Detrimental Effect of the Film Coat Chemistry and Thickness on the Physical Stability of Amorphous Solid Dispersions in Tablet Formulations.

Amorphous solid dispersions (ASDs) have been widely utilized to enhance the bioavailability of pharmaceutical drugs with poor aqueous solubility. The role of various excipients on the amorphous drug to crystalline form conversion in ASDs has been widely documented. However, there has been no published study to investigate the role of film coating material on the physical stability of an ASD based tablet formulation, to the best of our knowledge. Here we show that the film coating can potentially have a detrimental impact on the physical stability of spray dried intermediates (SDI) in tablet formulations. The impact of the film coating on the physical stability of SDI was found to be related to the film coat material composition, and an increase in the film coating thickness led to a reduction in the physical stability of SDI in tablets. Oral compressed tablets in which the film coat material was "mixed-in" with the formulation blend showed a similar or worse physical stability than film coated tablets, further underscoring the film coat material impact on physical stability, independent of the film coating process. This study demonstrates a need for careful consideration of the film coat material selection for ASD based pharmaceutical product development.

Mechanism and Impact of Excipient Incompatibility: Cross-Linking of Xanthan Gum in Pediatric Powder-for-Suspension Formulations.

Research on pharmaceutical pediatric powder-for-suspension formulations mainly focuses on chemical and physical stability of the active pharmaceutical ingredient. However, the chemical stability of excipients could also play a key role in governing the quality and performance of the product. The suspending agents that are added into formulations to suspend the active pharmaceutical ingredient particles are critical to ensure the suspension dose accuracy. In this article, we investigate the chemical stability of the suspending agent-xanthan gum-in the presence of other excipients, particularly commonly used acid modifiers (i.e., citric acid, malic acid, succinic acid, and fumaric acid) in pediatric powder-for-suspension formulations. We observed that some of the acid modifiers catalyze cross-linking of xanthan gum during accelerated stability studies in powder blends, which significantly decreases the viscosity of the corresponding constituted suspension, resulting in poor suspendability and dose inaccuracy. Furthermore, we found that the cross-linking of xanthan gum is acid-dependent and that a careful selection of acid modifiers can mitigate the degradation issues of xanthan gum. Finally, we characterized the cross-linked xanthan gum using Fourier transform infrared spectroscopy and solid-state nuclear magnetic resonance and discussed the possible degradation mechanisms.

Dispersive Raman Spectroscopy for Quantifying Amorphous Drug Content in Intact Tablets.

Process-induced inadvertent phase change of an active pharmaceutical ingredient in a drug product could impact chemical stability, physical stability, shelf life, and bioperformance. In this study, dispersive Raman spectroscopy is presented as an alternative method for the nondestructive, high-throughput, at-line quantification of amorphous conversion. A quantitative Raman method was developed using a multivariate partial least squares (PLS) regression calibration technique with solid-state nuclear magnetic resonance (ssNMR) spectroscopy as the reference method. Compositionally identical calibration tablets containing 20% w/w total MK-A drug in varying weight proportions (0%-50% w/w based on total MK-A) of amorphous and crystalline MK-A were compressed at 10-45 kN force. PLS predictions of amorphous content of tablets using Raman spectroscopy correlated well with ssNMR quantification. The predictive accuracy of this model led to a strong correlation (R2 = 0.987) with a root mean-squared error of prediction of 1.5% w/w amorphous MK-A in tablets up to 50% w/w amorphous conversion in compressive stress range of 60-320 MPa. Overall, these results suggest that dispersive Raman spectroscopy offers fast, sensitive, and high-throughput (<5 min/tablet) method for quantitating amorphous conversion.

A study of phenylalanine side-chain dynamics in surface-adsorbed peptides using solid-state deuterium NMR and rotamer library statistics.

Extracellular matrix proteins adsorbed onto mineral surfaces exist in a unique environment where the structure and dynamics of the protein can be altered profoundly. To further elucidate how the mineral surface impacts molecular properties, we perform a comparative study of the dynamics of nonpolar side chains within the mineral-recognition domain of the biomineralization protein salivary statherin adsorbed onto its native hydroxyapatite (HAP) mineral surface versus the dynamics displayed by the native protein in the hydrated solid state. Specifically, the dynamics of phenylalanine side chains (viz., F7 and F14) located in the surface-adsorbed 15-amino acid HAP-recognition fragment (SN15: DpSpSEEKFLRRIGRFG) are studied using deuterium magic angle spinning ((2)H MAS) line shape and spin-lattice relaxation measurements. (2)H NMR MAS spectra and T1 relaxation times obtained from the deuterated phenylalanine side chains in free and HAP-adsorbed SN15 are fitted to models where the side chains are assumed to exchange between rotameric states and where the exchange rates and a priori rotameric state populations are varied iteratively. In condensed proteins, phenylalanine side-chain dynamics are dominated by 180° flips of the phenyl ring, i.e., the "π flip". However, for both F7 and F14, the number of exchanging side-chain rotameric states increases in the HAP-bound complex relative to the unbound solid sample, indicating that increased dynamic freedom accompanies introduction of the protein into the biofilm state. The observed rotameric exchange dynamics in the HAP-bound complex are on the order of 5-6 × 10(6) s(-1), as determined from the deuterium MAS line shapes. The dynamics in the HAP-bound complex are also shown to have some solution-like behavioral characteristics, with some interesting deviations from rotameric library statistics.

Direct observation of phenylalanine orientations in statherin bound to hydroxyapatite surfaces.

Extracellular biomineralization proteins such as salivary statherin control the growth of hydroxyapatite (HAP), the principal component of teeth and bones. Despite the important role that statherin plays in the regulation of hard tissue formation in humans, the surface recognition mechanisms involved are poorly understood. The protein-surface interaction likely involves very specific contacts between the surface atoms and the key protein side chains. This study demonstrates for the first time the power of combining near-edge X-ray absorption fine structure (NEXAFS) spectroscopy with element labeling to quantify the orientation of individual side chains. In this work, the 15 amino acid N-terminal binding domain of statherin has been adsorbed onto HAP surfaces, and the orientations of phenylalanine rings F7 and F14 have been determined using NEXAFS analysis and fluorine labels at individual phenylalanine sites. The NEXAFS-derived phenylalanine tilt angles have been verified with sum frequency generation spectroscopy.

Toward a structure determination method for biomineral-associated protein using combined solid- state NMR and computational structure prediction.

Protein-biomineral interactions are paramount to materials production in biology, including the mineral phase of hard tissue. Unfortunately, the structure of biomineral-associated proteins cannot be determined by X-ray crystallography or solution nuclear magnetic resonance (NMR). Here we report a method for determining the structure of biomineral-associated proteins. The method combines solid-state NMR (ssNMR) and ssNMR-biased computational structure prediction. In addition, the algorithm is able to identify lattice geometries most compatible with ssNMR constraints, representing a quantitative, novel method for investigating crystal-face binding specificity. We use this method to determine most of the structure of human salivary statherin interacting with the mineral phase of tooth enamel. Computation and experiment converge on an ensemble of related structures and identify preferential binding at three crystal surfaces. The work represents a significant advance toward determining structure of biomineral-adsorbed protein using experimentally biased structure prediction. This method is generally applicable to proteins that can be chemically synthesized.

Trading sensitivity for information: Carr-Purcell-Meiboom-Gill acquisition in solid-state NMR.

The Carr-Purcell-Meiboom-Gill (CPMG) experiment has gained popularity in solid-state NMR as a method for enhancing sensitivity for anisotropically broadened spectra of both spin 1/2 and half integer quadrupolar nuclei. Most commonly, the train of CPMG echoes is Fourier transformed directly, which causes the NMR powder pattern to break up into a series of sidebands, sometimes called "spikelets." Larger sensitivity enhancements are observed as the delay between the pi pulses is shortened. As the duration between the pi pulses is shortened, however, the echoes become truncated and information about the nuclear spin interactions is lost. We explored the relationship between enhanced sensitivity and loss of information as a function of the product Omega 2tau, where Omega is the span of the anisotropic lineshape and 2tau is the pi pulse spacing. For a lineshape dominated by the nuclear shielding anisotropy, we found that the minimum uncertainty in the tensor values is obtained using Omega 2tau values in the range Omega 2tau approximately 12(-1)(+6) and Omega 2tau approximately 9(-3)(+3) for eta(s)=0 and eta(s)=1, respectively. For an anisotropic second-order quadrupolar central transition lineshape under magic-angle spinning (MAS), the optimum range of Omega 2tau approximately 9(-2)(+3) was found. Additionally, we show how the Two-dimensional One Pulse (TOP) like processing approach can be used to eliminate the cumbersome sideband pattern lineshape and recover a more familiar lineshape that is easily analyzed with conventional lineshape simulation algorithms.

The Role of Basic Amino Acids in the Molecular Recognition of Hydroxyapatite by Statherin using Solid State NMR.

Organisms use proteins such as statherin to control the growth of hydroxyapatite (HAP), which is the principal component of teeth and bone. Though much emphasis has been placed on the acidic character of these proteins, the role of their basic amino acids is not well understood. In this work, solid state nuclear magnetic resonance was used to probe the interaction of the basic arginine side chains with the HAP surface. Statherin samples were individually labeled at each arginine site, and the distance to the surface was measured using the Rotational Echo DOuble Resonance (REDOR) technique. The results indicate a strong coupling between the R9 and R10 residues and the phosphorus atoms on the surface, with internuclear distances of 4.62 ± 0.29 Å and 4.53 ± 0.16 Å, respectively. Conversely, results also indicate weak coupling between R13 and the surface, suggesting this residue is more removed from the surface than R9 and R10. Combining these results with previous data, a new model for the molecular recognition of HAP by statherin is constructed.

A (13)C{(31)P} REDOR NMR investigation of the role of glutamic acid residues in statherin- hydroxyapatite recognition.

The side chain carboxyl groups of acidic proteins found in the extra-cellular matrix (ECM) of mineralized tissues play a key role in promoting or inhibiting the growth of minerals such as hydroxyapatite (HAP), the principal mineral component of bone and teeth. Among the acidic proteins found in the saliva is statherin, a 43-residue tyrosine-rich peptide that is a potent lubricant in the salivary pellicle and an inhibitor of both HAP crystal nucleation and growth. Three acidic amino acids-D1, E4, and E5-are located in the N-terminal 15 amino acid segment, with a fourth amino acid, E26, located outside the N-terminus. We have utilized (13)C{(31)P} REDOR NMR to analyze the role played by acidic amino acids in the binding mechanism of statherin to the HAP surface by measuring the distance between the delta-carboxyl (13)C spins of the three glutamic acid side chains of statherin (residues E4, E5, E26) and (31)P spins of the phosphate groups at the HAP surface. (13)C{(31)P} REDOR studies of glutamic-5-(13)C acid incorporated at positions E4 and E26 indicate a (13)C-(31)P distance of more than 6.5 A between the side chain carboxyl (13)C spin of E4 and the closest (31)P in the HAP surface. In contrast, the carboxyl (13)C spin at E5 has a much shorter (13)C-(31)P internuclear distance of 4.25 +/- 0.09 A, indicating that the carboxyl group of this side chain interacts directly with the surface. (13)C T(1rho) and slow-spinning MAS studies indicate that the motions of the side chains of E4 and E5 are more restricted than that of E26. Together, these results provide further insight into the molecular interactions of statherin with HAP surfaces.

Separating chemical shift and quadrupolar anisotropies via multiple-quantum NMR spectroscopy.

Chemical shift anisotropy (CSA) has been an invaluable probe of structure and dynamics for a variety of systems in NMR spectroscopy. Unfortunately, the presence of strong quadrupolar couplings has severely limited the ability to measure CSA in nuclei with spins I > 1/2. Here we show that these two interactions can be refocused at different times in a 2D multiple-quantum NMR experiment on polycrystalline samples. Combining this experiment with appropriate affine transformations allows these interactions to be cleanly separated into orthogonal dimensions. The 1D projection onto each axis can be fit to extract the respective principal tensor components. These components can then be used to fit the 2D spectrum for the relative orientation between the CSA and quadrupolar-coupling tensors. The necessary affine transformation parameters are given for all possible I values. Illustrative examples of spectra and analyses are given for 63Cu in K3[Cu(CN)4], 59Co in K3[Co(CN)6], and 87Rb in RbCrO4.

Spectral editing in solid-state MAS NMR of quadrupolar nuclei using selective satellite inversion.

A sensitivity enhancement method based on selective adiabatic inversion of a satellite transition has been employed in a (pi/2)CT-(pi)ST1-(pi/2)CT spectral editing sequence to both enhance and resolve multisite NMR spectra of quadrupolar nuclei. In addition to a total enhancement of 2.5 times for spin 3/2 nuclei, enhancements up to 2.0 times is reported for the edited sites in a mixture of rubidium salts.

Solid-state NMR characterization of 69Ga and 71Ga in crystalline solids.

Gallium model systems containing four- and six-coordinate gallium sites have been investigated using solid-state NMR. Measurement of the isotropic chemical shift and electric field gradient (EFG) have been performed at 9.4 T on alpha-Ga2O3, beta-Ga2O3, LiGaO2, NaGaO2, KGaO2, Ga2(SO4)3, and LaGaO3 using a variety of techniques on both NMR active nuclei (69Ga and 71Ga) including static, high speed magic-angle spinning (MAS), satellite transition (ST) spectroscopy, and rotor-assisted population transfer (RAPT). The chemical shift is found to correlate well with the coordination number, with four-coordinate gallium having values of approximately 50 ppm and six-coordinate gallium having values near 225 ppm (referenced to 1 M gallium nitrate solution). The magnitude of the EFG is found to be correlated to the distortion of the gallium polyhedra, with the strained systems having EFGs of 3 x 10(21) Vm(-2) or more, while the less strained systems have values of 1.5 x 10(21) Vm(-2) or less. A plot of chemical shift versus EFG suggests that solid-state NMR of gallium oxyanions can be more discriminating than liquid state NMR chemical shifts alone.