(T1130-02-10) The Use of Raman Mapping and Hot-Stage Microscopy to Detect Micro-Domains of Crystallinity in Amorphous Solid Dispersions of Nilotinib

Purpose: Nilotinib, a marketed product for the treatment of chronic myeloid leukemia, exhibits minimal aqueous solubility which can negatively affect its bioavailability. In efforts to overcome this limitation, an amorphous solid dispersion (ASD) using HPMCP was prepared that exhibited a 2-fold increase in dissolution. It is thought that a salt is formed between nilotinib and the polymer, resulting in an increase in solubility (1). The current research describes further investigations into this ASD, in addition to a nilotinib-HPMCAS ASD. Traditional characterization techniques such as XRPD and DSC indicate that the ASDs are amorphous. To probe for micro-domains of crystalline API, the state-of-the-art analytical technique is pair distribution function (PDF) analysis, a technique that requires a synchrotron (2). Here, we use laboratory micro-methods such as Raman mapping and hot-stage microscopy to detect the micro-domains of crystalline nilotinib. The presence of crystalline micro-domains might be an indicator of future stability of the ASDs.

Methods: Nilotinib ASDs using both HPMCAS (AS) and HPMCP (P) polymers were prepared using a rotovap. Orthogonal techniques were then used to physically characterize the ASDs. The amorphous structure was confirmed by X-ray powder diffraction (XRPD), differential scanning calorimetry (DSC), and modulated differential scanning calorimetry (mDSC). XRPD patterns of were collected on a PANalytical Empyrean diffractometer using a Cu Ka incident beam radiation. Each sample was sandwiched between 3-µm-thick Etnom films and analyzed in transmission geometry at 2-40° 2è range. The glass transition temperature (Tg) of the ASDs was measured using a DSC Discovery 25 (TA instruments). Each sample was heated at an overall rate of 1 °C/min from -25 °C to 250 °C and modulated by 1 °C for 50 seconds. Micro techniques included both Raman mapping and Fourier-transform infrared microspectroscopy to provide molecular structural information, and hot-stage optical microscopy (HSOM) to evaluate thermal properties. A HORIBA XploRA Confocal Raman Microscope was used to collect spectra over a 25 x 25 micrometer area, 2 micrometer steps, a 785 nm laser, and a 1200 l/mm grating.  A Smiths Detection IlluminatIR 2.0™ Fourier-transform Infrared Microspectrometer accessory on an Olympus Series BX51TRF polarized light microscope was use in attenuated total reflection mode using a diamond ATR objective. Hot-stage optical microscopy was conducted using a Linkam LTS420 hot stage with a T95 LinkPad system controller on an Olympus Series BX51TRF polarized light microscope. Images were acquired using a with a Lumenera Series Infinity 3-3URC digital camera.  Image capture and image processing using Image-Pro®.

Results: Nilotinib ASDs displayed a broad halo in the XRPD indicative of amorphous material. DSC and mDSC confirmed the amorphous character of the ASDs with a single glass transition (Tg ~ 97 - 116 °C). FT-IR results of the Nilotinib ASDs support salt formation, however, the results suggested some free nilotinib might be present in the AS dispersion. Raman mapping of the ASDs suggested the nilotinib appears to be amorphous due to the presence of broadened peaks compared to the crystalline nilotinib reference material. However, regions in the Raman maps can be found in both dispersions where some residual crystalline nilotinib character remains. The profiles in Figure 1 show the intensity of the nilotinib peak at 1667 cm^-1 compared to the intensity of the ionized carboxylic acid group of the polymer at 1675 cm^-1 for the two ASDs. Spectra extracted from the red pixels revealed the presence of crystalline nilotinib. HSOM analysis of the two ASDs confirmed the detection of micro-domains of crystallinity. When the ASDs were annealed via heating, the strain birefringence disappeared, revealing small crystals interior to the glassy bulk (Figure 2).

Conclusion: Nilotinib ASDs were physically characterized using Raman microscopy, infrared microspectroscopy, Hot-stage microscopy, XRPD, DSC, and mDSC.  The macro techniques indicated the ASDs were amorphous. Micro techniques enabled the detection of micro-domains of crystallinity in the ASDs. These micro-techniques are a valuable tool for fully characterizing ASDs, as they can detect very small amounts of crystalline drug which could indicate which dispersions might be more stable than others. (3).

References: 1. Byrn, S.R., Smith, D., Zimmerman, A., Larsen, B.S., Tian, F. (2021). Nilotinib Compositions with Enhanced Solubility. (US 2021/026005 A1), Filing Date Feb 20, 2020, Issuance Date Aug 26, 2021.
2. V. Bezzon, F. Ferreira, P. Smith, C. Benmore, S. Byrn, G. de Araujo, International Journal of Pharmaceutics, “Amorphous Dispersions of Flubendazole in Hydroxypropyl Methylcellulose: Formulation Stability Assisted by Pair Distribution Function Analysis” Volume 600 (2021).
3. K. Chen, N. Swarnakar, S. Byrn, D. Engers, D. Purcell, P. Smith, S. Tiwari, “Copovidone-Based Spray Dried Amorphous Solid Dispersions: Investigation of Drug Loadings and the Limit of Congruency”, presented at AAPS PharmSci 360 (2024).

Figure 1: Raman mapping profiles for micro-domains of crystalline nilotinib (red pixels) for the nilotinib-HPMCAS ASD (left image) and the nilotinib-HPMCP ASD (right image)

Figure 2: HSOM images of micro-domains of crystallinity in the nilotinib-HPMCAS ASD (left image) and the nilotinib-HPMCP ASD (right image)