Excipient selection can significantly affect solid-state phase transformation in formulation during wet granulation

Phase transformations in formulations can lead to instability in physicochemical, biopharmaceutical, and processing properties of products. The influences of formulation design on the optimal dosage forms should be specified. The aim here was to investigate whether excipients with different water sorption behavior affect hydrate formation of nitrofurantoin in wet masses. Nitrofurantoin anhydrate was used as a hydrate-forming model drug, and 4 excipients with different water-absorbing potential (amorphous low-substituted hydroxypropylcellulose, modified maize starch, partially amorphous silicified microcrystalline cellulose, and crystalline α-lactose monohydrate) were granulated with varying amounts of purified water. Off-line evaluation of wet masses containing nitrofurantoin anhydrate and excipient (1∶1) was performed using an X-ray powder diffractometer (XRPD) and near-infrared spectroscopy, and drying phase was evaluated by variable temperature XRPD. Only amorphous excipient in the formulation retarded hydrate formation of an active pharmaceutical ingredient (API) at high water contents. Hygroscopic partially crystalline excipient hindered hydrate formation of API at low water contents. Crystalline excipient was unable to control hydrate formation of API. The character of excipient affects the stability of formulation. Thus, correct selection of excipients for the formulation can control processing-induced phase transitions and improve the storage stability of the final dosage form. Published: October 6, 2005     

Investigating Gabapentin Polymorphism Using Solid-State NMR Spectroscopy

Solid-state NMR spectroscopy (SSNMR), coupled with powder X-ray diffraction (PXRD), was used to identify the physical forms of gabapentin in samples prepared by recrystallization, spray drying, dehydration, and milling. Four different crystalline forms of gabapentin were observed: form I, a monohydrate, form II, the most stable at ambient conditions, form III, produced by either recrystallization or milling, and an isomorphous desolvate produced from desolvating the monohydrate. As-received gabapentin (form II) was ball-milled for 45 min in both the presence and absence of hydroxypropylcellulose (HPC). The samples were then stored for 2 days at 50°C under 0% relative humidity and analyzed by ¹³C SSNMR and PXRD. High-performance liquid chromatography was run on the samples to determine the amount of degradation product formed before and after storage. The ¹H T ₁ values measured for the sample varied from 130 s for the as-received unstressed material without HPC to 11 s for the material that had been ball-milled in the presence of HPC. Samples with longer ¹H T ₁ values were substantially more stable than samples that had shorter T ₁ values. Samples milled with HPC had detectable form III crystals as well. These results suggest that SSNMR can be used to predict gabapentin stability in formulated products.     

Characterisation of Crystalline-Amorphous Blends of Sucrose with Terahertz-Pulsed Spectroscopy: the Development of a Prediction Technique for Estimating the Degree of Crystallinity with Partial Least Squares Regression

The control of the amorphous and crystalline states of drugs and excipients is important in many instances of product formulation, manufacture, and packaging, such as the formulation of certain (freeze-dried) fast melt tablets. This study examines the use of terahertz-pulsed spectroscopy (TPS) coupled with two different data analytical methods as an off-line tool (in the first instance) for assessing the degree of crystallinity in a binary mixture of amorphous and polycrystalline sucrose. The terahertz spectrum of sucrose was recorded in the wave number range between 3 and 100 cm⁻¹ for both the pure crystalline form and for a mixture of the crystalline and amorphous (freeze-dried) form. The THz spectra of crystalline sucrose showed distinct absorption bands at ∼48, ∼55, and ∼60 cm⁻¹ while all these features were absent in the amorphous sucrose. Calibration models were constructed based on (1) peak area analysis and (2) partial least square regression analysis, with the latter giving the best LOD and LOQ of 0.76% and 2.3%, respectively. The potential for using THz spectroscopy, as a quantitative in-line tool for percent crystallinity in a range of complex systems such as conventional tablets and freeze-dried formulations, is suggested in this study.Key words: crystallinity, partial least squares, sugars, terahertz-pulsed spectroscopy     

In Situ Structural Studies of Anabaena Sensory Rhodopsin in the E.?coli Membrane

Magic-angle spinning nuclear magnetic resonance is well suited for the study of membrane proteins in the nativelike lipid environment. However, the natural cellular membrane is invariably more complex than the proteoliposomes most often used for solid-state NMR (SSNMR) studies, and differences may affect the structure and dynamics of the proteins under examination. In this work we use SSNMR and other biochemical and biophysical methods to probe the structure of a seven-transmembrane helical photoreceptor, Anabaena sensory rhodopsin (ASR), prepared in the Escherichia coli inner membrane, and compare it to that in a bilayer formed by DMPC/DMPA lipids. We find that ASR is organized into trimers in both environments but forms two-dimensional crystal lattices of different symmetries. It favors hexagonal packing in liposomes, but may form a square lattice in the E. coli membrane. To examine possible changes in structure site-specifically, we perform two- and three-dimensional SSNMR experiments and analyze the differences in chemical shifts and peak intensities. Overall, this analysis reveals that the structure of ASR is largely conserved in the inner membrane of E. coli, with many of the important structural features of rhodopsins previously observed in ASR in proteoliposomes being preserved. Small, site-specific perturbations in protein structure that occur as a result of the membrane changes indicate that the protein can subtly adapt to its environment without large structural rearrangement.     

Structural characterization of wheat starch granules differing in amylose content and functional characteristics

Small-angle X-ray scattering (SAXS) together with several complementary techniques, such as differential scanning calorimetry and X-ray diffraction, have been employed to investigate the structural features that give diverse functional properties to wheat starches (Triticum aestivum L.) within a narrow range of enriched amylose content (36–43%). For these starches, which come from a heterogeneous genetic background, SAXS analysis of duplicate samples enabled structural information to be obtained about their lamellar architecture where differences in lamellar spacing among samples were only several tenths of nanometer. The SAXS analysis of these wheat starches with increased amylose content has shown that amylose accumulates in both crystalline and amorphous parts of the lamella. Using waxy starch as a distinctive comparison with the other samples confirmed a general trend of increasing amylose content being linked with the accumulation of defects within crystalline lamellae. We conclude that amylose content directly influences the architecture of semi-crystalline lamellae, whereas thermodynamic and functional properties are brought about by the interplay of amylose content and amylopectin architecture.     

Solid state NMR studies on the structural and conformational properties of natural maize starches

Natural maize starches having a range of amylose contents have been characterised by CP/MAS NMR spectroscopy. Chemical shifts, relative resonance intensities, line-widths and spectral shapes were compared at different moisture contents. At 10% moisture content, these parameters showed few significant differences across a range of apparent amylose levels from 0 to 84%. After hydration of the granules to ≈30% moisture, it was found that the amylose content significantly affected the relative signal intensities and line-widths especially of C-1 and C-4 resonances. Narrower line-widths after hydration were attributed to (i) an increased degree of crystallinity, and (ii) disappearance of the signals of amorphous material which, on becoming more mobile, became invisible to the CP/MAS experiment. The enhanced resolution at higher moisture levels revealed signals which were assigned to the amylose–lipid complex, i.e. V-type amylose. The amount of V-amylose detected by NMR increased with both amylose content and lipid content of the granule. Prolonged treatment of the granules with iodine vapour significantly increased the amount of V-type amylose in the high amylose samples, but caused a decrease in their degree of crystallinity. Waxy-maize starch was barely affected by iodination. The results provide evidence that amylose tends to disrupt the structural order within amylopectin crystallities. This effect is enhanced by the formation of the amylose–iodine complex, indicating that V-amylose could be a major crystallite-disrupting agent in native starch granules.     

Structural parameters of amylopectin clusters and semi-crystalline growth rings in wheat starches with different amylose content

Small-angle X-ray scattering (SAXS) and scanning electron microscopy (SEM) were used to investigate the internal structure of wheat starch granules with different amylose content. Different approaches were used for treatment (interpretation) of SAXS data to assess the values of structural parameters of amylopectin clusters and the size of crystalline and amorphous lamella in different wheat starches. The average values of the semi-crystalline growth rings thickness in starches have been determined and the relationship between structural characteristics and thermodynamic melting parameters is discussed.     

New insights on the mechanism of acid degradation of pea starch

The degradation of pea starch granules by acid hydrolysis has been investigated using a range of chemical and structural methods, namely through measuring changes in amylose content by both the iodine binding and concanavalin A precipitation methods, along with small angle X-ray scattering (SAXS), wide angle X-ray diffraction (XRD) and field emission scanning electron microscopy (FE-SEM). The relative crystallinity, intensity of the lamellar peak and the low-q scattering increased during the initial stages of acid hydrolysis, indicating early degradation of the amorphous regions (growth rings and lamellae). In the first 2 days of hydrolysis, there was a rapid decline in amylose content, a concomitant loss of precipitability of amylopectin by concanavalin A, and damage to the surface and internal granular structures was evident. These observations are consistent with both amylose and amylopectin being located on the surface of the granules and attacked simultaneously in the early stages of acid hydrolysis. The results are also consistent with amylose being more concentrated at the core of the granules. More extensive hydrolysis resulted in the simultaneous disruption of amorphous and crystalline regions, which was indicated by a decrease in lamellar peak intensity, decrease in interhelix peak intensity and no further increase in crystallinity. These results provide new insights into the organization of starch granules.     

Determination of the polymorphic composition of smooth pea starch

A method for estimating the proportions of ‘A’ and ‘B’ polymorphs comprising a sample of ‘C’ type starch is proposed which uses established experimental techniques with commercially available spreadsheet and X-ray analysis software. Waxy maize, potato and smooth pea starches were used to provide X-ray diffraction patterns characteristic of the ‘A’, ‘B’ and ‘C’ starch polymorphs. Samples of amorphous starches were also prepared. The method initially involved subtraction of the amorphous phase and instrumental background from the X-ray diffraction patterns of each starch sample using the spreadsheet program, Lotus 1-2-3. The remainder of the pattern, representing the crystalline portion of the starch sample, was then analysed by profile fitting to elucidate the positions and areas of individual diffraction peaks. The ratio of the total peak area to the areas under peaks characteristic of ‘A’ and ‘B’ type starches, respectively, were used to calculate the relative proportions of these polymorphs in smooth pea starch. These proportions were found to be 56±3% ‘A’ polymorph to 44±3% ‘B’ polymorph. A ‘C’ type pattern was constructed by using Lotus 1-2-3 to combine diffraction patterns from the crystalline portions of ‘A’ and ‘B’ type starches in the proportions given above. Polymorph patterns were obtained by manipulation of the diffraction patterns from the crystalline portions of starches using Lotus 1-2-3. An ‘A’ type pattern was obtained by subtraction of a ‘B’ type pattern from that of a ‘C’ type. Similarly, a ‘B’ type pattern was obtained by subtraction of an ‘A’ type pattern from that of a ‘C’ type.     

Aqueous dissolution of crystalline and amorphous amylose-alcohol complexes

Complexes of amylose, the linear starch polysaccharide, with linear alcohols having chain lengths varying from 4 to 8 carbon atoms, were prepared. Either crystalline or amorphous complexes could be formed depending on preparation conditions. Crystalline complexes gave sharp X-ray diffraction patterns, characteristic of the V_H form of amylose, whereas no observable pattern was obtained from the amorphous form. Thermal dissociation of the complexes occurred at increasing temperatures with increasing alcohol chain length. Crystalline complexes dissociated at temperatures approximately 23°C higher than their amorphous counterparts and the enthalpy of dissociation was also greater for the crystalline samples. Enthalpy values were independent of alcohol chain length. Differences in thermal behaviour of the two types of complex may be described in terms of the polymer crystal lattice energy and may explain the variability of reported results for complex dissociation in the literature.     

Starch synthesis in Arabidopsis. Granule synthesis,composition, and structure

The aim of this work was to characterize starch synthesis, composition, and granule structure in Arabidopsis leaves. First, the potential role of starch-degrading enzymes during starch accumulation was investigated. To discover whether simultaneous synthesis and degradation of starch occurred during net accumulation, starch was labeled by supplying (14)CO(2) to intact, photosynthesizing plants. Release of this label from starch was monitored during a chase period in air, using different light intensities to vary the net rate of starch synthesis. No release of label was detected unless there was net degradation of starch during the chase. Similar experiments were performed on a mutant line (dbe1) that accumulates the soluble polysaccharide, phytoglycogen. Label was not released from phytoglycogen during the chase indicating that, even when in a soluble form, glucan is not appreciably degraded during accumulation. Second, the effect on starch composition of growth conditions and mutations causing starch accumulation was studied. An increase in starch content correlated with an increased amylose content of the starch and with an increase in the ratio of granule-bound starch synthase to soluble starch synthase activity. Third, the structural organization and morphology of Arabidopsis starch granules was studied. The starch granules were birefringent, indicating a radial organization of the polymers, and x-ray scatter analyses revealed that granules contained alternating crystalline and amorphous lamellae with a periodicity of 9 nm. Granules from the wild type and the high-starch mutant sex1 were flattened and discoid, whereas those of the high-starch mutant sex4 were larger and more rounded. These larger granules contained "growth rings" with a periodicity of 200 to 300 nm. We conclude that leaf starch is synthesized without appreciable turnover and comprises similar polymers and contains similar levels of molecular organization to storage starches, making Arabidopsis an excellent model system for studying granule biosynthesis.     

A Method to Evaluate the Effect of Contact with Excipients on the Surface Crystallization of Amorphous Drugs

Amorphous drugs are used to improve the solubility, dissolution, and bioavailability of drugs. However, these metastable forms of drugs can transform into more stable, less soluble, crystalline counterparts. This study reports a method for evaluating the effect of commonly used excipients on the surface crystallization of amorphous drugs and its application to two model amorphous compounds, nifedipine and indomethacin. In this method, amorphous samples of the drugs were covered by excipients and stored in controlled environments. An inverted light microscope was used to measure in real time the rates of surface crystal nucleation and growth. For nifedipine, vacuum-dried microcrystalline cellulose and lactose monohydrate increased the nucleation rate of the β polymorph from two to five times when samples were stored in a desiccator, while d-mannitol and magnesium stearate increased the nucleation rate 50 times. At 50% relative humidity, the nucleation rates were further increased, suggesting that moisture played an important role in the crystallization caused by the excipients. The effect of excipients on the crystal growth rate was not significant, suggesting that contact with excipients influences the physical stability of amorphous nifedipine mainly through the effect on crystal nucleation. This effect seems to be drug specific because for two polymorphs of indomethacin, no significant change in the nucleation rate was observed under the excipients.KEY WORDS: amorphous, drugs, growth rate, nucleation rate, tablet excipients     

Effect of enzymatic hydrolysis on native starch granule structure

Enzymatic digestion of six starches of different botanical origin was studied in real time by in situ time-resolved small-angle neutron scattering (SANS) and complemented by the analysis of native and digested material by X-ray diffraction, differential scanning calorimetry, small-angle X-ray scattering, and scanning electron microscopy with the aim of following changes in starch granule nanostructure during enzymatic digestion. This range of techniques enables coverage over five orders of length-scale, as is necessary for this hierarchically structured material. Starches studied varied in their digestibility and displayed structural differences in the course of enzymatic digestion. The use of time-resolved SANS showed that solvent-drying of digested residues does not induce any structural artifacts on the length scale followed by small-angle scattering. In the course of digestion, the lamellar peak intensity gradually decreased and low-q scattering increased. These trends were more substantial for A-type than for B-type starches. These observations were explained by preferential digestion of the amorphous growth rings. Hydrolysis of the semicrystalline growth rings was explained on the basis of a liquid-crystalline model for starch considering differences between A-type and B-type starches in the length and rigidity of amylopectin spacers and branches. As evidenced by differing morphologies of enzymatic attack among varieties, the existence of granular pores and channels and physical penetrability of the amorphous growth ring affect the accessibility of the enzyme to the substrate. The combined effects of the granule microstructure and the nanostructure of the growth rings influence the opportunity of the enzyme to access its substrate; as a consequence, these structures determine the enzymatic digestibility of granular starches more than the absolute physical densities of the amorphous growth rings and amorphous and crystalline regions of the semicrystalline growth rings.     

Structural and thermodynamic properties of rice starches with different genetic background Part 2. Defectiveness of different supramolecular structures in starch granules

High-sensitivity differential scanning microcalorimetry (HSDSC), small-angle X-ray scattering (SAXS), light (LM) and scanning electronic (SEM) microscopy techniques were used to study the defectiveness of different supramolecular structures in starches extracted from 11 Thai cultivars of rice differing in level of amylose and amylopectin defects in starch crystalline lamellae. Despite differences in chain-length distribution of amylopectin macromolecules and amylose level in starches, the invariance in the sizes of crystalline lamellae, amylopectin clusters and granules was established. The combined analysis of DSC, SAXS, LM and SEM data for native starches, as well as the comparison of the thermodynamic data for native and annealed starches, allowed to determine the structure of defects and the localization of amylose chains in crystalline and amorphous lamellae, defectiveness of lamellae, clusters and granules. It was shown that amylose “tie chains”, amylose–lipid complexes located in crystalline lamellae, defective ends of double helical chains dangling from crystallites inside amorphous lamellae (“dangling” chains), as well as amylopectin chains with DP 6–12 and 25–36 could be considered as defects. Their accumulation can lead to a formation of remnant granules. The changes observed in the structure of amylopectin chains and amylose content in starches are reflected in the interconnected alterations of structural organization on the lamellar, cluster and granule levels.     

A statistical selection strategy for normalization procedures in LC-MS proteomics experiments through dataset-dependent ranking of normalization scaling factors

Quantification of LC-MS peak intensities assigned during peptide identification in a typical comparative proteomics experiment will deviate from run-to-run of the instrument due to both technical and biological variation. Thus, normalization of peak intensities across an LC-MS proteomics dataset is a fundamental step in pre-processing. However, the downstream analysis of LC-MS proteomics data can be dramatically affected by the normalization method selected. Current normalization procedures for LC-MS proteomics data are presented in the context of normalization values derived from subsets of the full collection of identified peptides. The distribution of these normalization values is unknown a priori. If they are not independent from the biological factors associated with the experiment the normalization process can introduce bias into the data, possibly affecting downstream statistical biomarker discovery. We present a novel approach to evaluate normalization strategies, which includes the peptide selection component associated with the derivation of normalization values. Our approach evaluates the effect of normalization on the between-group variance structure in order to identify the most appropriate normalization methods that improve the structure of the data without introducing bias into the normalized peak intensities.     

Automated protein resonance assignments of magic angle spinning solid-state NMR spectra of β1 immunoglobulin binding domain of protein G (GB1)

Magic-angle spinning solid-state NMR (MAS SSNMR) represents a fast developing experimental technique with great potential to provide structural and dynamics information for proteins not amenable to other methods. However, few automated analysis tools are currently available for MAS SSNMR. We present a methodology for automating protein resonance assignments of MAS SSNMR spectral data and its application to experimental peak lists of the β1 immunoglobulin binding domain of protein G (GB1) derived from a uniformly ¹³C- and ¹⁵N-labeled sample. This application to the 56 amino acid GB1 produced an overall 84.1% assignment of the N, CO, CA, and CB resonances with no errors using peak lists from NCACX 3D, CANcoCA 3D, and CANCOCX 4D experiments. This proof of concept demonstrates the tractability of this problem.     

Hydrothermal treatment and iodine binding provide insights into the organization of glucan chains within the semi‐crystalline lamellae of corn starch granules

The importance of glucan chains that pass through both the amorphous and crystalline lamellae (tie chains) in the organization of corn starch granules was studied using heat‐moisture treatment (HMT), annealing (ANN), and iodine binding. Molecular structural analysis showed that hylon starches (HV, HVII, and HVIII) contained higher proportion of intermediate glucan chains (HVIII > HVII > HV) than normal corn (CN) starch. Wide angle X‐ray scattering revealed that on HMT, the extent of polymorphic transition in hylon starches decreased with increasing proportion of intermediate and long chains. Iodine treated hylon starches exhibited increased order in the V‐type polymorphism as evidenced by the intense peak at 20° 2θ and the strong reflection intensity at 7.5° 2θ and the extent of the change depended on the type of hylon starch. DSC results showed that the gelatinization enthalpy of CN and waxy corn starch (CW) remained unchanged after ANN. However, hylon starches showed a significant increase in enthalpy with more distinct endotherms after ANN. It can be concluded that tie chains influence the organization of crystalline lamellae in amylose extender mutant starches. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 871–885, 2014.     

Structural characteristics of low-glycemic response rice starch produced by citric acid treatment

The structural characteristics of citric acid-treated rice starch, which was subjected to autoclaving and stored at a high temperature under acidic conditions, were investigated by high performance anion-exchange chromatography, multi-angle laser-light scattering, X-ray diffraction, differential scanning calorimetry (DSC), nuclear magnetic resonance, and scanning electron microscopy. The citric acid-treated rice starch contained chains of different lengths (DP 7−70) and had an A + V type polymorphism. The DSC thermogram of acid-treated rice starch showed a broad endothermic peak, which indicated that the structure of the acid-treated rice starch contained a number of double helices with various melting temperatures. Microscopic observation showed that the internal structure of the acid-treated starch displayed more or less spherical lumps that could be composed of short chains and amylose–lipid complex. The digestive properties of acid-treated rice starch were altered by heat processing such as cooking and autoclaving because crystalline regions were converted to amorphous regions or lamellae.     

Solid State Characterization of Commercial Crystalline and Amorphous Atorvastatin Calcium Samples

Atorvastatin calcium (ATC), an anti-lipid BCS class II drug, is marketed in crystalline and amorphous solid forms. The objective of this study was to perform solid state characterization of commercial crystalline and amorphous ATC drug samples available in the Indian market. Six samples each of crystalline and amorphous ATC were characterized using X-ray powder diffractometry (XRPD), differential scanning calorimetry (DSC), thermogravimetric analysis, Karl Fisher titrimetry, microscopy (hot stage microscopy, scanning electron microscopy), contact angle, and intrinsic dissolution rate (IDR). All crystalline ATC samples were found to be stable form I, however one sample possessed polymorphic impurity, evidenced in XRPD and DSC analysis. Amongst the amorphous ATC samples, XRPD demonstrated five samples to be amorphous ‘form 27’, while, one matched amorphous ‘form 23’. Thermal behavior of amorphous ATC samples was compared to amorphous ATC generated by melt quenching in DSC. ATC was found to be an excellent glass former with T_g/T_m of 0.95. Residual crystallinity was detected in two of the amorphous samples by complementary use of conventional and modulated DSC techniques. The wettability and IDR of all amorphous samples was found to be higher than the crystalline samples. In conclusion, commercial ATC samples exhibited diverse solid state behavior that can impact the performance and stability of the dosage forms.