A Prolific Solvate Former, Galunisertib, under the Pressure of Crystal Structure Prediction, Produces Ten Diverse Polymorphs.

The solid form screening of galunisertib produced many solvates, prompting an extensive investigation into possible risks to the development of the favored monohydrate form. Inspired by crystal structure prediction, the search for neat polymorphs was expanded to an unusual range of experiments, including melt crystallization under pressure, to work around solvate formation and the thermal instability of the molecule. Ten polymorphs of galunisertib were found; however, the structure predicted to be the most stable has yet to be obtained. We present the crystal structures of all ten unsolvated polymorphs of galunisertib, showing how state-of-the-art characterization methods can be combined with emerging computational modeling techniques to produce a complete structure landscape and assess the risk of late-appearing, more stable polymorphs. The exceptional conformational polymorphism of this prolific solvate former invites further development of methods, computational and experimental, that are applicable to larger, flexible molecules with complex solid form landscapes.

Navigating the Waters of Unconventional Crystalline Hydrates.

Elucidating the crystal structures, transformations, and thermodynamics of the two zwitterionic hydrates (Hy2 and HyA) of 3-(4-dibenzo[b,f][1,4]oxepin-11-yl-piperazin-1-yl)-2,2-dimethylpropanoic acid (DB7) rationalizes the complex interplay of temperature, water activity, and pH on the solid form stability and transformation pathways to three neutral anhydrate polymorphs (Forms I, II°, and III). HyA contains 1.29 to 1.95 molecules of water per DB7 zwitterion (DB7z). Removal of the essential water stabilizing HyA causes it to collapse to an amorphous phase, frequently concomitantly nucleating the stable anhydrate Forms I and II°. Hy2 is a stoichiometric dihydrate and the only known precursor to Form III, a high energy disordered anhydrate, with the level of disorder depending on the drying conditions. X-ray crystallography, solid state NMR, and H/D exchange experiments on highly crystalline phase pure samples obtained by exquisite control over crystallization, filtration, and drying conditions, along with computational modeling, provided a molecular level understanding of this system. The slow rates of many transformations and sensitivity of equilibria to exact conditions, arising from its varying static and dynamic disorder and water mobility in different phases, meant that characterizing DB7 hydration in terms of simplified hydrate classifications was inappropriate for developing this pharmaceutical.

Lack of effect of the apolipoprotein E ε4 genotype on cognition during healthy aging.

BACKGROUND: The apolipoprotein E (APOE) ε4 genotype is associated with an increased risk of Alzheimer's disease. In community surveys, older adults with this genotype have been found to have lower scores on neuropsychological tests than those who do not. It is possible that this is the consequence of subclinical changes in cognition in those persons who later develop dementia. The aim of this research was to determine whether the effect of APOE genotype on cognition would remain if those who subsequently became demented were retrospectively removed from the analysis of the baseline test data from a sample of healthy adults. METHOD: A sample of 241 nondemented persons over the age of 65 for whom APOE genotyping was available were administered a range of neuropsychological tests at baseline and were followed up 10 years later. RESULTS: Significant differences between the ε4-present and ε4-absent groups were found for the delayed recall trial of the Rey Auditory Verbal Learning Test and the Trail Making Test. When those participants known to have developed dementia during the follow-up period were excluded from the analysis of the baseline data these differences disappeared. A total of 113 nondemented survivors from the original sample were retested, and no difference was found in the rate of decline on any measure between the ε4-present and ε4-absent groups. CONCLUSIONS: It is likely that the reported effect of the ε4 APOE genotype on cognition is the consequence of the ε4-present group containing persons whose cognition is subtly affected by the early stages of a dementing process. It is also unlikely that the presence of the ε4 allele by itself leads to a significantly accelerated rate of cognitive decline in the nondemented elderly.

Differences in erythrocyte folate concentrations in older adults reached steady-state within one year in a two-year, controlled, 1 mg/d folate supplementation trial.

Daily supplementation with folate increases erythrocyte folate concentrations; however, the time to reach steady-state concentrations has not been empirically demonstrated. Previous predictions of time to steady state or time to 90% steady-state concentration, based on modeling changes in erythrocyte folate during short-term trials, range widely from 40 to 86 wk. We sought to determine the time to steady-state erythrocyte folate concentrations following the initiation of daily folate supplementation using data collected from a 2-y, double-blind, placebo-controlled, randomized trial involving 276 participants aged 65 y or older. The daily supplement contained 1 mg of folate. Erythrocyte folate concentrations were measured, using a microbiological assay, at baseline and at 6, 12, 18, and 24 mo. The mean plasma and erythrocyte folate concentrations in the folate-supplemented group were higher than in the placebo group at 6, 12, 18, and 24 mo (P < 0.001). Adjusted for baseline differences, the difference in erythrocyte folate concentrations between the folate and placebo group at 6 mo was 1.78 µmol/L (95% CI: 1.62-1.95 µmol/L). The difference increased significantly to 2.02 µmol/L (95% CI: 1.85-2.18 µmol/L) at 12 mo. This difference (between the folate and placebo groups) did not significantly change after a further year of folate supplementation; at 18 mo, it was 2.09 µmol/L (95% CI: 1.92-2.27 µmol/L) and at 24 mo it was 1.98 µmol/L (95% CI: 1.18-2.15 µmol/L). Twelve months of daily folate supplementation with 1 mg is sufficient time to cause erythrocyte folate concentrations to reach a new steady state.

Homocysteine-lowering vitamins do not lower plasma S-adenosylhomocysteine in older people with elevated homocysteine concentrations.

Elevated plasma total homocysteine (tHcy) is a risk factor for vascular disease but lowering tHcy with B-vitamins, including folate, has generally not reduced vascular events in secondary prevention trials. Elevated plasma S-adenosylhomocysteine (AdoHcy) concentration may be a more sensitive indicator of vascular disease than plasma tHcy. However, unlike tHcy, plasma AdoHcy did not correlate with folate concentration in one study indicating that folate supplementation may not lower AdoHcy. Our aim was to determine whether providing B-vitamin supplements to healthy older people with elevated tHcy (>13 micromol/l) affects plasma AdoHcy and S-adenosylmethionine (AdoMet) concentrations. Healthy older participants (n 276; > or = 65 years) were randomised to receive a daily supplement containing folate (1 mg), vitamin B12 (500 microg) and vitamin B6 (10 mg), or placebo, for 2 years. Of these participants, we selected the first fifty participants in each treatment group and measured plasma AdoHcy and AdoMet. Plasma tHcy was 4.4 (95 % CI 3.2, 5.6; P < 0.001) micromol/l lower at 2 years in the vitamins group compared with the placebo group. At 2 years, there were no significant differences in plasma AdoMet (+4 % (95 % CI - 2, 11); P = 0.19), AdoHcy ( - 1 % (95 % CI - 10, 8); P = 0.61) or the AdoMet:AdoHcy ratio (0.22 (95 % CI - 0.04, 0.49); P = 0.10) between the two groups. In conclusion, B-vitamin supplementation of older people lowered plasma tHcy but had no effect on plasma AdoMet or AdoHcy concentration. If elevated plasma AdoHcy is detrimental, this may explain why B-vitamins have generally failed to reduce vascular events in clinical trials.

Lowering plasma homocysteine concentrations of older men and women with folate, vitamin B-12, and vitamin B-6 does not affect the proportion of (n-3) long chain polyunsaturated fatty acids in plasma phosphatidylcholine.

There is evidence to suggest that folate, homocysteine, or both affect the (n-3) long chain PUFA composition of tissues; however, this evidence is derived largely from experiments with animals and small observational studies in humans. Results from randomized controlled trials are needed. The objective of this study was to determine whether homocysteine lowering with a B vitamin supplement affects the proportion of (n-3) long-chain PUFA in plasma phosphatidylcholine. We conducted a double-blind, placebo-controlled, randomized clinical trial involving 253 participants, 65 y or older, with plasma homocysteine concentrations of at least 13 micromol/L. Participants in the vitamin group (n = 127) took a daily supplement containing 1000 microg folate, 500 microg vitamin B-12, and 10 mg vitamin B-6 for 2 y. The fatty acid composition of plasma phosphatidylcholine was measured at baseline and at 2 y. Plasma homocysteine concentrations during the course of the study were 4.4 micromol/L lower in the vitamin group than in the placebo group. The proportions of eicosapentaenoic, docosapentaenoic, and docosahexaenoic acids in plasma phosphatidylcholine did not differ between the vitamin and placebo groups at 2 y; the mean differences after adjusting for baseline values and sex were -0.03 (99% CI: -0.22, 0.16), 0.03 (99% CI: -0.03, 0.09), and -0.02 (99% CI: -0.27, 0.24) mol%, respectively. Lowering plasma homocysteine concentrations of older men and women with folate, vitamin B-12, and vitamin B-6 had no effect on the proportion of (n-3) long-chain PUFA in plasma phosphatidylcholine.

Lowering homocysteine with B vitamins has no effect on blood pressure in older adults.

An elevated circulating homocysteine concentration is associated with the risk of cardiovascular disease. The mechanism by which an elevated homocysteine increases cardiovascular risk is unclear but may be mediated in part by elevating blood pressure. It is well established that supplements containing folate, vitamins B-12, and B-6 lower homocysteine concentrations. However, the effect of homocysteine-lowering vitamins on blood pressure has not been well studied. We sought to determine whether lowering homocysteine with B vitamins lowers blood pressure in healthy older people with elevated homocysteine concentrations. Two hundred seventy-six healthy older participants (> or = 65 y) with a homocysteine > or = 13 micromol/L were randomized to receive a daily supplement containing folate (1 mg), vitamin B-12 (500 microg), and vitamin B-6 (10 mg), or a placebo, for 2 y. Plasma homocysteine was lower in the Vitamins group than the Placebo group at both 1 [-4.3 micromol/L (95% CI; -4.9, -3.7)] and 2 y [-4.4 micromol/L (95% CI: -5.3, -3.6)]. Systolic and diastolic blood pressures as well as pulse pressure in the Vitamins group did not differ from the Placebo group over the duration of the trial. The mean differences in blood pressures, adjusted for baseline values, did not exceed 1 mm Hg. Supplemental B-vitamins lowered plasma homocysteine but had no effect on blood pressure in older people with elevated baseline homocysteine concentrations.

Lowering homocysteine with B vitamins has no effect on biomarkers of bone turnover in older persons: a 2-y randomized controlled trial.

BACKGROUND: In recent prospective studies, higher homocysteine concentrations were shown to be a risk factor for osteoporotic fractures in older persons. Supplements containing folate and vitamins B-12 and B-6 lower homocysteine concentrations. OBJECTIVE: The objective of the study was to determine in healthy older persons whether lowering homocysteine with B vitamins affects plasma biomarkers of bone turnover. DESIGN: Healthy older persons (n = 276; aged >or=65 y) were randomly assigned to receive either a daily supplement containing folate (1 mg), vitamin B-12 (500 microg), and vitamin B-6 (10 mg) or a placebo for 2 y. Of these participants, we selected 135 with baseline homocysteine concentrations >15.0 micromol/L, and we measured serum bone-specific alkaline phosphatase, a marker of bone formation, and bone-derived collagen fragments, a marker of bone resorption, at baseline and 2 y later. RESULTS: At 2 y, plasma homocysteine concentrations were 5.2 mumol/L (95% CI: 3.9, 6.6 micromol/L; P < 0.001) lower in the vitamin than in the placebo group. No significant differences were found in either serum bone-specific alkaline phosphatase (-0.3 microg/L; 95% CI: -2.8, 2.1 microg/L; P = 0.79) or bone-derived collagen fragments (-0.0 microg/L; 95% CI: -0.1, 0.1 microg/L; P = 0.76) between the vitamin and placebo groups, respectively, with 2 y of supplementation. CONCLUSION: Supplementation with folate and vitamins B-6 and B-12 lowered plasma homocysteine but had no beneficial effect on bone turnover at the end of 2 y, as assessed by biomarkers of bone formation and resorption.

Staphylococcus aureus genotyping using novel real-time PCR formats.

One approach to microbial genotyping is to make use of sets of single-nucleotide polymorphisms (SNPs) in combination with binary markers. Here we report the modification and automation of a SNP-plus-binary-marker-based approach to the genotyping of Staphylococcus aureus and its application to 391 S. aureus isolates from southeast Queensland, Australia. The SNPs used were arcC210, tpi243, arcC162, gmk318, pta294, tpi36, tpi241, and pta383. These provide a Simpson's index of diversity (D) of 0.95 with respect to the S. aureus multilocus sequence typing database and define 61 genotypes and the major clonal complexes. The binary markers used were pvl, cna, sdrE, pT181, and pUB110. Two novel real-time PCR formats for interrogating these markers were compared. One of these makes use of "light upon extension" (LUX) primers and biplexed reactions, while the other is a streamlined modification of kinetic PCR using SYBR green. The latter format proved to be more robust. In addition, automated methods for DNA template preparation, reaction setup, and data analysis were developed. A single SNP-based method for ST-93 (Queensland clone) identification was also devised. The genotyping revealed the numerical importance of the "South West Pacific" and "Queensland" community-acquired methicillin-resistant S. aureus (MRSA) clones and the clonal complex 239 "Aus-1/Aus-2" hospital-associated MRSA. There was a strong association between the community-acquired clones and pvl.

A controlled trial of homocysteine lowering and cognitive performance.

BACKGROUND: The results of observational studies suggest that plasma homocysteine concentrations are inversely related to cognitive function in older people. Our objective was to test the hypothesis that lowering the plasma homocysteine concentration improves cognitive function in healthy older people. METHODS: We conducted a two-year, double-blind, placebo-controlled, randomized clinical trial involving 276 healthy participants, 65 years of age or older, with plasma homocysteine concentrations of at least 13 micromol per liter. Homocysteine-lowering treatment was a daily supplement containing folate (1000 microg) and vitamins B12 (500 microg) and B6 (10 mg). Tests of cognition were conducted at baseline and after one and two years of treatment. Treatment effects were adjusted for baseline values, sex, and education. RESULTS: On average, during the course of the study, the plasma homocysteine concentration was 4.36 micromol per liter (95 percent confidence interval, 3.81 to 4.91 micromol per liter) lower in the vitamin group than in the placebo group (P<0.001). Overall, there were no significant differences between the vitamin and placebo groups in the scores on tests of cognition. CONCLUSIONS: The results of this trial do not support the hypothesis that homocysteine lowering with B vitamins improves cognitive performance. (Australian Clinical Trials registry number, ACTR NO 12605000030673.).

The aesthetic appeal of minimal structures: judging the attractiveness of solutions to traveling salesperson problems.

Ormerod and Chronicle (1999) reported that optimal solutions to traveling salesperson problems were judged to be aesthetically more pleasing than poorer solutions and that solutions with more convex hull nodes were rated as better figures. To test these conclusions, solution regularity and the number of potential intersections were held constant, whereas solution optimality, the number of internal nodes, and the number of nearest neighbors in each solution were varied factorially. The results did not support the view that the convex hull is an important determinant of figural attractiveness. Also, in contrast to the findings of Ormerod and Chronicle, there were consistent individual differences. Participants appeared to be divided as to whether the most attractive figure enclosed a given area within a perimeter of minimum or maximum length. It is concluded that future research in this area cannot afford to focus exclusively on group performance measures.

Pharmaceutical co-crystals.

Crystal engineering has evolved in such a manner that it is now synonymous with the paradigm of supramolecular synthesis, that is, it invokes self-assembly of existing molecules to generate a wide range of new solid forms without the need to break or form covalent bonds. This review addresses how crystal engineering has been applied to active pharmaceutical ingredients, API's, with emphasis upon how pharmaceutical co-crystals, a long known but little explored alternative to the four traditionally known forms of API, can be generated in a rational fashion. Case studies on Carbamazepine (CBZ) and Piracetam are presented which illustrate the relative ease with which pharmaceutical co-crystals can be prepared and their diversity in terms of composition and physical properties.

The predictably elusive form II of aspirin.

The elusive form II of aspirin has been obtained during co-crystallization experiments with levetiracetam or acetamide, and it has been characterized by IR, DSC, HPLC, and single-crystal X-ray diffraction.

Crystal engineering of pharmaceutical co-crystals from polymorphic active pharmaceutical ingredients.

The carboxylic acid-primary amide supramolecular heterosynthon is exploited for the generation of pharmaceutical co-crystals that contain two active pharmaceutical ingredients that are polymorphic in their pure forms.