In Quest for Improved Drugs against Diabetes: The Added Value of X-ray Powder Diffraction Methods

Abstract

Human insulin (HI) is a well-characterized natural hormone which regulates glycose levels into the blood-stream and is widely used for diabetes treatment. Numerous studies have manifested that despite significant efforts devoted to structural characterization of this molecule and its complexes with organic compounds (ligands), there is still a rich diagram of phase transitions and novel crystalline forms to be discovered. Towards the improvement of drug delivery, identification of new insulin polymorphs from polycrystalline samples, simulating the commercially available drugs, is feasible today via macromolecular X-ray powder diffraction (XRPD). This approach has been developed, and is considered as a respectable method, which can be employed in biosciences for various purposes, such as observing phase transitions and characterizing bulk pharmaceuticals. An overview of the structural studies on human insulin complexes performed over the past decade employing both synchrotron and laboratory sources for XRPD measurements, is reported herein. This review aims to assemble all of the recent advances in the diabetes treatment field in terms of drug formulation, verifying in parallel the efficiency and applicability of protein XRPD for quick and accurate preliminary structural characterization in the large scale.

Applications of X-ray Powder Diffraction in Protein Crystallography and Drug Screening

“…Ligands such as phenol and resorcinol derivatives which led to the formation of more than one monoclinic symmetry polymorphs are of particular interest [31,32] (Figure 11). The previously referenced P2 1(γ) crystal polymorph (a = 87.0749 7 Table 2, while in pH: 6.7 to 8.6 and 6.2 to 8.1, the complexes adopt the rhombohedral R3 symmetry, with R 6 and T 3 R 3 f HI conformation accordingly [26,31]. In the remarkable case of 4-ethytlresorsinol, monoclinic symmetry was observed throughout the whole pH range (4.95 to 8.05) for repeated crystallization experiments [32].…”

“…XRPD has proved its applicability as the most suitable tool for high throughput and accurate characterization of numerous microcrystalline suspensions by virtue of the simplicity of XRPD data collection and the uniqueness of each polymorph's diffraction pattern. To date, research reports on HI microcrystals exhibit fascinating polymorphism, occurring upon physicochemical modifications of their environment, namely pH, temperature, and relative humidity, or ligand binding and further expanding the phase diagram of the molecule [26]. Further advantages from the use of XRPD measurements include homogeneity and purity control of the precipitates, whereas, even in cases of challenging samples, powders can easily lead to the extraction of accurate lattice parameters which allow for the detection of structural modifications.…”

“…However, a steadily increasing number of studies has underlined efficiency of the powder diffraction method in a wide spectrum of fields, including structure determination of zeolites, small organic molecules [8,[17][18][19], and, more recently, biological macromolecules [9,[20][21][22][23][24][25][26][27]. The use of high-resolution synchrotron data, along with new analytical procedures, have stimulated exciting progress in XRPD efficacy, expanding the variety of proteins under structural examination and constituting the technique as a strong, complementary tool to already well-established methods [23].…”

“…Although the use of polycrystalline specimens has some disadvantages, as this approach may provide low or medium resolution diffraction data (3-10Å), these samples are produced more easily and quicker than a single crystal. XRPD methods allow for the examination of low-quality crystals as an ensemble, protein polymorph screening becomes a common procedure, while time-resolved studies are also possible [23,26,28,29]. Preliminary structural features extracted from these experiments can provide crucial information toward their complete structural characterization.…”

“…The use of powder diffraction measurements additionally allow for homogeneity and purity control studies of the precipitates, revealing crucial information for the pharmaceutical industry that cannot be obtained easily via SCXD measurements, as one crystal may not be representative of the whole batch [26]. In addition, crystallite size comprising the powder can be estimated from the peak widths present in XRPD profiles [39].…”