The influence of solution environment on the nucleation kinetics and crystallisability of para-aminobenzoic acid.

The influence of solvent type on the solution thermodynamics, nucleation-kinetics and crystal growth of alpha para-aminobenzoic acid (PABA) crystallising from supersaturated ethanol, acetonitrile and water solutions, is examined using poly-thermal analysis of the metastable zone width. Application of a recently proposed model for analysis of crystallisation kinetics (J. Cryst. Growth, 2010, 312, 698-704) indicates a solvent and concentration dependence of the nucleation mechanism and key nucleation parameters for the alpha form of PABA. The mechanism of nucleation is found to change from instantaneous to progressive with decreasing concentration and also when changing the solvent from ethanol to acetonitrile to water. The dependence of the nucleation mechanism is correlated to the kinetic component of the nucleation rate through calculated values of instantaneously nucleated crystallites, which increase from 1.40 × 109 m-3 in ethanol to 1.08 × 1010 m-3 in acetonitrile to 2.58 × 1010 m-3 in water. This in combination with low calculated number concentrations of interfacial tension between 1.13 and 2.71 mJ m-2, supports the conclusion that the kinetic component of the nucleation rate is more limiting when crystallising PABA from ethanol solutions in comparison to water solutions. This finding is further supported by molecular dynamics simulations of the solvation free energy of PABA, which is found to be greatest in water, -42.4 kJ mol-1 and lowest in ethanol, -58.5 kJ mol-1.

Towards an understanding of the nucleation of alpha-para amino benzoic acid from ethanolic solutions: a multi-scale approach.

The molecular assembly and subsequent nucleation of para-amino benzoic acid (PABA) from ethanolic solutions is probed using a multi-scale and multi-technique approach. This is applied by examining and interrelating information regarding the molecular, solution-state, cluster, solid-state and surface structures to understand why the alpha form of PABA is crystallised in preference to its low temperature beta form. Calculations suggest that conformational changes within the solute molecule play little or no role in directing the nucleation of either the alpha or beta crystal forms. Combined ab initio and molecular dynamics calculations of the stability of small clusters in solution suggests that the hydrogen-bonded carboxylic acid dimers, present in the alpha structure, are the most stable in solution and play a major role in the self-assembly and polymorphic expression of the alpha form in ethanol in preference to the beta form. These calculations are in good agreement with X-ray small-angle scattering analysis which reveals the presence of PABA clusters in ethanol which are consistent with the size and shape of a carboxylic acid dimer. SAXS studies also reveal the presence of larger cluster structures in a size range 10-40 nm which appear to grow, perhaps reflecting a change in the balance between monomers and dimers within the solution during the nucleation process. The results of crystallisation-kinetics experiments indicate an instantaneous nucleation mechanism where the number of instantaneously nucleated crystallites is calculated to be 1360-660 nuclei per ml and the subsequent growth is found to be only rate limited by diffusion of the growth unit to the crystallite surface. A linear dependence of growth rate with respect to supersaturation is observed for the (0 1 -1) capping face, which is associated with strong π-π stacking interactions. This is consistent with a solid-on-solid mechanism associated with surface roughened growth and concomitant poor lattice-perfection. Conversely, the side (1 0 -1) surface has a growth mechanism consistent with a 2D nucleation birth and spread mechanism. Hence, these mechanisms result in very fast growth along the b-axis and the needle-like morphology that is observed for alpha-PABA.

Effect of enzyme therapy in juvenile patients with Pompe disease: a three-year open-label study.

Pompe disease is a rare neuromuscular disorder caused by deficiency of acid α-glucosidase. Treatment with recombinant human α-glucosidase recently received marketing approval based on prolonged survival of affected infants. The current open-label study was performed to evaluate the response in older children (age 5.9-15.2 years). The five patients that we studied had limb-girdle muscle weakness and three of them also had decreased pulmonary function in upright and supine position. They received 20-mg/kg recombinant human α-glucosidase every two weeks over a 3-year period. No infusion-associated reactions were observed. Pulmonary function remained stable (n = 4) or improved slightly (n = 1). Muscle strength increased. Only one patient approached the normal range. Patients obtained higher scores on the Quick Motor Function Test. None of the patients deteriorated. Follow-up data of two unmatched historical cohorts of adults and children with Pompe disease were used for comparison. They showed an average decline in pulmonary function of 1.6% and 5% per year. Data on muscle strength and function of untreated children were not available. Further studies are required.

Identification of critical regions for clinical features of distal 10q deletion syndrome.

Array comparative genomic hybridization studies were performed to further characterize cytogenetic abnormalities found originally by karyotype and fluorescence in situ hybridization in five clinical cases of distal 10q deletions, including several with complex cytogenetic rearrangements and one with a partial male-to-female sex-reversal phenotype. These results have enabled us to narrow the previously proposed critical regions for the craniofacial, urogenital, and neuropsychiatric disease-related manifestations associated with distal 10q deletion syndrome. Furthermore, we propose that haploinsufficiency of the DOCK1 gene may play a crucial role in the pathogenesis of the 10q deletion syndrome. We hypothesize that alteration of DOCK1 and/or other genes involved in regulation and signaling of multiple pathways can explain the wide range of phenotypic variability between patients with similar or identical cytogenetic abnormalities.

Recombinant human acid [alpha]-glucosidase: major clinical benefits in infantile-onset Pompe disease.

BACKGROUND: Pompe disease is a progressive metabolic neuromuscular disorder resulting from deficiency of lysosomal acid alpha-glucosidase (GAA). Infantile-onset Pompe disease is characterized by cardiomyopathy, respiratory and skeletal muscle weakness, and early death. The safety and efficacy of recombinant human (rh) GAA were evaluated in 18 patients with rapidly progressing infantile-onset Pompe disease. METHODS: Patients were diagnosed at 6 months of age and younger and exhibited severe GAA deficiency and cardiomyopathy. Patients received IV infusions of rhGAA at 20 mg/kg (n = 9) or 40 mg/kg (n = 9) every other week. Analyses were performed 52 weeks after the last patient was randomized to treatment. RESULTS: All patients (100%) survived to 18 months of age. A Cox proportional hazards analysis demonstrated that treatment reduced the risk of death by 99%, reduced the risk of death or invasive ventilation by 92%, and reduced the risk of death or any type of ventilation by 88%, as compared to an untreated historical control group. There was no clear advantage of the 40-mg/kg dose with regard to efficacy. Eleven of the 18 patients experienced 164 infusion-associated reactions; all were mild or moderate in intensity. CONCLUSIONS: Recombinant human acid alpha-glucosidase is safe and effective for treatment of infantile-onset Pompe disease. Eleven patients experienced adverse events related to treatment, but none discontinued. The young age at which these patients initiated therapy may have contributed to their improved response compared to previous trials with recombinant human acid alpha-glucosidase in which patients were older.

Enhancement in immune function and growth using E-JUR-94013 supplementation.

Animal studies suggest that fish oils are capable of modulating the cell functions of immune system and there is some evidence that the effects of fish oils on immune function are due to fatty acids rather than trace elements or antioxidants. The major objectives of this study were: i) to identify a fish species with high nutritional value able to improve pig feeding conditions; ii) to utilize diets that modulate the immune system early in life in pigs and; iii) to enhance growth rate on a physiological basis. With the aim of maximizing feeding intake after weaning in order to reduce stress and increase growth rate, a study was carried out on 300 pigs supplemented with different fish extracts obtained by advanced biotechnological methods. The results of this work suggest that the lipoproteins obtained from the Trachurus trachurus (E-JUR-94013) species may have a great effect as both an immunomodulating compound (acting mainly on the regulation of IgA synthesis and/or release) and as a hypocholesterolemic compound, reducing the total cholesterol level in the serum of treated pigs. Both effects resulted in better pig growth, demonstrating that E-JUR-94013 can also be used as a natural growth promoter and an immune enhancer.

Double-blind, randomized, placebo-controlled pilot study with anapsos in senile dementia: effects on cognition, brain bioelectrical activity and cerebral hemodynamics.

The aim of this study was to evaluate the effects of two doses of anapsos in comparison with placebo on cognitive performance, brain bioelectrical activity pattern and cerebral hemodynamic parameters in patients with mild to moderate senile dementia of vascular type and Alzheimer type. Forty-five patients (age 73.8 +/- 7.6 years; range 56-89 years) with mild to moderate senile dementia (Global Deterioration Scale: stages 3-5) of the vascular (VD; n = 22) or the Alzheimer type (AD; n = 23) were included in a double-blind randomized placebo-controlled clinical trial. After a 2-week period of drug washout, patients were treated with placebo (n = 15; age 72.7 +/- 7.5 years), 360 mg/day of anapsos (n = 15; age 75.5 +/- 7.2 years), or 720 mg/day of anapsos (n = 15; age 73 +/- 7.7 years) for 4 weeks (28 days). At baseline and after the 4-week period of double-blind treatment, cognitive performance, brain bioelectrical activity power and blood flow hemodynamics in the middle cerebral arteries were evaluated with ADAScog, brain mapping and transcranial Doppler ultrasonography, respectively. Patients receiving 360 mg/day of anapsos showed a significant improvement in cognitive performance after treatment (ADAScog scores: p < 0.05) that was not observed in patients treated with placebo or 720 mg/day of anapsos. As compared to placebo, anapsos (360 mg/day) induced a significant improvement in ADAScog scores in mild senile dementia patients (p < 0.01) and in the subset of patients with AD (p < 0.05). Anapsos (360 mg/day) also increased cerebral blood flow velocities in left and right middle cerebral arteries in the subgroup of AD patients, whereas with the dose of 720 mg/kg this increase was only observed in the left side. Patients treated with anapsos (360 mg/day) showed a decrease in relative delta power and an increase in relative theta and alpha brain bioelectrical activity frequencies, indicating an acceleration of the EEG pattern. The present results show that anapsos (360 mg/day) improves cognitive performance, cerebral blood perfusion and brain bioelectrical activity in patients with senile dementia. These effects of anapsos were more marked in demented patients with mild mental deterioration and/or with dementia of the Alzheimer type.

Tumor necrosis factor constellation polymorphism and clozapine-induced agranulocytosis in two different ethnic groups.

Genes of the major histocompatibility complex (MHC) are associated with susceptibility to different immune and nonimmune mediated diseases. We had reported that the drug adverse reaction, clozapine-induced agranulocytosis (CA), is associated with different HLA types and HSP70 variants in Ashkenazi Jewish and non-Jewish patients, suggesting that a gene within the MHC region is associated with CA. This study was designed to find common genetic markers for this disorder in both ethnic groups. The tumor necrosis factor (TNF) microsatellites d3 and b4 were found in higher frequencies in both Jewish and non-Jewish patients: 51 of 66 (77%) and 48 of 66 (57%), respectively. Comparisons of these frequencies with those of controls, 28 of 66 (42%) and 18 of 66 (27%), were statistically significant (corrected P value = .001 for the d3 allele and .0005 for the b4 allele). On the other hand, the TNF microsatellite b5 was underrepresented in the group of patients, 9 of 66 (14%), when compared with the control subjects, 43 of 66 (65%) (corrected P value = .0005), probably related to protection from CA. Our results show a strong association of some genetic variants of the TNF loci with susceptibility to CA in two different ethnic groups suggesting involvement of TNF and/or associated gene(s) products in the pathogenesis of this hematologic-drug adverse reaction.

Citicoline improves memory performance in elderly subjects.

Citicoline is a choline donor involved in the biosynthesis of brain phospholipids and acetylcholine extensively used in the treatment of neurodegenerative diseases. In this study we investigated the effects of the oral administration of citicoline alone (C1000:1000 mg/day; C500:500 mg/day) or in combination with nimodipine (C +NI:300 + 90 mg/day) during 4 weeks on memory performance in elderly subjects with memory deficits and without dementia (N = 24; age = 66.12 +/- 10.78 years; MMS score = 31.69 +/- 2.76). Results indicated that citicoline in comparison with placebo improves memory in free recall tasks, but not in recognition tests. A significant improvement in word recall (5.17 +/- 1.1 vs. 3.95 +/- 1.2 omissions; p < 0.005), immediate object recall (6.5 +/- 1.6 vs. 5.5 +/- 1.2 omission; p < 0.05) and delayed object recall (8.5 +/- 2.1 vs. 6.7 +/- 2.4 omissions; p < 0.005) was observed after citicoline treatment. Similar results were found in the three subgroups of treatment (8 subjects per group), suggesting that citicoline possesses memory-enhancing activity at doses of 300-1000 mg/day. A decrease in systolic blood pressure and minor changes in lymphocyte cell counting were also observed in old subjects after receiving citicoline. These effects are consistent with the vasoregulatory and neuroimmune actions of citicoline and suggest that this compound may improve memory by acting on mechanisms of brain neurotropism and cerebrovascular regulation. According to the present results, showing that citicoline improves memory performance in elderly subjects, we concluded that this molecule is suitable for the treatment of memory deficits in old people.

The major histocompatibility complex region marked by HSP70-1 and HSP70-2 variants is associated with clozapine-induced agranulocytosis in two different ethnic groups.

Genes of the major histocompatibility complex (MHC) have been associated with susceptibility to drug-induced adverse reactions. We previously found that clozapine-induced agranulocytosis (CA) is associated with the HLA-DRB1*0402, DRB4*0101, DQB1*0302, DQA1*0301 haplotype in Ashkenazi Jewish patients and with the HLA-DRB1*1601, DRB5*02, DQB1*0502, DQA1*0102 haplotype in non-Jewish patients. In the present study, we tested the hypothesis that the variants of the heat-shock protein 70 (HSP-70) encoded by the HSP-70 loci located within the MHC region and known to be involved in apoptosis and regulation of cell proliferation could play an important role in molecular mechanisms of CA. First, we analyzed HSP70-2 polymorphism in risk-associated haplotypes from HLA homozygous cells and normal individuals and confirmed that the HSP70-2 9-kb variant was associated invariably with DR4 (HLA-DRB1*0402, DQB1*0302) and DR2 (HLA-DRB1*01601, DQB1*0502, DQA1*0102 and HLA-DRB1*1501, DQB1*0602) haplotypes, which were the haplotypes found increased in Jewish and non-Jewish patients with CA, respectively. The 9.0-kb variant was also found to be associated with HLA-B44, DRB1*0401 and HLA-B44, DRB1*07 haplotypes. Second, in patients with CA (12 Ashkenazi Jewish and 20 non-Jewish patients), HSP70-1 A and HSP70-2 9.0-kb variants were associated with the MHC haplotypes found by us to be markers of susceptibility to CA. The clozapine-treated control group had an excess number of HSP70-1 C and HSP70-2 8.5-kb variants, consistent with genetic resistance to CA associated with those variants. This finding supports our hypothesis that a dominant gene within the MHC region (marked by HSP70-1 and HSP70-2), but not necessarily HLA, is associated with CA in two different ethnic groups.

HLA associations in clozapine-induced agranulocytosis.

We previously reported preliminary results of association of clozapine-induced agranulocytosis (CA) with HLA-B38, DR4, DQ3 in five Ashkenazi Jewish patients and with HLA-DR2, DQ1 in four non-Jewish patients. In the present study, 31 additional patients with CA, 10 Ashkenazi Jewish, and 21 of non-Jewish ancestry, were studied. HLA alleles and haplotypes were compared among 52 patients (33 Ashkenazi Jewish, 19 non-Jewish) matched for ethnic background and clinical status. Our results show two associations and define the HLA allele markers for the Ashkenazi Jewish and non-Jewish haplotypes associated with CA. The most important markers for susceptibility for CA in Ashkenazi Jewish patients were DRB1*0402, DQB1*0302, and DQA1*0301, and in non-Jewish patients, HLA-DR*02, DQB1*0502, and DQA1*0102. HLA-DRB1*011 and DQB1*0301 were underrepresented in Ashkenazi Jewish patients when compared with controls. We hypothesize that genes of the major histocompatability complex, other than class I and class II, are responsible for CA; among them are the variants of the heat-shock proteins 70 or the tumor necrosis factor loci.

Advances in HLA genetics.

The major histocompatibility complex (MHC) is a genetic system of over 70 known genes that occupies the midportion of the short arm of the sixth chromosome (C6p) and spans about 4 million base pairs of DNA. The high-resolution typing of class I and class II MHC genes and the identification of genes between and near them has increased the definition of the genetic basis of immune responses and diseases of unknown etiology such as autoimmune diseases in man. Although there are many more genetic systems that participate in the rejection of tissues and in the immune response, the MHC plays a central role in tissue compatibility and immune response against cancer and infectious diseases. In this paper, the authors review evidence about the role of HLA polymorphism in the pathogenesis and development of cancer, infectious diseases, autoimmune diseases and transplantation.

HSP70-2 9.0 kb variant is in linkage disequilibrium with the HLA-B and DRB1* alleles associated with clozapine-induced agranulocytosis.

In order to extend the analysis of the association between class I and class II HLA markers and HSP-70 alleles, we studied the genetic polymorphism of HSP70-2 genes by restriction fragment length polymorphism analysis in a panel of HLA-homozygous cell lines carrying the HLA-B alleles known to be associated with clozapine-induced agranulocytosis. We have found a linkage disequilibrium between the 9.0 kb variant of HSP70-2 with the class I antigens HLA-B7, B38, and B44 and with the class II antigens HLA-DR2 and DR4. We discuss the importance of analyzing the variants of HSP70-2 in patients with agranulocytosis to confirm that the 9.0 kb allele is a genetic marker for the disease. If that is the case, HSP-70 variants could explain the different associations of HLA alleles in individuals of Jewish and non-Jewish ancestry because the HLA alleles are in linkage disequilibrium with the 9.0 kb band. We postulate that HSP-70 molecules could also play a significant role in determining the molecular mechanisms that induce agranulocytosis by clozapine.