 Considering the concentration of plasticizer present in the amorphous SD formulations, it was estimated that the % crystallinity present in the 5% P407 formulations was within the range of 10.12% to 17.61The XRPD% in its di crystallineffractograms form ofPL-S630 and 27.33 and% to PVP 54.39 VA64% for showed the 15% that P407 they formulations. were amorphous It is inimportant nature dueto tonote slight that amorphous the formulations halo raised with abovethe higher the baseline. P407 loadings The presence had a degree of two Braggof crystallinity peaks of P407that was at 19significantly and 24◦ respectively higher than indicates formulations that P407 with was low not P407 converted loading to since the amorphousthe APIs can form be incorporated as reported byinto Hurleythe core et al.of P407 (2018) micelle [5]. The at drughigh peaksloads associated[24]. with crystalline INM were completely absent in the melt extrudatesIt is important with various that cooling drug–polymer also had ratios, a significant indicating effect that INM on the was degree successfully of crystallinity. converted to For itsexample, amorphous SD2 form-NT had as shown the highest in Figure % 7crystall. Hyperinity DSC,, indicating like the XRPD that using di ffractograms, a high P407 also loading confirmed with a thatslow INM cooling was successfully process increases converted the % to crystallinity its amorphous. Whereas form and SD2 P407-Liq remained N2 which semi-crystalline. was a rapid cooling methodHyper, had DSC a waslow performed% crystallinity. to characterize the solid state of the SD formulations and to determine the Tg Forof the all SD SD formulations. formulations, The a single DSC thermogramsTg was observed of the which pure confirms components that andINM SD was formulations converted to are its shownamorphous in Figures form6b,, indicating8 and9. The that % crystallinitythe polymers of and P407 drug for eachwere of present the samples in a two was-phase also determined.system as P407 was still semi-crystalline (see Figures 8 and 9).

FigureFigure 8. 8.Hyper Hyper DSC DSC thermograms thermograms of of air-cooled air-cooled SD SD formulations. formulations. Hyper Hyper DSC DSC indicates indicates that that INM INM was was Pharmaceuticsconverted 2020 to, 1 its2, x amorphous FOR PEER REVIEW form. The area of interest for T is marked by the straight lines. 14 of 24 converted to its amorphous form. The area of interest for gTg is marked by the straight lines.

FigureFigure 9.9.Hyper Hyper DSC DSC thermograms thermograms of selected of selected SD formulations SD formulations used toused investigate to investigate the eﬀect the of effect cooling. of P407cooling. was P407 still w presentas still inpresent its crystalline in its crystalline form in allform formulations in all formulations (AC = Air (AC cooled, = Air cooled, NT = Normal NT = Normal room

temperatureroom temperature and Liq and N 2Liq= LiquidN2 = Liquid Nitrogen). Nitrogen). Hyper Hyper DSC DSC indicates indicates that that INM INM was was converted converted to itsto

amorphousits amorphous form. form. The The area area of interestof interest for forTg isTg marked is marked by by straight straight lines. lines.

The thermogram of crystalline INM showed an endothermic peak at 165.47 C corresponding to The experimental glass transition temperatures were significantly different◦ to the predicted Tgs thecalculated melting using endotherm the Couchman of the stable–Karaszγ INM equat form.ion Thermograms, as shown in of Table all the 7, SDas drug formulations–polymer characterized loading can have a significant effect on the Tg. The effect of drug and polymer loading on the glass transition of SD formulations was predicted using the model reported by Couchman and Karasz [25,26]. The theoretical glass transition of the SD formulations was calculated using the following Equation (5):

( 푥1훥퐶P1푇푔1+푥2훥퐶P2푇푔2) Tgmix (HME System) = (5) (푥1훥퐶P1+ 푥2훥퐶P2)

where Tg1 and Tg2, are the Tgs of API and polymeric carriers, respectively, X1 and X2 correspond to the weight fractions of each of the pure materials and ΔCp1 and ΔCp2 corresponds to the change in the specific heat capacity at each of the glass transition temperature, respectively [27]. This analysis confirmed that P407 was not solubilized and is present in its semi-crystalline form with some level of compatibility with PL-S630/PVP VA64-INM system due to the reduction of the enthalpy of fusion and melting temperature in the various SD formulations. This was consistent with higher Bragg peak intensities observed in the XRPD data from the 15% P407 sample with 30% drug loading. The cooling method used had very little effect on the Tg. However, the degree of crystallinity was also affected by (1) the cooling method used (2) % P407 loading and (3) the polymeric carrier used. For example, SD4 had a higher % crystallinity compared to SD6. This is because PL-S630 and PVP VA64 possess different solubility properties with PVP VA64-INM SD formulations having a significantly greater than INM-PL-S630 SDs. There was a significant difference in the Tgs between SD4 and SD6 due to the different solid-state properties of PL-S630 and PVP VA64. However, the inclusion of P407 improved the processability and fragility of the extrudates, and also lowered the processing temperatures. The HSPs alongside XRPD and hyper DSC confirmed that the drug and polymer were indeed miscible. XRPD and hyper DSC confirmed successful conversion of INM to the amorphous form as mentioned before, hyper DSC also confirmed that although P407 remained semi-crystalline, as only one single Tg was present in all SD formulations. According to Couchman and Kasasz, (1978) if a SD formulation only contains one Tg then the drug and polymer are indeed miscible. ATR-FTIR and Raman spectroscopy confirmed hydrogen bonding interaction between PVP VA64/PL-S630 and INM due to the interaction between the amide carbonyl C=O of the polymer and the free carboxylic acid

Pharmaceutics 2020, 12, 212 13 of 24 showed the complete disappearance of the melting endotherm, indicating that INM was completely converted to its amorphous state, SD formulations existed as a two-phase system due to the presence of the semi-crystalline P407. In the low-temperature region of the DSC scan, the glass transition temperature [Tg] of aINM was detected at 45.5 ◦C[2]. Pezzoli et al. (2019) also reported a Tg of 45.5 ◦C for aINM when processed using HME [2]. PVP VA64/PL-S630 were indeed amorphous with a Tg at 109 and 107 ◦C respectively, as expected, based on the XRPD results. A separate Tg attributable to INM was not present in the hyper DSC thermograms of the SD formulations, suggesting a complete absence of a separated phase rich in amorphous INM and a good level of mixing. P407 also displayed an endothermic peak at 58.16 ◦C due to its semi-crystalline structure with an enthalpy of fusion (∆Hm) of 117.1 J/g which corresponds to its crystalline fraction. Therefore, assuming that 100% semi-crystalline P407 has an enthalpy of fusion of 117.1 J/g, the degree of crystallinity of P407 within SD formulations was calculated. The P407 enthalpy of fusion (J/g) was calculated by dividing the area of the endothermic (melt) peak by the heating rate, which was 100◦C/min. The % crystallinity of each of the SD formulations was calculated by dividing the P407 enthalpy of fusion (J/g) of each of the formulations by the theoretical enthalpy of fusion (J/g) of both the 5% and 15% P407 formulations. For SD1-AC to SD6-AC, the P407 enthalpy of fusion (J/g) in Table7 was divided by 5.86 J /g for the 5% P407 formulations and 17.56 J/g for the 15% P407 formulations and multiplied by 100 to determine the % crystallinity in each of the formulations. For example, for SD1-AC, 0.593 J/g was divided by 5.86 J/g and multiplied by 100 to give 10.12 % P407 crystallinity. Table7 displays an overview of the degree of crystallinity exhibited by the various extrudates and glass transition temperatures (experimental vs predicted) relative to the pure crystalline drug. After careful analysis of the hyper DSC thermograms of the SD formulations, the endotherm attributed to the melting of the P407 fraction was detected in all formulations at 52.63 ◦C, with a mean ∆Hm of 0.862 J/g for the 5% P407 SD formulations and 6.68 J/g for the 15% P407 SD formulations. When calculated, ∆Hm of 100% crystalline P407 in 5% SD formulations was 5.86 J/g which corresponds to 14.7% crystalline P407. Similarly, 15% SD formulations contain in theory 38.0% P407.

Table 7. Experimental Tg, Predicted Tg, P407 enthalpy of fusion (J/g) and % crystallinity of various SD formulations and selected SD formulations used to investigate the eﬀect of cooling on the solubility of INM.

Predicted Tg P407 Enthalpy of Identiﬁer No Experimental Tg % Crystallinity (Couchman-Karasz) fusion (J/g) Crystalline INM 45.56 - - - P407 67.00 - 117.1 - − SD1-AC 74.91 83.54 0.593 10.12 SD2-AC 67.42 84.70 9.551 54.39 SD3-AC 71.82 80.14 0.963 16.43 SD4-AC 60.54 78.26 5.697 32.44 SD5-AC 74.58 86.94 1.032 17.61 SD6-AC 67.42 84.71 4.799 27.33 SD2-NT 69.24 84.70 11.90 67.78

SD2-Liq N2 70.19 84.70 9.478 53.97

Considering the concentration of plasticizer present in the amorphous SD formulations, it was estimated that the % crystallinity present in the 5% P407 formulations was within the range of 10.12% to 17.61% in its crystalline form and 27.33% to 54.39% for the 15% P407 formulations. It is important Pharmaceutics 2020, 12, 212 14 of 24 to note that the formulations with the higher P407 loadings had a degree of crystallinity that was significantly higher than formulations with low P407 loading since the APIs can be incorporated into the core of P407 micelle at high loads [24]. It is important that cooling also had a significant effect on the degree of crystallinity. For example, SD2-NT had the highest % crystallinity, indicating that using a high P407 loading with a slow cooling process increases the % crystallinity. Whereas SD2-Liq N2 which was a rapid cooling method, had a low % crystallinity. For all SD formulations, a single Tg was observed which confirms that INM was converted to its amorphous form, indicating that the polymers and drug were present in a two-phase system as P407 was still semi-crystalline (see Figures8 and9). The experimental glass transition temperatures were significantly different to the predicted Tgs calculated using the Couchman–Karasz equation, as shown in Table7, as drug–polymer loading can have a significant effect on the Tg. The effect of drug and polymer loading on the glass transition of SD formulations was predicted using the model reported by Couchman and Karasz [25,26]. The theoretical glass transition of the SD formulations was calculated using the following Equation (5):

(x1∆CP1Tg1 + x2∆CP2Tg2) Tgmix (HME System) = (5) (x1∆CP1 + x2∆CP2) where Tg1 and Tg2, are the Tgs of API and polymeric carriers, respectively, X1 and X2 correspond to the weight fractions of each of the pure materials and ∆Cp1 and ∆Cp2 corresponds to the change in the specific heat capacity at each of the glass transition temperature, respectively [27]. This analysis confirmed that P407 was not solubilized and is present in its semi-crystalline form with some level of compatibility with PL-S630/PVP VA64-INM system due to the reduction of the enthalpy of fusion and melting temperature in the various SD formulations. This was consistent with higher Bragg peak intensities observed in the XRPD data from the 15% P407 sample with 30% drug loading. The cooling method used had very little effect on the Tg. However, the degree of crystallinity was also affected by (1) the cooling method used (2) % P407 loading and (3) the polymeric carrier used. For example, SD4 had a higher % crystallinity compared to SD6. This is because PL-S630 and PVP VA64 possess different solubility properties with PVP VA64-INM SD formulations having a significantly greater than INM-PL-S630 SDs. There was a significant difference in the Tgs between SD4 and SD6 due to the different solid-state properties of PL-S630 and PVP VA64. However, the inclusion of P407 improved the processability and fragility of the extrudates, and also lowered the processing temperatures. The HSPs alongside XRPD and hyper DSC confirmed that the drug and polymer were indeed miscible. XRPD and hyper DSC confirmed successful conversion of INM to the amorphous form as mentioned before, hyper DSC also confirmed that although P407 remained semi-crystalline, as only one single Tg was present in all SD formulations. According to Couchman and Kasasz, (1978) if a SD formulation only contains one Tg then the drug and polymer are indeed miscible. ATR-FTIR and Raman spectroscopy confirmed hydrogen bonding interaction between PVP VA64/PL-S630 and INM due to the interaction between the amide carbonyl C=O of the polymer and the free carboxylic acid C=O of INM. For successful conversion to the amorphous form interaction between drug and polymer is necessary.

3.2. Solubility Studies

3.2.1. Phase Solubility Studies It is well reported in literature that the API INM due to it being a weak acid (pKa 4.5) (INM can partially dissociate into ions within aqueous solution). Its solubility is aﬀected by pH [28], meaning that the solubility of INM increases with increasing polymer concentration and pH. In this work, Pharmaceutics 2020, 12, 212 15 of 24 phase solubility studies were carried out in buﬀer of pH 1.2. PL-S630 and PVP VA64 are non-ionic polymeric carriers and display a pH-independent solubility. The INM aqueous solubility from the several ternary and quaternary SD formulations are shown in Table8. For the SD formulations, after 24 h the quaternary SD formulations had the highest aqueous solubility with a value of 34.17 µg/mL (Quart SD1) with 30% INM loading as shown in Table8. The SD formulations after 3 h had a higher solubility for INM compared to the physical mixtures. This may have been due to the conversion to the amorphous form, drug–polymer interaction and drug–polymer miscibility. Crystalline INM is present in the physical mixtures resulting in recrystallization.

Table 8. Comparison of the aqueous solubility of INM and standard deviation (STD) from various quaternary and ternary SD formulations and physical blends in pH buﬀer 1.2 after 3 and 24 h respectively. Physical blends contain crystalline INM.

Solubility of INM (µg/mL) after 3 h Solubility of INM (µg/mL) after 24 h Identiﬁer No. Physical Physical SD STD STD STD SD STD Blend Blend SD1 5.50 0.40 3.27 0.03 26.13 0.01 23.47 0.03 SD2 13.00 0.15 3.30 0.00 27.30 0.02 34.17 0.00 SD3 7.80 0.69 2.74 0.00 21.93 0.00 4.00 0.00 SD4 20.73 0.65 2.59 0.01 26.50 0.01 6.13 0.01 SD5 8.40 1.20 0.97 0.01 7.77 0.01 6.43 0.01 SD6 10.60 1.20 1.47 0.01 11.57 0.015 10.60 0.01 SD2-AC 13.00 0.15 3.03 0.00 24.30 0.03 32.17 0.00 SD2-NT 14.03 0.26 4.03 0.01 27.30 0.02 34.00 0.01

SD2-Liq N2 12.00 1.35 2.03 0.00 15.12 0.04 20.00 0.00

After 24 h, the physical mixtures had a significantly higher solubility as polymeric particles hydrated rapidly resulting in the increased wettability of the drug particles [5,29]. However, this depends on the % composition of drug and polymer used as shown in Table8. The kinetic solubility of the mixed copovidone systems was considerably higher than the ternary systems (Table4) possibly because of the greater shift in the vinyl acetate carbonyl of PVP VA64/PL-S630 due to hydrogen bonding as shown in Figure5. However higher shifts do not guarantee a higher kinetic solubility. This is true when the SD comes into contact with water causing a change in the kinetics and thermodynamics of the system. The decrease in INM solubility was due to recrystallization of the API. The cooling method used also had a significant effect on the solubility of INM. For example, SD2-NT had a significantly higher solubility than SD2-AC and SD2-Liq N2. The effect of cooling on the SD formulations with PVP VA64 and PL-S630 had a significant effect on the dissolution properties. After 24 h, the SD formulation cooled to normal room temperature (NT) and air-cooled formulations had the highest aqueous solubility of INM as slow cooling of aINM increases physical stability as reported by Baghel et al. (2015) [11]. As SD2-NT had the highest degree of crystallinity due to the slow cooling process, it increased the solubility of INM significantly. There was also a significant improvement in the kinetic solubility of INM after 24 h with a maximum kinetic solubility of 34.17 µg/mL, compared to 30 µg/mL after 72 h reported by Chokshi et al. (2008) [10]. SD2-NT had the highest solubility with a value of 34.17 µg/mL after 24 h, which again shows that slow cooling increases solubility. Like the degree of crystallinity, there was a significant difference between SD4 and SD6 due to different solubility properties of PVP VA64 and PL-S630, as mentioned previously.

3.2.2. In-Vitro Dissolution Studies The in vitro dissolution rates of INM and selected SD formulations, shown in Tables9 and 10, were performed using non-sink conditions in pH buﬀer 1.2. Table9 shows the concentration of INM (µg/mL) at various time intervals over 3 h. Control of the particle size of each formulation was vital in Pharmaceutics 2020, 12, 212 16 of 24 this study, as it can aﬀect dynamic solubility of each formulation. The particle size of each formulation was 200 microns to ensure uniformity. The formulation equal to 100 mg of INM was placed within the dissolution vessels.

Table 9. Comparison of the aqueous solubility of INM from various quaternary and ternary SD formulations in pH buﬀer 1.2 taken at various time intervals over 24 h.

Con. of Conc. of Conc. of Conc. of Conc. Conc. of Conc. of Conc. of INM INM INM INM Of INM INM INM INM Identiﬁer (µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL) (µg/mL) at 0 h at 0.2 h at 0.3 h at 0.4 h at 0.75 h at 1 h at 2 h at 3 h SD1 0.00 2.27 2.73 2.70 2.60 3.07 5.23 5.50 SD2 0.00 4.13 7.46 9.90 9.90 11.00 11.20 13.00 SD3 0.00 4.23 5.30 9.27 7.77 8.63 8.27 7.80 SD4 0.00 10.40 10.17 10.63 16.13 10.80 20.83 20.73 SD5 0.00 4.17 5.87 6.13 6.40 6.10 8.03 8.40 SD6 0.00 5.80 8.00 6.93 8.03 9.07 8.57 10.60 SD2-AC 0.00 4.13 7.46 9.90 9.90 11.00 11.20 13.00 SD2-NT 0.00 15.27 15.83 17.10 15.57 11.60 13.03 14.03 SD2-Liq 0.00 10.17 10.90 11.60 11.17 14.90 11.43 12.00 N2

Table 10. Comparison of the aqueous solubility of INM from various quaternary and ternary SD formulations in pH buﬀer 1.2 after 3 and 24 h. Supersaturation parameter [SP] was calculated for SD formulations after 3 h.

Identiﬁer No. of Conc. of INM, 3 Conc. of INM, SP of INM STD STD No. Phases h. (µg/mL) 24 h. (µg/mL) after 3 h SD1 2 5.50 0.40 23.47 0.01 0.97 SD2 2 13.00 0.15 34.17 0.02 0.98 SD3 2 7.80 0.69 4.00 0.00 0.49 SD4 2 20.73 0.65 6.13 0.01 0.24 SD5 2 8.40 1.20 6.43 0.01 0.56 SD6 2 10.60 1.20 10.60 0.015 0.45 SD2-AC 2 13.00 0.15 32.17 0.03 0.49 SD2-NT 2 14.03 0.26 34.00 0.02 0.70 SD2-Liq N2 2 12.00 1.35 20.00 0.04 0.30

It is important to note that there was no interference from the polymers which therefore did not aﬀect the value of UV absorption. The λ max for both PL-S630 and PVP VA64 is 440 nm for vinyl-pyrrolidone and 258 nm for vinyl acetate as stated in the literature [30,31]. INM has a λ max of 320 nm. The aqueous solubility of crystalline and amorphous INM was 1.2 and 2.4 µg/mL respectively [5]. Non-sink conditions were used for this study as crystallization of APIs and nucleation within supersaturated dissolution conditions can be observed [32]. An illustration of non-sink conditions is shown in Figure 10. In Figure 10, the SD has no initial crystallinity due to the conversion to the amorphous state, but several crystals grow during the dissolution test as the supersaturation generated results in phase separation and nucleation [33]. The dissolution of dissolved INM depended upon the dissolution rate comparative to the rate of crystallization. If the dissolution rate is rapid relative Pharmaceutics 2020, 12, 212 17 of 24

to crystallization, then 100% release may be achieved, with a subsequent decrease in the solution Pharmaceutics 2020, 12, x FOR PEER REVIEW 17 of 24 Pharmaceuticsconcentration 2020, 12 as, x FOR crystallization PEER REVIEW occurs (shown in Figure 10). If the rate of crystallization17 is of rapid 24 compared to the dissolution rate, then 100% drug release will not be achieved and will display a is is rapid rapid compared compared to tothe the dissolution dissolution rate, rate, then then 100% 100% dru drug releaseg release will will not not be be achieved achieved and and will will ‘’spring” and ‘’parachute” eﬀect which is the case in this study. displaydisplay a ‘’spring’’ a ‘’spring’’ and and ‘’parachute’’ ‘’parachute’’ effect effect which which is theis the case case in inthis this study. study.

FigureFigure 10. 10. 10.Illustration IllustrationIllustration of of nonof nonnon-sink-sink-sink conditions conditions used used in in the the the dissolution dissolution dissolution test. test. test. Dissolution Dissolution Dissolution profile profile profile 1: 1: dissolutiondissolution of ofthe the crystalline crystalline API API;API; profile; profileprofile 2: dissolution 2: dissolution of aof “spring” a “spring” effect effect effect of ofthe the API API without without the the presenpresencepresence ceof ofdissolutionof dissolutiondissolution enhance enhancers enhancers profilers profile profile 3: 3: dissolution3: dissolution dissolution of of aof “spring”“aspring “spring” andand” and ‘’parachute”‘’parachute’’ ‘’parachute’’ form form form of of the theof APIthe API in the presence of dissolution enhancers act as a “parachute.” Ceq represents equilibrium APIin in the the presence presence of dissolution of dissolution enhancers enhancers act as aact “parachute.” as a “parachute.”Ceq represents Ceq represents equilibrium equilibrium solubility. solubility.solubility. The dissolution profiles for melt extrudates containing pure drug and polymers are shown in FiguresTheThe dissolution 11 dissolution and 12. profiles The profiles aqueous for for melt solubility melt extrudates extrudates of dissolved containing containing INM pure inpure SD drug formulationsdrug and and polymers polymers increased are are shown comparedshown in in FigurestoFigures aINM 11 11and and and 12 pure .12 The. The INM aqueous aqueous respectively. solubility solubility Pure of ofdissolved and dissolved aINM INM INM had in aninSD SD aqueous formulations formulations solubility increased increased of 1.2 comparedµ comparedg/mL and to 2.4aINMto aINMµg/ andmL and aspure expected,pure INM INM respectively. which respectively. is similar Pure Pure toand theand aINM values aINM hadreported had an aaqueousn aqueous in the solubility literature solubility of [28 of1.2]. 1.2 Amorphousµg/ µmg/Lm andL and 2.4 INM 2.4 µg/mhadµg/mL aasL kinetic asexpected, expected, solubility which which of is 2.4 similaris µsimilarg/mL to astothe expectedthe values values reported due reported to the in transformationinthe the literature literature [28] to [28].the Amorphous. Amorphous amorphous INM state.INM hadThehad a kinetic increasea kinetic solubility in solubility the aqueous of of2.4 2.4 µ solubilityg/mL µg/mL as asexpected was expected dependent due due to upontothe the transformation the transformation concentration to tothe of the polymeramorphous amorphous used. state. state. TheThe increase increase in inthe the aqueous aqueous solubility solubility was was dependent dependent upon upon the the concentration concentration of ofpo polymerlymer used. used.

FigureFigure 11 .11 11.In.- InvitroIn-vitro-vitro dissolution dissolution profiles profiles proﬁles of airof -air air-cooledcooled-cooled SD SDformulations formulations performed performed in buffer in bubufferﬀ erof ofpHof pHpH 1.2. 1.2.1.2.

Pharmaceutics 2020, 12, 212 18 of 24 Pharmaceutics 2020, 12, x FOR PEER REVIEW 18 of 24

Figure 12.12. In-vitroIn-vitro dissolution profiles proﬁles of quaternary SD formulations performed in in buffer buﬀer of pH 1.2. The maximummaximum aqueousaqueous solubilitysolubility isis 20.7320.73 µµg/mg/mLL afterafter 33 h.h. (AC(AC == AirAir cooled,cooled, NTNT == Normal room

temperature andand LiqLiq NN22 = LiquidLiquid Nitrogen). Nitrogen).

In this this study study there there was was a a tendency tendency that that asas the the concentration concentration of of P407 P407 increased, increased, the drug concentration also increased increased (Figure(Figure 1212).). The The aqueous solubility of dissolved INM significantlysignificantly increased comparedcompared toto thethe valuesvalues reportedreported inin literature.literature. Chokshi et al.al. (2008) [10] [10] prepared INM-PVPINM-PVP VA64 binarybinary mixturesmixtures andand recordedrecorded a kinetic solubility of 10 µgg/mL/mL and 30 µµg/mg/mLL in pH bubufferffer 1.2 after 24 and 72 h using 30%30% INM.INM. Table 1010 showsshows thethe SPSP ofof INMINM afterafter 33 hh inin pHpH bubufferffer 1.21.2 underunder non-sinknon-sink conditions.conditions. For the purposes of this work,work, only the supersaturationsupersaturation parameter (SP) of SD formulations after 3 h was selected and calculated. According to to Chen Chen et et al. al (2015),. (2015), SP apparently SP apparently is adimensionless is a dimensionless parameter parameter with a range with between a range zerobetween and zero one. and Zero one. means Zero nomeans supersaturation no supersaturation power, power, while onewhile indicates one indicates the tendency the tendency of drug of precipitationdrug precipitation to occur to isoccur reduced is reduced [34]. SP [34] is a. function SP is a offunction the chemical of the structurechemical of structure the drug, of initial the drug, drug concentration,initial drug concentration, type of polymer type andof polymer concentration and concentration of polymer. of polymer. Table 1010 showsshows thatthat allall formulationsformulations hadhad aa SPSP valuevalue ofof 0.24 or above indicating that the INM was resilient againstagainst drugdrug precipitation, precipitation, however however the the quaternary quaternary formulation formulation SD1 SD1 contained contained the the highest highest SP valueSP value indication indication that that polymer polymer type type and concentrationand concentration of polymer of polymer had a had significant a significant effect oneffect the on ability the ofability the SD of the to inhibit SD to inhibit drug precipitation drug precipitation and recrystallization. and recrystallization. During the the extrusion extrusion process, process, INM INM was transformed was transformed to the amorphous to the amorphous state in all SDstate formulations. in all SD Therefore,formulations. as aTherefore, result, the as aqueous a result solubility, the aqueous of INM solubility was significantly of INM greater was significantly compared to greater both amorphouscompared to and both crystalline amorphous INM and with crystalline a maximum INM aqueous with solubilitya maximum of 20.73 aqueousµg/mL. solubility This study of 20.73 also showedµg/mL. This that thestudy cooling also showed method usedthat the for cooling extrusion method had a significantused for extrusion impact on had the a kinetic significant solubility impact of INM.on the The kinetic highest solubility aqueous of solubilityINM. The observed highest aqueo was 34.00us solubilityµg/mL for observed SD2 NT was containing 34.00 µg/m 30% INML for afterSD2 24NT h. containing The dissolution 30% INM concentration after 24 h. The increased dissolution by 10 concentrati times as comparedon increased to pure by and10 times amorphous as compared drug. to pureAll and SD formulations amorphous drug. with highest P407 loading (15%) had a significantly higher solubility compared to theAll 5% SD P407 formulations formulations with after highest 3 h. TheP407 significant loading (15%) increase had in a aqueous significantly solubility higher was solubility related tocompared the degree to the of crystallinity,5% P407 formulations cooling process after 3 usedh. The and significant interaction increase between in aqueous polymeric solubility carrier andwas API,related including to the degree polymer–drug of crystallinity, miscibility. cooling The process P407 micelleused and core interaction contains between propylene polymeric oxide which carrier is hydrophobicand API, including which polymer when incorporated–drug miscibility. into INMThe P407 resulted micelle in increasedcore contains solubility propylene of INM. oxide P407 which in natureis hydrophobic is a unimer which which when when incorporated placed into into solution INM canresult formed in micelles increased via self-assemblysolubility of INM. [35]. P407 in According to Suksiriworapong et al. (2013) the critical micelle concentration of P407 is 2.8 10 8 nature is a unimer which when placed into solution can form micelles via self-assembly [35]. × − M (0.03Accordingµg/mL), to as Suksiriworapong the concentration et of al drug. (2013) and the SD critical formulations micelle in concentration this study was of greaterP407 is than2.8 × 0.0310−8 µMg /(0.03mL it µg/m resultedL), as in the micelle concentration formation of as drug shown andin SD Table formulations4[ 36]. Moreover, in this study the degree was greater of crystallinity than 0.03 µg/mL it resulted in micelle formation as shown in Table 4 [36]. Moreover, the degree of crystallinity was significantly lower in the higher P407 loadings, increasing solubility. P407 can result in many

Pharmaceutics 2020, 12, x FOR PEER REVIEW 19 of 24 benefits such as greater wetting, increasing surface area and decreasing interfacial tension between API and the dissolution medium. It is stated in the literature that reducing the interfacial tension decreases nucleation [37]. The recrystallization of amorphous INM in solid solutions was dependent upon three factors; % P407 loading, degree of P407 crystallinity and cooling method used. It is well reported in literature that high P407 loading can lead to recrystallization due to the hydrophobic P407 propylene oxide core [35]. Slow cooling is ideal for a process that does not require high energy removal. It is less expensive regarding extrusion, it is easier to maintain, has lower operating costs compared to liquid nitrogen and requires less space compared to fluid cooling. The AUC of pure and aINM was compared to the AUC of each SD formulation using statistical Pharmaceuticsanalysis. The2020 statistical, 12, 212 tests used were Tukey Kramer post hoc test and 1-way ANOVA (Figure19 of13 24). For both the quaternary and ternary SD formulations with the exception of SD2, SD4, and SD6 there waswas significantlyno statistical lowerdifference in the between higher P407amorphous loadings, and increasing crystalline solubility. INM. The P407 overall can resulteffect inof manydrug, benefitspolymer such concentration as greater wetting,and % wt. increasing of P407 surfacedid have area a significan and decreasingt effect interfacial on the solubility tension between of INM APIand andAUC the in dissolutionsolution. medium. It is stated in the literature that reducing the interfacial tension decreases nucleationFigure [ 3713]. shows that the formulations containing the highest P407 loading were statistically different to crystalline INM which shows that the increase in P407 increased the solubility of INM The recrystallization of amorphous INM in solid solutions was dependent upon three factors; % [5]. Figure 13 also shows that cooling had a significant effect on solubility. SD2-NT had the highest P407 loading, degree of P407 crystallinity and cooling method used. It is well reported in literature that solubility after 3 and 24 h with values of 14.03 µg/mL and 34.00 µg/mL. Whereas the rapid cooling high P407 loading can lead to recrystallization due to the hydrophobic P407 propylene oxide core [35]. method SD2-Liq N2 had a solubility of 12.00 µg/mL and 20.00 µg/mL respectively. This corresponds Slow cooling is ideal for a process that does not require high energy removal. It is less expensive to the values reported for the degree of crystallinity, with SD2-NT having the highest degree of regarding extrusion, it is easier to maintain, has lower operating costs compared to liquid nitrogen and crystallinity. This shows that the greater the % crystallinity of P407 (see Table 7), the greater the requires less space compared to fluid cooling. solubility. SD2-NT had the highest aqueous solubility due to the slow cooling process. Therefore, The AUC of pure and aINM was compared to the AUC of each SD formulation using statistical cooling influenced the solubility of INM and AUC in solution. analysis. The statistical tests used were Tukey Kramer post hoc test and 1-way ANOVA (Figure 13). For The solubility also depended upon the polymeric carrier used as mentioned already. There was both the quaternary and ternary SD formulations with the exception of SD2, SD4, and SD6 there was a significant difference in solubility between SD4 and SD6 which contain PVP VA64 and PL-S630 no statistical difference between amorphous and crystalline INM. The overall effect of drug, polymer respectively. SD4 had a kinetic solubility of 20.73 µg/mL compared to 10.60 µg/mL for SD6. It is concentration and % wt. of P407 did have a significant effect on the solubility of INM and AUC important to note that the different solid-state and dissolution properties of PVP VA64 and PL-S630 in solution. significantly affect the solubility of INM achieved.

FigureFigure 13.13. Graphical representations representations of of AUC AUC of of quaternary quaternary and and ternary ternary SD SD formulations formulations in inpH pH 1.2. 1.2. ** **and and * *represents represents the the statistical statistical difference diﬀerence ( (pp << 0.05)0.05) between between ASD, ASD, amorphous INM and pure INMINM respectively,respectively, for for thethe one-way one-way ANOVA ANOVA and and Tukey–Kramer Tukey–Kramer post post hoc hoc test. test. (AC (AC= = Air Air cooled, cooled, NT NT ==

NormalNormal roomroom temperaturetemperature andand LiqLiq NN22 == Liquid Nitrogen).

Figure 13 shows that the formulations containing the highest P407 loading were statistically 3.3. Accelerated Stability Studies different to crystalline INM which shows that the increase in P407 increased the solubility of INM [5]. Figure 13 also shows that cooling had a significant effect on solubility. SD2-NT had the highest solubility after 3 and 24 h with values of 14.03 µg/mL and 34.00 µg/mL. Whereas the rapid cooling method

SD2-Liq N2 had a solubility of 12.00 µg/mL and 20.00 µg/mL respectively. This corresponds to the values reported for the degree of crystallinity, with SD2-NT having the highest degree of crystallinity. This shows that the greater the % crystallinity of P407 (see Table7), the greater the solubility. SD2-NT had the highest aqueous solubility due to the slow cooling process. Therefore, cooling influenced the solubility of INM and AUC in solution. The solubility also depended upon the polymeric carrier used as mentioned already. There was a significant difference in solubility between SD4 and SD6 which contain PVP VA64 and PL-S630 respectively. SD4 had a kinetic solubility of 20.73 µg/mL compared to 10.60 µg/mL for SD6. It is important to note that the different solid-state and dissolution properties of PVP VA64 and PL-S630 significantly affect the solubility of INM achieved. Pharmaceutics 2020, 12, x FOR PEER REVIEW 20 of 24

Accelerated stability studies were performed at 40 °C/75% RH using the Amebis stability testing system. After 5 months it was noted that some samples had adsorbed significant quantities of water

Pharmaceuticsand that all2020 samples, 12, 212 had attained a darker yellow colour. Samples were removed from the humidity20 of 24 chambers and dried overnight under vacuum to remove adsorbed water. This was because water acts as a plasticizer, thereby enhancing the molecular mobility of drugs and polymers [38]. They were 3.3.dried Accelerated for 24h in Stability a desiccator Studies over phosphorous pentoxide. XRPD and hyper DSC confirmed that after Pharmaceutics 2020, 12, x FOR PEER REVIEW 20 of 24 5 months INM remained amorphous for all SD formulations (Figures 14, 15 and 16). The Bragg peaks Accelerated stability studies were performed at 40 ◦C/75% RH using the Amebis stability testing system.associatedAccelerated After with 5 months crystallinestability it studies was INM noted werewere that performedcompletely some samples at absent 40 ° hadC/75% in adsorbedall RH XRPD using significantdiffractograms the Amebis quantities stability of SDs of testing after water 5 andsystem.months. that After all samples 5 months had it attained was noted a darker that some yellow samples colour. had Samples adsorbed were significant removed quantities from the humidity of water chambersand that all and samples dried had overnight attained under a darker vacuum yellow to colour. remove Samples adsorbed were water. removed This wasfrom because the humidity water actschambers as a plasticizer, and dried therebyovernight enhancing under vacuum the molecular to remove mobility adsorbed of drugs water. and This polymers was because [38]. water They wereacts as dried a plasticizer for 24h, inthereby a desiccator enhancing over the phosphorous molecular mobility pentoxide. of drugs XRPD and and polymers hyper DSC[38]. confirmedThey were thatdried after for 24h 5 months in a desiccator INM remained over phosphorous amorphous pentoxide. for all SD XRPD formulations and hyper (Figures DSC confirmed 14–16). The that Bragg after peaks5 months associated INM remained with crystalline amorphous INM for were all completelySD formulations absent (Figures in all XRPD 14, 15 di andffractograms 16). The Bragg of SDs peaks after 5associated months. with crystalline INM were completely absent in all XRPD diffractograms of SDs after 5 months.

Figure 14. XRPD diffractograms of SD formulations and XRPD diffractograms of the SD formulation used to investigate the effect of cooling after 5 months of a stability study under accelerated conditions of 40 °C and 75% RH. P407 was still present in its crystalline form in all formulations (AC = Air cooled,

NT = Normal room temperature and Liq N2 = Liquid Nitrogen).

In the XRPD diffractograms shown in Figure 14 after 5 months stability, the Bragg peaks associated with crystalline INM were completely absent. Hyper DSC also confirmed that INM was stable after 5 months stability studies. For hyper DSC the samples were dried as the moisture was Figure 14.14. XRPD diffractograms diffractograms of SD formulations and XRPD diffract diffractogramsograms of the SD formulation coming off at the same temperature that the Tg occurred as reported by Hurley et al. (2018) [5]. After used to investigate the effect effect of cooling after 5 months of a stability stability study study under under accelerated accelerated conditions 5 months stability, all SD formulations remained amorphous and there was no reduction in Tg due to of 40 °C◦C and 75% RH. P407 was still present in its crystalline form in all formulations (AC = AirAir cooled, cooled, moisture removal. Also, there was no significant difference in Tg prior to and after 5 months stability NT = NormalNormal room room tem temperatureperature and Liq N 2 == LiquidLiquid Nitrogen). Nitrogen). (See Figures 14, 15 and 16.). 2 In the XRPD diffractograms shown in Figure 14 after 5 months stability, the Bragg peaks associated with crystalline INM were completely absent. Hyper DSC also confirmed that INM was stable after 5 months stability studies. For hyper DSC the samples were dried as the moisture was coming off at the same temperature that the Tg occurred as reported by Hurley et al. (2018) [5]. After 5 months stability, all SD formulations remained amorphous and there was no reduction in Tg due to moisture removal. Also, there was no significant difference in Tg prior to and after 5 months stability (See Figures 14, 15 and 16.).

FigureFigure 15.15. HyperHyper DSCDSC thermogramsthermograms ofof air-cooledair-cooled SDSD formulationsformulations afterafter 55 monthsmonths ofof aa stabilitystability studystudy underunder acceleratedaccelerated conditionsconditions ofof 4040◦ °CC andand 75%75% RH.RH. P407P407 waswas stillstill presentpresent inin itsits crystallinecrystalline formform inin allall formulations.formulations. TheThe areaarea ofof interestinterest forfor crystallinecrystalline INMINM meltingmelting endothermendotherm isis markedmarked byby straightstraight lines.lines. T is indicated by a tick. Tgg is indicated by a tick.

Figure 15. Hyper DSC thermograms of air-cooled SD formulations after 5 months of a stability study under accelerated conditions of 40 °C and 75% RH. P407 was still present in its crystalline form in all formulations. The area of interest for crystalline INM melting endotherm is marked by straight lines.

Tg is indicated by a tick.

Pharmaceutics 2020, 12, x FOR PEER REVIEW 21 of 24

This was also shown by Hurley et al. (2018), [5], who prepared SDs using INM as a model drug. All SDs remained amorphous as a result of conversion from the crystalline to amorphous state and as a result of the high molar attraction constant of INM, hydrogen bonding and drug–polymer miscibility. This shows that all ASD formulations are miscible as predicted by the HSPs in Table 1. ThePharmaceutics results 2020 also, 12 confirm, 212 that the cooling method used did not have a significant impact on21 of the 24 amorphous stability and Tg of INM.

FigureFigure 16.16. Hyper DSCDSC thermograms thermograms of of selected selected SD SDformulations formulations used used to investigate to investigate the eﬀ ect the of effect cooling of coolingafter 5months after 5 months of a stability of a stability study under study accelerated under accelerated conditions conditions of 40 ◦C of and 40 75%°C and RH. 75% P407 RH. iswas P407 still is waspresent still in present its crystalline in its crystalline form in all form formulations in all formulations (AC = Air cooled, (AC = AirNT = cooNormalled, NT room = Normal temperature room

temperatureand Liq N2 = andLiquid Liq Nitrogen). N2 = Liquid The area Nitrogen). of interest The for area crystalline of interest INM formelting crystalline endotherm INM is melting marked by straight lines. T is indicated by a tick. endotherm is markedg by straight lines. Tg is indicated by a tick.

In the XRPD diffractograms shown in Figure 14 after 5 months stability, the Bragg peaks associated 4. Conclusion with crystalline INM were completely absent. Hyper DSC also confirmed that INM was stable after 5 monthsThe stability HSPs, alongside studies. For XRPD hyper and DSC hyper the DSC samples, confirmed were dried that as the the drug moisture and polymer was coming were o ffindeedat the miscible.same temperature XRPD and that hyper the TDSCg occurred confirmed as reported successful by Hurleyconversion et al. of (2018) INM [to5]. the After amorphous 5 months form stability, as mentionedall SD formulations before, hyper remained DSC amorphousalso confirmed and that there although was no reductionP407 remained in Tg duesemi to-crystalline, moisture removal. as only oneAlso, single there T wasg was no present significant in all di thefference SD formulations. in Tg prior to According and after 5to months Couchman stability and (See Kasasz, Figures (1978) 14– if16 a). SD formulationThis was also only shown contains by Hurleyone Tg then et al. the (2018), drug [5 and], who polymer prepared are SDsindeed using miscible. INM as ATR a model-FTIR drug. and RamanAll SDs spectroscopy remained amorphous confirmed as hydrogen a result of bonding conversion interaction from the between crystalline PVP to amorphousVA64/PL-S630 state and and INM as a dueresult to ofthe the interaction high molar between attraction the constant amide carbonyl of INM, C=O hydrogen of the bonding polymer and and drug–polymer the free carboxylic miscibility. acid C=OThis shows of INM. that For all ASD successful formulations conversion are miscible to the as amorphous predicted by form the HSPsinteraction in Table between1. The results drug also and polymerconfirm thatis necessary. the cooling ATR method-FTIR used spectroscopy did not have also a confir significantmed that impact P407 on does the amorphous not interact stability with INM and atTg aof molecular INM. level. The cooling method used did not have any effect on the Tg when air cooled, cooled to normal room4. Conclusions temperature and cooled via liquid nitrogen. However, cooling and the type of polymeric carrier used had a significant effect on the degree of crystallinity and solubility of INM. There was a The HSPs, alongside XRPD and hyper DSC, confirmed that the drug and polymer were indeed significant difference in the enthalpy of fusion in the formulation chosen to investigate the effect of miscible. XRPD and hyper DSC confirmed successful conversion of INM to the amorphous form as cooling. It is also important to note that in the formulations with the higher P407 loadings, the degree mentioned before, hyper DSC also confirmed that although P407 remained semi-crystalline, as only of crystallinity was within the range of 27.33% to 54.39%, compared to 10.12% to 17.61% for the 5% one single Tg was present in all the SD formulations. According to Couchman and Kasasz, (1978) if SD formulations. a SD formulation only contains one Tg then the drug and polymer are indeed miscible. ATR-FTIR Solubility studies of INM showed a significant increase in the kinetic solubility of INM compared and Raman spectroscopy confirmed hydrogen bonding interaction between PVP VA64/PL-S630 and to crystalline INM at 37 °C in pH 1.2. The SD formulations showed a significantly higher dissolution INM due to the interaction between the amide carbonyl C=O of the polymer and the free carboxylic rate of 20.63 µg/ml. The XRPD and hyper stability data showed that the cooling method used did not acid C=O of INM. For successful conversion to the amorphous form interaction between drug and have a significant effect on the amorphous stability of INM as all SD formulations remained polymer is necessary. ATR-FTIR spectroscopy also confirmed that P407 does not interact with INM at a amorphous after 5 months stability testing. molecular level. The results of the hyper DSC and solubility studies show that the greater the % crystallinity of The cooling method used did not have any effect on the Tg when air cooled, cooled to normal P407, the greater the solubility. However, the cooling process used, significantly affected the degree room temperature and cooled via liquid nitrogen. However, cooling and the type of polymeric carrier used had a significant effect on the degree of crystallinity and solubility of INM. There was a significant difference in the enthalpy of fusion in the formulation chosen to investigate the effect of cooling. It is also important to note that in the formulations with the higher P407 loadings, the degree of crystallinity was within the range of 27.33% to 54.39%, compared to 10.12% to 17.61% for the 5% SD formulations. Pharmaceutics 2020, 12, 212 22 of 24

Solubility studies of INM showed a significant increase in the kinetic solubility of INM compared to crystalline INM at 37 ◦C in pH 1.2. The SD formulations showed a significantly higher dissolution rate of 20.63 µg/mL. The XRPD and hyper stability data showed that the cooling method used did not have a significant effect on the amorphous stability of INM as all SD formulations remained amorphous after 5 months stability testing. The results of the hyper DSC and solubility studies show that the greater the % crystallinity of P407, the greater the solubility. However, the cooling process used, significantly affected the degree of crystallinity and solubility also as SD2-NT had the highest aqueous solubility for dissolved INM and the highest crystallinity due to the slow cooling process. Therefore, slow cooling enhances physical stability and solubility, as reported by Baghel et al. (2015) [11]. This work illustrates the significance of examining the cooling method used in order to improve the aqueous solubility, amorphous stability and solid-state properties of BCS class II drugs.

Author Contributions: Conceptualization, D.H., and C.L.H. Funding acquisition, C.L.H.; investigation, D.H.; methodology, D.H. and M.D.; supervision, C.L.H., J.G.L. and G.M.W.; writing—original draft, D.H.; writing—review and editing, D.H. and C.L.H. All authors have read and agreed to the published version of the manuscript. Funding: Financial assistance from the Synthesis and Solid State Pharmaceutical Centre, funded by Science Foundation Ireland under Grant Number 12/RC/2275 is gratefully acknowledged. Conﬂicts of Interest: The authors declare no conﬂict of interest.

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