A New Approach to Supramolecular Structure Determination in Pharmaceutical Preparation of Self-Assembling Peptides: A Case Study of Lanreotide Autogel

Abstract

The supramolecular structure in peptides' prolonged-released gel formulations is the most critical parameter for the determination of the pharmaceutical profile of the drug. Here, we report our investigation on lanreotide Autogel as a case study. For the first time, we describe the use of the pulsed field gradient (PFG) diffusion-ordered spectroscopy (DOSY) magic-angle spinning NMR to characterize the supramolecular self-assembly and molecular mobility of different samples of lanreotide Autogel formulations prepared according to different formulation protocols. The diffusion coefficient was used to calculate the hydrodynamic radii of supramolecular assemblies and build relative molecular models. DOSY data were integrated with NMR imaging (MRI) measurements and atomic force microscopy (AFM) imaging.

Keywords: MRI; NMR; atomic force microscopy; delivery systems; peptides.

Figure 1

Somatostatin, lanreotide and octreotide peptide structures and sequences.

Figure 2

Schematic representation of (1) preparation of Somatuline analogs with different amounts of AcOH as indicated by the color bar on the left, and (2) the techniques applied for the characterization of supramolecular structures assumed by the obtained formulations.

Figure 3

The 2D-DOSY experiments acquired at 300 K on a 500 MHz spectrometer equipped with HR-MAS probe. Spectra reporting the distinctive peaks of Somatuline Autogel^®, AK1841045 (11% of AcOH), and AK1841052 (5.7% of AcOH), obtained in DMSO-d₆. The diffusion values were calculated as an average of each peptide peak diffusion value (m²/s) and were reported as D values (on the dashed lines) and as logD (ordinate on the right).

Figure 4

Diffusion experiments acquired using 300 MHz spectrometers equipped with MicWB40 Probe in combination with the Micro2.5 Gradient System. Diffusion maps of axial slices obtained on syringes containing each sample: (a) Somatuline Autogel^®, (b) AK1841045, (c) AK1841047, (d) AK1841050, and (f) AK1841053; diffusion maps of the sagittal slice of the syringe containing sample (e) AK1841052. The color map on the right indicates D values (m²/s): yellowish color indicates a value closer to 8 × 10⁻¹⁰, while blueish color indicates a value closer to 0.

Figure 5

Gaussian distribution of the D values obtained from each pixel in the MRI DWI images of the samples: (A) Somatuline Autogel^®, (B) AK1841045, (C) AK1841047, (D) AK1841050, (E) AK1841052, and (F) AK1841053. The black lines represent the histogram distributions, and the blue lines report the Gaussian fit. For each sample, the D values are reported, which correspond to the maximum peak of the Gaussian distribution.

Figure 6

Topographic AFM images (0.6 × 0.6 µm²) acquired on mica at room temperature in dry conditions. For each sample, we acquired two concentrations (40 μg/μL and 8 μg/μL). AFM measurements were performed in tapping mode configuration using silicon nitride cantilevers with an elastic constant of 0.6 N/m (MSNL Bruker) and a nominal tip radius of 10 nm.

Figure 7

Topographic AFM images (1.5 × 1.5 μm² (a), 0.6 × 0.6 μm² (b) and 3D (c)) of Somatuline Autogel^® (d,e). Graphs showing the height line profiles measured on the topographies indicated by the blue lines. AFM images show the presence of tubular structures that self-organized in ribbons. Graphs display the height and width of ribbons (d) and tubular structures (e). AFM measurements were performed in tapping mode on dry samples.

Figure 8

AFM height image (1.5 × 1.5 μm²) of AK1841045 (a) and height line profile measured on the topographies indicated by blue and pink lines (b,c). AFM images show the presence of tubular structures which self-organized in ribbons. Graphs display the height and width of tubular structures (b) and ribbons (c). AFM measurements were performed in tapping mode on dry samples.

Figure 9

AFM height image (0.6 × 0.6 μm²) of AK1841052 (a) and height line profile (b) measured on the topography indicated by the blue line. AFM measurements were performed in tapping mode on dry samples.