Recent Advances in Nonlinear Optical Analyses of Pharmaceutical

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Recent Advances in Nonlinear Optical Analyses of Pharmaceutical Materials in the Solid State Paul D. Schmitt Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00809 • Publication Date (Web): 26 Jan 2017 Downloaded from http://pubs.acs.org on February 4, 2017

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Molecular Pharmaceutics

Recent Advances in Nonlinear Optical Analyses of Pharmaceutical Materials in the Solid State Paul D. Schmitt Department of Chemistry, Wabash College, Crawfordsville, IN 47933, United States Abstract: The past decade has seen an increase in the use of nonlinear optical (NLO) techniques such as second harmonic generation, coherent anti-stokes Raman scattering, stimulated Raman scattering, and two-photon fluorescence for the solid-state characterization of pharmaceutical materials. These combined techniques offer several advantages (e.g., speed, selectivity, quantitation) of potential interest to the pharmaceutical community, as decreased characterization times in formulation development and testing could help decrease the time required to bring new, higher quality drugs to market. The large body of literature recently published in this field merits a review. Literature will be discussed in order of drug development, starting with applications in initial therapeutic molecule crystallization and polymorphic analysis, followed by final dosage form characterization, and ending with drug product performance testing. Key Words: Pharmaceuticals, formulation development, nonlinear optics, second harmonic generation, coherent anti-stokes Raman spectroscopy, stimulated Raman spectroscopy, polymorphism, amorphous solid dispersions, dissolution testing, chemical imaging. Introduction: According to the most recent estimates, the total costs of bringing a new drug to market are approximately $2.87 billion (2013 dollars), requiring approximately 11 years of research and development from initial synthesis to new drug application (NDA) approval.1 Of this total, an estimated $430 million in out-of-pocket costs and an average of 31.2 months are required to progress a new chemical entity (NCE) from synthesis to initial human/ clinical testing.1 Among the multitude of tasks required during this period of pre-clinical research, the development, validation, and continued testing of the drug formulation can each be time consuming steps. Formulations development refers to the process of creating drug delivery vehicle (e.g., tablets) that ensures the prolonged stability and eventual bioavailability of the drug product. While the costs associated with formulation development and testing alone are difficult to estimate, this phase of research is seeing increased expenditures due to the formulations challenges found in the majority of emerging NCEs.2–6 Additionally, modern regulations demand increasingly thorough knowledge of drug stability and behavior within the final dosage form.7 Given the time and overall costs required to bring novel therapeutics to patients, new technologies capable of providing earlier feedback during these initial phases of product validation and testing have the potential to decrease the costs associated with final drug approval. One field undergoing increased use in the characterization of drug products and formulation behavior is nonlinear optics (NLO), including such techniques as second harmonic generation (SHG) and coherent anti-stokes

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Raman scattering (CARS), among others. Arguably, this increase is largely due to the decreasing cost and complexity of ultrafast lasers required for such measurements, together with a maturing of the methods as applied to pharmaceutical materials.8–10 Several properties of NLO measurements provide advantages for such analyses.11–13 These include, but are not limited to: crystal-specific imaging (e.g., SHG is only bulk allowed in non-centrosymmetric samples such as chiral crystals), chemical specificity (e.g., CARS provides contrast directly linked to vibrational signatures), optical sectioning (inherent confocal nature of all NLO techniques), measurement speed (e.g., CARS vs. spontaneous Raman), and label-free imaging (no external labels required as in labelling-based fluorescent techniques). A large body of recently published literature highlights the applications and utility of these methods to the characterization of pharmaceutical materials, meriting a thorough review. This review will proceed in order of formulation development. Following a discussion of initial API crystallization and polymorphism, the potential of NLO techniques for amorphous solid dispersion (ASD) development will be discussed. Drug product/ excipient chemical mapping in tablets and other solid dosage forms will then be discussed. Finally, the relevance of NLO measurements to drug product testing, including monitoring the dissolution and phase behavior of solid dosage forms, will be explored. The ability to perform these measurements selectively and rapidly is critically assessed within the current landscape of quality by design (QbD)7 demanded in the modern pharmaceutical industry. Background: A Brief Description Nonlinear Optical (NLO) Techniques: A detailed discussion of the theoretical principles giving rise to nonlinear optical effects is beyond the scope of this review. For readers seeking further information, many excellent reviews and texts detailing the quantitative and qualitative origins of nonlinear optics have been published previously.9,14,15 However, a brief introduction to the primary methodologies included within this review is merited. Second harmonic generation is the frequency doubling of light. A common classical model for the origins of SHG is the damped, driven anharmonic oscillator in which a high amplitude driving field yields nonzero contributions from nonlinear scattering effects.14 Most generally, second-order nonlinear light scattering (dependent upon the square of the incident field amplitude) is known as sum frequency generation (SFG), in which two incident fields of energies h ω1 and h ω2 combine in energy to yield light with energy h ω1 + h ω2 = h ω3 . SHG is simply a specific case of SFG in which the two incident fields are degenerate. A Jablonski diagram for a general (non-resonant) SHG process is shown in Figure 1 (left). As a coherent second order NLO effect, SHG is forbidden in bulk materials with inversion symmetry and is therefore selective for molecular boundaries/ interfaces and other noncentrosymmetric assemblies, including the large majority of chiral crystals. Generally speaking, coherent SHG is a background-free measurement, as coherent contributions from centrosymmetric or disordered media are symmetry forbidden. Chemical selectivity and information content of this method is correspondingly low, as the spectral content of the frequency doubled light is primarily dictated by the spectral content of the driving field. While SHG intensity can also be measured spectroscopically (e.g., as a function of the fundamental or second harmonic frequencies),16 the works cited here-in do not assume this paradigm. In the assumption that the driving field does not show appreciable overlap with a vibrational or electronic resonance in the material, no such transition is probed in the course of the measurement


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(hence the use of dotted lines to represent virtual states in Figure 1). Both single point SHG measurements and beam-scanning SHG microscopy will be discussed in this review. Coherent anti-stokes Raman scattering (CARS) is a third order NLO process yielding spectral information on the vibrational dynamics and symmetry of the material being studied. In contrast with SFG/ SHG, CARS selectively probes vibrational transitions in a sample based on the energy differences between the two incident fields, typically referred to as the pump and Stokes beams. Vibrational transitions degenerate with this difference frequency are driven by the incident fields. The anti-Stokes shift of additional photons within the Stokes beam following a vibrational relaxation in the molecule is detected. Tuning the difference frequency of the incident fields to match characteristic vibrations of analytes enables chemical/ bond selectivity. A Jablonski diagram for a general CARS process is shown in Figure 1 (middle).Recently, broadband CARS (BCARS) has also been developed.17 In BCARS, the Stokes beam exhibits a broad spectral bandwidth that simultaneously encompasses whole spectral features. Finally, a Jablonski diagram for a typical stimulated Raman scattering (SRS) process is also shown in Figure 1 (right). Similar to CARS, SRS uses two incident beams (pump and Stokes) tuned such that the difference frequency is in resonance with a vibrational transition of interest.18–20 Either the stimulated Raman loss (SRL) in the pump beam or the stimulated Raman gain (SRG) in the Stokes beam can be used in the measurement. While SRS is a stronger effect than spontaneous Raman, the magnitude of the SRG/ SRL is still smaller than that of the laser noise, such that fast (~MHz) modulation of an input beam and detection via lock-in amplification is typically employed.19,21 Initial crystallization and Polymorphism Polymorphism refers to the ability of a molecule to crystallize into multiple crystal structures.22,23 In the development of an API, identification of polymorphic forms and confirmation of the desired form within the product is exhaustively evaluated to ensure API stability, dissolution kinetics, bioavailability, and intellectual property rights. The most common methods used to characterize or validate the polymorphic form of an API include Raman spectroscopy and powder X-ray diffraction (PXRD). Unfortunately, characterization of a large number of samples is complicated due to the relatively long integration times (generally a minimum of 30-60 seconds) required for analysis such methods.24–29 This is particularly evident in early stage high-throughput screening for polymorphic form discovery, where the number of samples needing to be characterized can routinely reach several thousand.30,31 Decreasing both the time and amount of material required for characterization could help alleviate these bottlenecks in the drug development process. Several manuscripts have demonstrated the ability to detect or discriminate different polymorphic crystal forms by SHG or CARS. Early studies have used differences in the integrated SHG intensities to distinguish two polymorphic forms of a small molecule. Rawle et al. used single-point SHG measurements to distinguish two pure polymorphs of ranitidine hydrochloride (forms I and II), as well as to distinguish a 50:50 mixture of the two structures.32 The observed sample response at the doubled frequency showed a correlation with the relative % (w/w) of Rantidine Hydrochloride form II, with a 50/50 (w/w) mixture of the polymorphic forms showing a response equal to an equally-weighted linear combination of the pure component SHG intensities. A similar analysis was also shown with enalapril maleate forms I and II by Strachan et al.33



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More recently, Schmitt et al. used nonlinear optical Stokes ellipsometry34 (NOSE) to rapidly discriminate two polymorphic forms of the small molecule D-mannitol.35 NOSE combines SHG microscopy with fast (8MHz) polarization modulation to measure the polarization-dependent SHG efficiency on a per-crystal basis. Together with an iterative least-squares analysis and linear discriminant analysis (LDA), NOSE enabled the Boolean discrimination of orthorhombic crystal forms of D-mannitol. Discrimination of orthorhombic structures by statistical tests (t test) was achieved with >99.99% confidence based on the raw polarization dependence and intensity discrepancy of the SHG. However, the highest confidence in discriminatory capability was achieved by removing the effects of crystal orientation from the polarization-dependent response via the iterative, nonlinear least-squares fitting algorithm. CARS microscopy has also been used to identify the polymorphic form of an API within a final dosage product. Hartshorn et al. used broadband CARS to identify three different polymorphic forms of the drug indomethacin together with multiple excipients within a tablet.17 The chemical structure of indomethacin is given in the supporting information, Figure SI-1. Figure 2 shows broadband CARS images and per-pixel spectra from tablets composed of different polymorphic ratios, reproduced from reference 17. γ-indomethacin, α-indomethacin, and croscarmellose sodium are shown in green, blue, and red, respectively. Individual spectra are from the pixels indicated with arrows in the bottom panels. In addition to the ability to accurately quantify the relative loadings of the polymorphic forms, broadband CARS images found the occurrence of small quantities of amorphous Indomethacin within the tableted form. Likely formed during tablet pressing, the amorphous Indomethacin would have been challenging to detect using alternative techniques such as spontaneous Raman scattering alone, due to the relatively low total concentration of amorphous material for bulk analysis and the long integration times required for spontaneous Raman imaging. These two approaches differ in their potential applications, complexity, and information content. Relative to NOSE measurements, SHG studies based on simple signal integration are more straightforward to obtain, but are also more prone to artifacts in quantitative analyses due to additional variables such as phase matching in the SHG-active material (assuming a collimated source), unknown or highly variable particle size distributions, and preferred orientation effects. Recently, SHG microscopy (as distinct from single-point measurements) has been shown to address many of these limitations by directly using the size distribution generated from the micrographs as part of the quantitative analysis.36 In this implementation, SHG microscopy can be performed without requiring a calibration reference. NOSE microscopy further extends the quantitative advantages of SHG microscopy by providing additional access to the high sensitivity of SHG to the polarization state of the driving field. While the single-point measurements of powders generally suggest experiments in polymorph identification in the context of quality/ formulation control, NOSE microscopy targets early stage experiments in polymorphic form discovery. In principle, however, single-point measurements could be applicable to high throughput screening and crystal form discovery. Single-point measurements provide a relatively simple approach as opposed to imaging, as the instrumentation requires no scanning mirrors, generally has less internally-timed components, and requires simpler data analysis approaches for the scalar outputs. However, the information content is correspondingly lower in single point measurements. NOSE images allow assessment of crystal habit and number density, and have been shown to enable the recovery of individual crystal orientations,35 potentially a benefit for subsequent analyses that may


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suffer from preferred orientation effects (PXRD, Raman). Additionally, NOSE measurements require no calibration of the instrument. While the work by Hartshorn et al. focused on the analysis of a final dosage form, here again the technique could, in principle, be applied to early stage crystal form discovery.17 Relative to measurements based on SHG, the available spectral information in CARS arguably yields a more direct and robust ability for polymorph identification. However, this increased spectral information content comes at the cost of instrument complexity. Narrow-band CARS microscopy requires the spatial and temporal overlap of two incident beams, one of which is independently tunable, while broadband CARS requires a broad-band infrared source as well as the use of a spectrometer. CARS microscopes are just now becoming commercially available, with the instrumentation costs representing a significant barrier for widespread adoption. In principle, SHG can be done concurrently with CARS. However, in practice, ps pump pulses are typically used for CARS in order to limit bandwidth and selectively target particular vibrational resonances, while SHG is most efficient with significantly shorter pulses. Additionally, as implemented in Hartshorn et al., CARS provides contrast based solely on vibrational transitions rather than symmetry, which may be subtle between certain polymorphic forms. Polarization-resolved CARS has recently been shown to be sensitive to the symmetry of the probed vibrational resonances.37 Nonresonant background (e.g., from coherent anti-stokes two-photon scattering) can interfere, complicating image analysis to isolate just the vibrational changes. The costs and potential measurement challenges associated with CARS are poised to decrease as the technology develops and becomes more established. Trace crystallinity and amorphous solid dispersions An increasing number of drug molecules under development are in danger of being abandoned due to poor aqueous solubility.2,3 While the development of these active ingredients as amorphous solid dispersions (ASDs) has been shown as a promising route to kinetically overcome the thermodynamic barriers of solubility, the tendency of the drug product to crystallize over time threatens shelf life, formulation stability, and overall efficacy.6,38 The ability to detect trace levels of crystallinity within bulk amorphous materials is highly advantageous for proper quality control and stability testing of amorphous formulations. For formulations at low drug loadings (≤10%), even samples that have completely crystallized may show levels of crystallinity approaching the limits of detection of common characterization methods such as Raman, DSC, and PXRD (commonly ~1-5%).39–41 Solid-state NMR (ssNMR) can achieve an order of magnitude lower detection limits (~0.1%) but routinely requires several hours of measurement time.42–45 Such occurrences of residual crystallinity may compromise the therepeutic dose of the drug within the formulation. SHG microscopy has been shown to enable the selective detection of trace levels of crystalline small molecules within a nominally amorphous matrix.46 Multiple manuscripts have addressed various applications of these measurements. In 2010, Wanapun et al. used SHG microscopy to monitor crystal nucleation and growth in pure melt-quenched amorphous drugs.47 The decrease in detection limits of SHG microscopy for crystalline material as compared to common techniques was quantified, suggesting a detection limit for SHG microscopy as low as ~3 ppb under favorable conditions. SHG microscopy also revealed inconsistencies in the preparation of amorphous drug materials, as repeated preparations varied widely in the presence or absence of small (~several µm) nuclei within the melt-quenched product. Subsequent crystallization times were observed by SHG and optical microscopies to be much faster for samples containing trace



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nuclei in the melt, consistent with classical nucleation theory. Similar conclusions were found in a later study by the same author, in which cryomilled griseofulvin was observed to crystallize ~40x faster than SHG-amorphous melt-quenched griseofulvin.48 These experiments suggested the formulation of nanocrystalline nuclei, below the detection limit of SHG, that were still large enough to allow subsequent crystal growth. These combined results have implications for assessment and prediction of the stability and shelf life of amorphous formulations. In the event that an amorphous material contains hard-to-detect micro or nanocrystalline domains, it is likely that the active ingredient may crystallize more rapidly than its truly amorphous counterpart. Such behavior could drastically decrease the shelflife of the drug product. In contrast to the pure amorphous drug products examined in the previous two studies, solid dispersions seek to imbed the active ingredient in a polymer matrix to increase the stability of the final dosage form. While often present at lower levels as compared to pure drug products, crystal nucleation is still a concern in such formulations. Kestur et al. demonstrated the utility of SHG microscopy for quantifying trace crystallinity in model amorphous solid dispersions.46 SHG is symmetry forbidden from the polymer chains (negligible long-range order), allowing selective imaging for noncentrosymmetric crystalline moieties within the dispersion/ physical mixture. Figure 3 shows a volume rendering of the SHG images for both pure crystalline naproxen and 0.1% crystalline naproxen in hydroxypropyl methylcellulose (HPMCAS), together with a depth-scan over an individual SHG-active crystallite, reproduced from reference 46. The chemical structure of naproxen is given in the supporting information, Figure SI-1. For relatively high crystalline contents (0.1% to 100%) in which crystallinity could be independently validated using PXRD and Raman spectroscopy, SHG microscopy was found to exhibit a linear response from in powdered blends of naproxen in HPMCAS. Calibration curves were also prepared via PXRD and Raman analyses. These same three techniques were also used to monitor crystallization of a 25% DL solid dispersion of naproxen in HPMCAS. The observed kinetics of crystallization from SHG microscopy matched those found from PXRD and Raman. Moreover, SHG showed lower limits of detection (~10 ppm) as compared to both Raman and PXRD (>1%). Hsu et al. used SHG microscopy together with SEM, AFM, and PXRD to characterize the phase behavior of drop-printed solid dispersions of naproxen in polyvinylpyrrolidone (PVP).49 Drop printing solid dispersions, in which small (~3 µL) volumes of drug/ polymer solutions are printed on edible substrates to facilitate rapid and reproducible solvent removal, has the potential to circumvent scale-up complications in the production of amorphous products by other methods (e.g., spray-drying, hot-melt extrusion). SHG microscopy enabled crystalline-specific imaging of the drop-printed solids, yielding assessments of overall crystallinity as well as crystal size and shape. Combined analyses by SEM, AFM, PXRD, and SHG showed differences in the size, shape, and number of naproxen crystals depending on the substrate chosen for drop-printing (chitosan vs. HPMC) and the storage temperature (25°C vs. 40°C). Among other findings, larger crystals were observed in samples stored at 40°C over those stored at 25° C (chitosan substrates), although the overall crystallinity in the 25°C samples was observed to be higher. These results were found to be consistent with classical nucleation theory, whereby the samples stored at 40°C will experience increased mass transport over those stored at 25°C. However, the decreased rate of solvent evaporation for samples stored at 25°C allows increased crystallization time relative to the 40°C samples, yielding higher overall crystallinity in the former. However, minimal differences in


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dissolution rates between these two samples were observed, suggesting that the increased crystalline content of the 25°C samples (lower dissolution rates anticipated) was at least partially offset by the decreased average crystal size within the same sample. While SHG enables detection of trace crystallinity, it is limited to homochiral API preparations, as racemic co-crystals are symmetry forbidden for SHG. Additionally, different API crystal structures may have very disparate (by several orders of magnitude) SHG activities. While this fact can be advantageous in determination of new crystal forms as discussed in the preceding section, it can complicate quantitative analysis of polymorphic mixtures or high symmetry (low SHG activity) crystal forms. While the cost of SHG is significantly lower than in CARS, it may still represent a significant expense. Fortunately, fiber lasers are emerging as low-cost, low-maintenance sources. Chemical mapping of final dosage forms The therapeutic behavior and stability of a final dosage form is affected by the spatial distribution and phase (e.g., crystalline, amorphous) of the API and excipients. Validation of a uniform distribution of ingredients within the tablet helps ensure reliable product performance. NLO spectroscopic imaging techniques such as CARS and stimulated Raman scattering (SRS) microscopy have been used to chemically map the contents and ingredient distributions within final dosage forms. Hartshorn et al. used broadband CARS (BCARS) microscopy to map the distribution of two polymorphs of indomethacin together with a variety of excipients and manufacturing compounds, including lactose monohydrate, microcrystalline cellulose, croscarmellose sodium, and magnesium stearate.17 BCARS images were compared to wide-field spontaneous Raman measurements for validation. BCARS accurately recovered the relative concentrations of each different pharmaceutical component within the tablets. BCARS enabled imaging with as little as 100 ms/ pixel dwell time, with a SNR improvement of ~102 over wide-field spontaneous Raman measurements. Quantification of the relative amounts of pharmaceutical components by BCARS was also validated via ensemble-averaged confocal Raman measurements. Slipchenko et al. used SRS to chemically map amlodipine besylate tablets from a variety of manufacturers.50 SRS mapping was able to distinguish the spatial distribution of the API together with a variety of excipients, including microcrystalline cellulose, dibasic calcium phosphate anhydrous, sodium starch glycolate, and magnesium stearate. The chemical structure of amlodipine besylate is given in the supporting information, Figure SI-1. Large area SRS images of tablets from six different manufacturers are shown in Figure 4, reproduced from reference 50. SRS was also compared to spontaneous Raman and CARS imaging techniques. SRS showed an imaging speed ~104 times faster than spontaneous Raman, requiring only 53 seconds per 512 x 512 image. Replicate analyses of a single FOV allowed a comparison of SRS and CARS imaging modalities. CARS images exhibited higher SNR than SRS images when on-resonance with a vibrational transition in the amlodipine besylate (1650 cm-1). However, offresonance CARS images (ωp - ωs = 2200 cm-1) matched those obtained at 1650 cm-1 even after desynchronizing the input beams. These results were contributed to significant background from autofluorescence in the CARS images. SRS images at the same resonance (2200 cm-1) yielded only small puncta of signal that exhibited minimal spatial overlap with features in the on-resonance SRS image. As these puncta persisted with desynchronizing of the input beams, they were tentatively attributed as arising from the photo-induced heating of dust particles on the tablet or coverslip surface.



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Toth et al. used UV-SHG (532 nm incident, 266 nm detection) together with two-photon excited ultraviolet fluorescence (TPE-UVF) to image the API griseofulvin within a variety of common excipients including HPMCAS, PVP, and lactose.51 Griseofulvin was found to produce a combined UV-SHG/ TPE-UVF signal at least three orders of magnitude greater than the brightest excipient (anhydrous lactose), enabling selective imaging of the spatial distribution of the API. Conventional SHG (800 nm incident, 400 nm detection) and two-photon excited fluorescence (TPEF) were also employed as imaging modalities. Combined conventional SHG/ TPEF signals for griseofulvin were ~200 times greater than for anhydrous lactose. In addition to imaging griseofulvin in a variety of excipients, UV-SHG and TPE-UVF were also used to image a commercial tadalafil tablet (Cialis). While the chemical information within SHG/ two photon fluorescence images is rather low, such methodologies are potentially powerful due to the relatively fast imaging rates (~2 seconds/ frame). For formulations in which the composition is known to facilitate selective imaging of the API by these methods (i.e. no SHG-active excipients), mapping of the drug distribution within a final dosage form could be performed very rapidly as compared to orthogonal methods such as spontaneous Raman (~hours/ frame). While SHG generally yields the highest frame rates for imaging, chemical mapping can be made difficult due to the presence of SHG-active excipients (e.g., lactose). Fortunately, the difference in brightness and/or polarization-dependence may be used to allow discrimination. These differences are significantly enhanced for UV-SHG, due to the resonant SHG enhancement present in many APIs but not in common excipients. TPE-UVF can also help overcome this barrier, as it provides information the spatial distribution of aromatic APIs (crystalline or amorphous), and can be performed simultaneously with UV-SHG. While chemical identification may be more direct using CARS, imaging can be complicated by non-resonant background interferences. Heterodyne detection and other approaches may remove this interference, but with significant increases in instrumental complexity. SRS is another option, but can still be challenging to discriminate between changes in spectral vs. number density, since only one frequency at a time is typically recorded. Hyphenated techniques using SHG to guide locations for orthogonal analysis are attractive compromises, as detailed in a subsequent section (vide infra). In-situ chemical imaging during dissolution Dissolution testing is ubiquitously performed in drug product development and quality control, as it allows a direct assessment of active ingredient dissolution kinetics under conditions designed to mimic those encountered in vivo. In amorphous solid dispersions, dissolution testing is particularly challenging to predictively monitor, as poorly soluble APIs can selectively partition within amorphous materials and/or crystallize following initial rapid dissolution.52,53 In addition to monitoring the solutionphase concentration of the active ingredient (most commonly by simple UV-vis absorbance), imaging/ spectroscopic monitoring of changes in final dosage forms in the presence of the dissolution medium can also be performed. Traditionally, these studies are problematic when using conventional techniques, either due to chemical interferences (e.g., water interference in IR), long measurement times (e.g., spontaneous Raman), or simply due to incompatibility of the dissolution platform with simultaneous analysis by other methods (e.g. solid-state NMR). Windbergs et al. used CARS microscopy to monitor the dissolution and phase behavior of theophylline (anhydrous and monohydrate forms) in tableted physical mixtures and extrudates with the lipid tripalmitin.54,55 The chemical structure of theophylline monohydrate is given in the supporting


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information, Figure SI-1. In-situ CARS allowed real-time monitoring of the drug product during dissolution, enabling direct visualization of the disappearance of API crystals and retention of the lipid matrix over the course of dissolution. CARS images of theophylline monohydrate tablets (top row), anhydrous theophylline tablets (middle row), and tablets of anhydrous theophylline extrudate (bottom row) after various lengths of time in the dissolution medium are shown in Figure 5, reproduced from reference 54. The lipid (tripalmitin) is shown in red and the active ingredient is shown in green. In the tableted physical mixtures, theophylline anhydrate was found to transition to the monohydrate form within ~5 minutes following the start of the dissolution experiment (water used as the dissolution medium), with dissolution of the monohydrate form requiring an additional ~90 minutes of dissolution time. In contrast, both tablets and raw powders of theophylline anhydrate/ tripalmitin extrudate were not observed to transition to the monohydrate form. These results were attributed to the purported solution-mediated mechanism for transition from the anydrate to monohydrate forms, with discrepancies in formulation behavior attributed to reduced local porosity and exposure of the active ingredient within the extrudates. Fussell et al. used hyperspectral CARS to monitor phase changes in theophylline anhydrate tablets upon dissolution.56 Changes in the solid dosage form before and after dissolution were observed, with spectral information consistent with the conversion of theophylline anhydrate from its anhydrous to monohydrate form. Single-frequency CARS (faster imaging times than were required for full hyperspectral characterization) was used to monitor the dosage form in situ during dissolution (water as the dissolution medium). Conversion of the majority mass fraction of anhydrous theophylline to its monohydrate form took place in approximately four minutes. In combination with visible absorbance, CARS microscopy was able to correlate a drop in drug dissolution rate with the transition of the active ingredient from its anhydrous to its monohydrate form. Additionally, single-frequency CARS was used to image the same tablets with a methyl cellulose solution as the dissolution medium. Formation of the monohydrate form was delayed relative to the water-based experiments, with incomplete transformation of the anhydrous form and monohydrate crystal habit differences observed over the experiment life time (15 min). These results are attributed to crystal face-specific adsorption of methyl cellulose to the anhydrous crystal surface, limiting the contact of the monohydrate crystals with water molecules on certain crystal faces. These results were correlated with increased dissolution rates found via UV-vis when a methyl cellulose solution was used as the dissolution medium. Kang et al. used CARS to chemically map the anti-cancer drug paclitaxel in polyethylene glycol (PEG) and poly(lactic-co-glycolic acid) (PLGA) films, and monitor the behavior of the drug product during dissolution.57 The chemical structure of paclitaxel is given in the supporting information, Figure SI-1. CARS images identified paclitaxel partitioning selectively into PEG domains in 90:10 and 80:20 PLGA/ PEG films and allowed visualization of paclitaxel release upon exposure of the film to an aqueous dissolution medium. CARS images were obtained at a variety of depths in the formulation, enabling 3D imaging of the drug product during dissolution. Pore and ring structures were found to form ~3 minutes after exposure of the formulation to the dissolution medium. Formation of these structures was delayed in deeper cross sections of the formulation. These results are summarized in Figure 6, reproduced from reference 57. CARS images of PEG are shown, with the vertical dimension corresponding to various depths within the film and the horizontal dimension showing the temporal behavior of a given field of view over the course of the dissolution experiment. Spectral results from the ring structures indicate the



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redistribution of paclitaxel at PEG/ PLGA interfaces. To further study the ring and pore structures, fluorescein was added as a solution-phase marker for simultaneous CARS and two-photon excited fluorescence (TPEF) imaging. TPEF images showed strong fluorescein signal from the PLGA matrix following 30 minutes of immersion in the dissolution medium. It was theorized that PEG forms a gel-like structure at the PEG/ PLGA interface upon introduction of water, thereby begetting partitioning of paclitaxel into this interfacial area. These results corroborate previous efforts to quantify the dissolution rate of PTX from polymer films. Additional studies monitoring PTX release from other polymer films such as PEG/ polyethylene vinyl acetate (PEVA) and poly(styrene-b-isobutylene-b-styrene) were also published.58,59 SHG microscopy, combined with in situ small-angle/ wide-angle X-ray scattering (SAXS, WAXS), was used to correlate overall crystallinity and crystal surface area of naproxen/ polyethylene glycol solid dispersions with drug product dissolution rates.60 5/95 naproxen/ PEG solid dispersions (w/w) crystallized at 25° C (kinetics of crystallization and overall crystallinity confirmed by SAXS, WAXS) were found to exhibit dissolution rates ~2 times faster than identical dispersions crystallized at 40° C. These results correlated well with surface area analysis obtained from SHG images, in which the ratio of surface areas for the 25° C to 40° C samples was found to be ~1.7, indicating a smaller mean crystal size within the dispersions crystallized at the lower temperature (constant drug mass). Visual inspection of the SHG images is consistent with these conclusions. Additionally, the 5/95 naproxen/ PEG dispersions crystallized at 25° C were found to exhibit a greatly increased dissolution rate as compared to 10/ 90 and 20/ 80 (w/w) dispersions crystallized at the same temperature. These studies demonstrate the ability of SHG to rapidly assess formulation crystallinity and crystal size distribution/presentation, correlated with corresponding differences in drug dissolution rates. To date, most measurements have been done using buffer solutions – measurements in more complex media more closely matched to stomach contents are more challenging, due to the scattering of light by the dissolution medium. These studies are particularly important for APIs showing low water solubility, as crystallization of the drug following ingestion but prior to entrance into the blood stream could diminish therapeutic effects. Fortunately, the problems of scattering are less pronounced in NLO imaging compared to optical absorbance monitoring of dissolution. Coupling NLO imaging to orthogonal methods While SHG microscopy is well-suited for quantitation (typical SNR yields imaging with just a few ns integration time per pixel), common observables associated with this measurement, namely intensity per unit volume and polarization dependence of the frequency doubled light, are significantly lower in qualitative information content compared to spectroscopic methods. Fortunately, the spatial information identifying crystalline domains within SHG images can be used to inform alternative measurements. By restricting more time-consuming measurements to only areas of interest preidentified by SHG, the timeframes required for analysis are decreased. Additionally, such an approach adds chemical information to SHG images, allowing orthogonal confirmation of crystallite identity. This SHG-guided analysis approach has been demonstrated with both Raman spectroscopy and synchrotron X-ray diffraction. Schmitt et al. used SHG microscopy together with confocal Raman microscopy to characterize trace crystallinity within lyophilized Abraxane powder.61 Abraxane, or nanoparticle albumin bound (nab)


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paclitaxel, is a novel amorphous anti-cancer formulation for injectable suspension. The use of SHG to selectively target measurements by Raman microscopy to trace areas of crystallinity decreased sample characterization times for Raman by approximately two orders of magnitude. Figure 7 shows an SHG image of Abraxane powder together with select Raman spectra from SHG active (red) and inactive (blue) domains, along with Raman spectra of pure paclitaxel and human serum albumin, reproduced from reference 61. Measurements took place on two independent instruments, with fiducial markers used to facilitate sample positioning on each instrument. SHG images also enabled direct observation of the particle size distribution within the lyophilized powder, as well as confirmation of crystal insolubility in the suspended state. Such analyses provide both quantitative and qualitative characterization. Similar measurements were performed by Newman et al., in which synchrotron X-ray diffraction was used for chemical analysis of 100 ppm (w/w) physical mixtures of crystalline ritonavir in HPMC. Experiments were performed on a custom NLO platform that has been integrated directly into a synchrotron beamline. The development of click-to-center algorithms enabled automated placement of the SHG-active area of the sample for subsequent diffraction studies. Diffraction experiments with a 5 µm synchrotron beam enabled positive chemical identification of ritonavir microcrystals via indexing of the individual diffraction spots. Positive chemical identification was realized with a signal to noise >5000 and a 1s data acquisition time for the X-ray. This study yielded higher confidence (increased SNR) relative to the SHG-guided Raman studies. The integration of spontaneous Raman spectroscopy with SHG microscopy affords both qualitative and quantitative information together with relatively simple (relative to synchrotron XRD) instrumentation. Furthermore, it addresses the major limitations of both spontaneous Raman in conventional analysis (long imaging times) and the limitations of CARS/ SRS (more expensive and complex instrumentation relative to SHG and spontaneous Raman). Characterization by spontaneous Raman is ubiquitous in pharmaceutical development, lowering the barrier for widespread implementation of this combined analysis approach. While SHG-guided synchrotron XRD is a more complex measurement, the existing combined platform62 removes the barriers associated with sample transfer/ fiducial marking present in the SHG-guided Raman experiments. It should also be noted that the user-based architecture of synchrotron light sources facilitates the use of a single instrument by many parties, such that many duplications of the instrument should not be necessary for relatively widespread use. Outlook Emerging NLO technologies for evaluating drug products and materials meet with several barriers on the road to widespread adoption in industry and regulatory science. In addition to the transition of instrumentation from academic prototypes to commercially viable platforms (a large barrier in and of itself), a sufficiently large body of knowledge, demonstrated applications, and interim standards must be demonstrated and developed for the acceptance of new technologies as part of regulatory standards in the pharmaceutical industry.63 Building on the large body of literature presented in this review, a brief discussion of progress towards the goal of widespread industrial and regulatory adoption is warranted. A number of barriers exist in the adoption of new technologies on the level of the company and/or the individual researcher. Perhaps the most obvious of these is the opportunity cost associated



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with training researchers in the use of new technologies and proper interpretation of the resulting data/ product. Divergence between the targeted (on the part of the manufacturer) and actual (on the part of the end user) application of the technology, as well as its general ease of use, can both impact the time required for adoption. Properly vetting emerging technologies for potential points of failure can also be challenging, particularly at the stage of initial commercialization. These collective barriers may work to create a general cultural and industrial reticence in the adoption of emerging technologies. These factors may be compounded for emerging NLO methods due to the general perception of these techniques as difficult and costly. Fortunately, recent advances in NLO analyses are poised to help mitigate these risks. The advent of turn-key fiber laser systems is decreasing the cost and complexity of ultrafast sources, while simultaneously increasing their reliability so as to minimize time lost on account of instrument malfunction. Commercial platforms for both SHG microscopy and CARS are now available. Beyond novel hardware, user-friendly software capable of executing a variety of widely-used algorithms (e.g., multivariate curve resolution for CARS) is also becoming more commonplace. Continuation of the current trends to this effect will continue to decrease the cost of these commercial systems and should work to help overcome the general perception of NLO analyses as prohibitively costly and complex. Additionally, a wide variety of mechanisms for commercial development and regulatory acceptance are available to researchers. As commercialization progresses, industrial pharmaceutical companies are encouraged to include data from emerging technologies as part of QbD.7 To this end, the FDA has recently published guidelines for industry regarding the inclusion of data from new manufacturing and characterization technologies within investigational new drug (IND) applications, new drug applications (NDA), and biologic license applications (BLA).64 This mechanism should facilitate scientific communication of the theory, applications, and viability of emerging technologies between the FDA and industry. Additionally, the FDA regularly calls on formal Advisory Committees for the purposes of evaluating new science for implementation in regulatory policy.65 Communication of and advocacy for new technologies on the part of these committees should be encouraged. A tight focus on QbD should be maintained by both regulatory and industrial partners, helping to catalyze the adoption of new technologies as well as increase the quality and decrease the cost of emerging drug products. test


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Acknowledgement PDS would like to acknowledge Garth J. Simpson of Purdue University for helpful discussions and advice. References (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12)

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Figure 1: Jablonski diagrams for three common nonlinear optical processes: second harmonic generation (SHG, left), coherent anti-stokes Raman scattering (CARS, middle), and stimulated Raman scattering (SRS, right).



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Figure 2: Broadband CARS images (top row) and single-pixel spectra (bottom row) from tablets of γ/ α indomethacin at various compositions (listed above). Images are pseudocolored RGB, with red corresponding to spectral components at 2880 cm-1 (croscarmellose sodium), green corresponding to spectral components from 1668-1700 cm-1 (γ indomethacin), and blue corresponding to spectral components at 1682 cm-1 (α indomethacin). Individual spectra in the bottom row follow the same color scheme, and are from the pixels indicated by the black arrows. The per-pixel dwell time was 500 ms. Reproduced from reference 17.


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Figure 3: Volume-rendered SHG images from 100% crystalline naproxen (left) and 0.1% crystalline naproxen in hydroxypropyl methylcellulose acetate succinate (w/w) (middle). The line scan at right shows the SHG intensity for an individual crystallite as a photon counting rate across the depth of the sample. Reproduced from reference 46.



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Figure 4: Pseudocolored SRS images of amlodipine besylate tablets with a variety of excipients from six different manufacturers, under 10x (0.40 NA) magnification. Red, green, blue, yellow/ orange and magenta correspond to amlodipine besylate, microcrystalline cellulose, dibasic calcium phosphate anhydrous, sodium starch glycolate and magnesium stearate, respectively. The yellow/ orange channel in the SRS image of the Apotex tablet (top row, middle) corresponds to a combination of lactose monohydrate and corn starch, rather than sodium starch glycolate. The scale bar is 200 µm. Reproduced from reference 50.


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Figure 5: Dissolution behavior of tablets of theophylline monohydrate/ tripalmitin (top row), anhydrous theophylline/ tripalmitin (middle row), and anhydrous theophylline/ tripalmitin extrudate (bottom row) as monitored by CARS microscopy. Tripalmitin is shown in red and the active ingredient is shown in green, with the immersion time in the given dissolution medium (purified water) indicated in each image. Reproduced from reference 54.



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Figure 6: CARS images (PEG spectral channel, 2890 cm-1) of films of 15% (w/w) paclitaxel in 80/20 PLGA/PEG during dissolution testing. The vertical dimension shows images at various depths within the film, while the horizontal dimension shows the change in a given field of view over the course of the dissolution experiment. Each image represents a 1.12 second integration. Reproduced from reference 57.


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Figure 7: SHG image of Abraxane powder (left) and accompanying Raman spectra after digital highpass filtering (right). The Raman spectrum shown in red is from the SHG active area of the image (point indicated by “II” at left), while the spectrum shown in blue is from an SHG inactive area of the image (point indicated by “I”). Black and green spectra are from pure paclitaxel (PTX) and pure human serum albumin (HSA), respectively. Spectral overlap between the red and black traces is observed, with increased SNR at 1007.1 cm-1 and 1604.3 cm-1 consistent with an increased PTX concentration at point “II” in the SHG image. Reproduced from reference 61.



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For TOC use only


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Recent Advances in Nonlinear Optical Analyses of Pharmaceutical

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