Enhancement of the physical stability of amorphous indomethacin by mixing it with octaacetylmaltose. inter and intra molecular studies.

PURPOSE: To demonstrate a very effective and easy way of stabilization of amorphous indomethacin (IMC) by preparing binary mixtures with octaacetylmaltose (acMAL). In order to understand the origin of increased stability of amorphous system inter- and intramolecular interactions between IMC and acMAL were studied. METHODS: The amorphous IMC, acMAL and binary mixtures (IMC-acMAL) with different weight ratios were analyzed by using Dielectric Spectroscopy (DS), Differential Scanning Calorimetry (DSC), Raman Spectroscopy, X-ray Diffraction (XRD), Infrared Spectroscopy (FTIR) and Quantitative Structure-Activity Relationship (QSAR). RESULTS: Our studies have revealed that indomethacin mixed with acetylated saccharide forms homogeneous mixture. Interestingly, even a small amount of modified maltose prevents from recrystallization of amorphous indomethacin. FTIR measurements and QSAR calculations have shown that octaacetylmaltose significantly affects the concentration of indomethacin dimers. Moreover, with increasing the amount of acMAL in the amorphous solid dispersion molecular interactions between matrix and API become more dominant than IMC-IMC ones. Structural investigations with the use of X-ray diffraction technique have demonstrated that binary mixture of indomethacin with acMAL does not recrystallize upon storage at room temperature for more than 1.5 year. Finally, it was shown that acMAL can be used to improve solubility of IMC. CONCLUSIONS: Acetylated derivative of maltose might be very effective agent to improve physical stability of amorphous indomethacin as well as to enhance its solubility. Intermolecular interactions between modified carbohydrate and IMC are likely to be responsible for increased stability effect in the glassy state.

A new way of stabilization of furosemide upon cryogenic grinding by using acylated saccharides matrices. The role of hydrogen bonds in decomposition mechanism.

Recently it was reported that upon mechanical milling of pure furosemide significant chemical degradation occurs (Adrjanowicz et al. Pharm. Res.2011, 28, 3220-3236). In this paper, we present a novel way of chemical stabilization amorphous furosemide against decomposing that occur during mechanical treatment by preparing binary mixtures with acylated saccharides. To get some insight into the mechanism of chemical degradation of furosemide induced by cryomilling, experimental investigations supported by density functional theory (DFT) computations were carried out. This included detailed studies on molecular dynamics and physical properties of cryoground samples. The main thrust of our paper is that we have shown that furosemide cryomilled with acylated saccharides forms chemically and physically stable homogeneous mixtures with only one glass transition temperature, Tg. Finally, solubility measurements have demonstrated that furosemide cryomilled with acylated saccharides (glucose, maltose and sucrose) is much more soluble with respect to the crystalline form of this active pharmaceutical ingredient (API).

Dielectric studies on molecular dynamics of two important disaccharides: sucrose and trehalose.

Broadband dielectric measurements were carried out in the supercooled as well as in the glassy state of two very important disaccharides: trehalose and sucrose. Multiple relaxation processes were observed. Above the glass transition temperatures of examined disaccharides structural relaxation of cooperative origin was detected, where in the glassy state more local motions (secondary modes) appeared. Our data were discussed in light of the findings reported by other groups. We pointed out that sample preparation might impact mobility and, thus, dielectric loss spectra in a significant way. Consequently, it may lead to misinterpretation of the dielectric relaxation processes. Moreover, impact of physical aging and pressure on dynamics of two secondary relaxation processes observed in the glassy state of trehalose and sucrose has been investigated. Additionally, we have demonstrated that, in contrast to the calorimetric measurements (DSC), activation energies of the ß- and γ-relaxation processes observed in the glassy state of sucrose and trehalose do not change as a result of physical aging. Finally, we found out that the ß-relaxation process slows down as pressure increases. We interpreted this fact in view of increasing rigidity of the structures of disaccharides.

Influenza virus-specific cytotoxic T lymphocytes recognize HLA-molecules. Blocking by monoclonal anti-HLA antibodies.

The effect of monoclonal anti-HLA antibodies on lysis of influenza virus-infected target cells by sensitized cytotoxic T lymphocytes was measured. Lysis was blocked, provided the antibody was present in a concentration sufficient to saturate the HLA determinants on the target cells. Antibodies to monomorphic HLA determinants and to beta 2-microglobulin inhibited. Antibody to HLA A2 and B17 blocked lysis of target cells that shared these antigens with the effector cell. It was concluded that the restriction elements for influenza virus-specific cytotoxic T cells are the HLA molecules themselves.

Identification of the slower secondary relaxation's nature in maltose by means of theoretical and dielectric studies.

Dielectric relaxation measurements on maltose were performed at ambient and increasing pressure. The loss spectra collected below glass transition of this disaccharide revealed presence of two well separated secondary relaxations. Activation energies determined for both modes are E(a)=73 kJ/mol and 47 kJ/mol for the slower (beta) and faster (gamma) relaxation, respectively. From high pressure measurements activation volume DeltaV=15.6 ml/mol for the slower secondary relaxation was estimated. Both quantities: activation energy and activation volume for alpha-process derived from dielectric data, were compared to those obtained from the conformational calculations with use of density functional theory (DFT). We found out satisfactory agreement between both quantities for the molecular motion related to the rotation of the two monosaccharide units around glycosidic linkage in this disaccharide.

Subpopulations of normal and leukemic human thymocytes: an analysis with the use of monoclonal antibodies.

Combinations of antibodies to membrane antigens and to terminal deoxynucleotidyl transferase (TdT) were used to study human thymocyte and bone marrow subpopulations and leukemia cells. Cortical thymocytes were TdT+ and expressed T-cell antigens (HuTLA+), a thymocyte-specific antigen (HTA-1+), and a leukocyte antigen (HLe-l++) but lacked detectable HLA-A,B,C and la (HLA-D) antigens. In contrast, medullary thymocytes were TdT-, HuTLA+, HTA-1-, HLe-l++. A small subpopulation of larger, probably immature, thymocytes were strongly TdT+, HuTLA+, la-, HTA-1-, HLe-l +/-. Many blast cells from cases of thymic acute lymphoblastic leukemia (Thy-ALL) showed the phenotype of this small subset, and only a proportion of Thy-ALL blast cells exhibited HTA-1 and HLe-l antigens as strongly as was observed on normal cortical thymocytes. In contrast, TdT+ cells observed in normal juvenile bone marrow were HuTLA, HTA-1-, HLA+, la+. This phenotype corresponded to the phenotype of the common form of ALL (non-T, non-B) and indicated that further studies are necessary to analyze the differentiation of bone marrow precursors to thymic cells.

Grain-resolved analysis of localized deformation in nickel-titanium wire under tensile load.

The stress-induced martensitic transformation in tensioned nickel-titanium shape-memory alloys proceeds by propagation of macroscopic fronts of localized deformation. We used three-dimensional synchrotron x-ray diffraction to image at micrometer-scale resolution the grain-resolved elastic strains and stresses in austenite around one such front in a prestrained nickel-titanium wire. We found that the local stresses in austenite grains are modified ahead of the nose cone-shaped buried interface where the martensitic transformation begins. Elevated shear stresses at the cone interface explain why the martensitic transformation proceeds in a localized manner. We established the crossover from stresses in individual grains to a continuum macroscopic internal stress field in the wire and rationalized the experimentally observed internal stress field and the topology of the macroscopic front by means of finite element simulations of the localized deformation.

Physical and developmental phenotype analyses in a boy with Wolf-Hirschhorn syndrome.

Wolf-Hirschhorn syndrome (WHS) is a rare genetic condition with characteristic facial traits, organ malformations, functional impairment and developmental delay due to partial short arm monosomy of chromosome 4. Although several hundreds of cases have been published to date, a systematic collection of its clinical symptoms and anthropological traits is missing in the literature, and reports on abilities and needs of children with WHS are scanty. Results of detailed physical and developmental phenotype analyses in a 1 10/12-year-old boy with monosomy 4p15.2-pter are presented. Physical analyses were based on systematic data acquisition. They disclosed a total of 32 clinical symptoms and 46 anthropological traits. Developmental analyses were based on the child's interactive play in an environment structured according to Montessori principles. They disclosed a total of 44 abilities and a number of needs to be satisfied by the environment for the support of the child's psychic and intellectual growth. While the physical phenotype is important for the diagnostic process, the developmental phenotype is essential for parental counseling.

Clinical and molecular-cytogenetic studies in seven patients with ring chromosome 18.

We report the results of detailed clinical and molecular-cytogenetic studies in seven patients with ring chromosome 18. Classical cytogenetics and fluorescence in situ hybridization (FISH) analysis with the chromosome 18 painting probe identified five non-mosaic and two complex mosaic 46,XX,dup(18)(p11.2)/47,XX,dup(18)(p11.2),+r(18) and 46,XX,dup(18)(p11.32)/47,XX,dup(18)(p11.32),+r(18) cases. FISH analysis was performed for precise characterization of the chromosome 18 breakpoints using chromosome 18-specific short-arm paint, centromeric, subtelomeric, and a panel of fifteen Alu- and DOP-PCR YAC probes. The breakpoints were assessed with an average resolution of approximately 2.2 Mb. In all r(18) chromosomes, the 18q terminal deletions ranging from 18q21.2 to 18q22.3 ( approximately 35 and 9 Mb, respectively) were found, whereas only in four cases could the loss of 18p material be demonstrated. In two cases the dup(18) chromosomes were identified as inv dup(18)(qter-->p11.32::q21.3-->qter) and inv dup(18)(qter-->p11.32::p11.32-->p11.1: :q21.3-->qter)pat, with no evidence of an 18p deletion. A novel inter-intrachromatid mechanism of formation of duplications and ring chromosomes is proposed. Although the effect of "ring instability syndrome" cannot be excluded, the phenotypes of our patients with characteristic features of 18q- and 18p- syndromes are compared and correlated with the analyzed genotypes. It has been observed that a short neck with absence of cardiac anomalies may be related to the deletion of the 18p material from the r(18) chromosome.

Severe VA/Q mismatch in perfused lungs evoked by sequential challenge with endotoxin and E. coli hemolysin.

Escherichia coli hemolysin (ECH), an important pathogenicity factor in extraintestinal E. coli infections, provokes pulmonary hypertension and microvascular leakage in buffer-perfused rabbit lungs. We investigated gas exchange abnormalities in response to low doses of ECH, lipopolysaccharides (LPS), and sequential and combined application of these bacterial agents by using the multiple inert gas elimination technique. In control lungs and after admixture of 100 ng/ml of LPS, unimodal narrow distribution of perfusion and ventilation to midrange ventilation-perfusion (VA/Q) areas was noted. ECH [0.08 hemolytic units (HU)/ml] caused a moderate increase in pulmonary arterial pressure (< 10 mmHg), progressive lung edema formation (approximately 10 g within 20 min), and a broadening of perfusate and gas flow dispersion. Application of 0.08 HU/ml of ECH in lungs "primed" with 100 ng/ml of LPS in a preceding 125-min perfusion period provoked a large increase in pulmonary arterial pressure (> 50 mmHg within 5 min), rapid edema formation (approximately 10 g within 10 min), and severe VA/Q mismatch with predominance of shunt flow. Vasoconstrictor response and VA/Q mismatch, but not edema formation, were largely inhibited by pretreatment of lungs with acetylsalicylic acid or the thromboxane receptor antagonist BM-13.505. In addition, "rescue" application of BM-13.505 rapidly reversed pressure rise and shunt flow due to sequential LPS and/or ECH stimulation, whereas edema formation was not affected. We conclude that the marked pulmonary hypertension in response to low doses of ECH in LPS-primed lungs is paralleled by severe gas exchange abnormalities with predominance of shunt flow. Both the vasoconstrictor response and the development of shunt are closely related to toxin-induced thromboxane generation.

Staphylococcal alpha-toxin induced ventilation-perfusion mismatch in isolated blood-free perfused rabbit lungs.

Gas exchange conditions in blood-free perfused isolated rabbit lungs were assessed by the use of the multiple inert gas elimination technique. Under baseline conditions, unimodal narrow distribution of perfusion and ventilation to midrange-ventilation-perfusion (VA/Q) areas was noted. Intravascular challenge with staphylococcal alpha-toxin caused a rapid increase in pulmonary arterial pressure (to > 40 mmHg within approximately 15 min) and delayed-onset (> 10-15 min) lung edema formation, with unaltered ventilation pressures. The vasoconstrictor response was paralleled by a progressive, severe leftward shift of perfusion to areas with low-VA/Q ratios, accompanied by a minor fraction of shunt flow. At pulmonary arterial pressures > 40 mmHg, extreme VA/Q mismatch with near absence of perfusate flow to midrange-VA/Q areas was registered. Vasoconstrictor response and VA/Q mismatch, but not the progressive edema formation, were virtually completely suppressed in lungs pretreated with acetylsalicylic acid or the thromboxane receptor antagonist BM 13505. Moreover, "rescue" application of BM 13505 after onset of alpha-toxin-induced pressor response and gas exchange abnormalities completely reversed pressure elevation and loss of VA/Q matching. We conclude that the marked vasoconstrictor response to staphylococcal alpha-toxin is paralleled by severe VA/Q mismatch with predominant perfusion of low-VA/Q areas independent of lung edema formation. Pressor response and VA/Q mismatch, but not vascular leakage, are suppressed by thromboxane inhibition.

Lymphocytes, neutrophils and serum immunoglobulins in patients with precancerous states of the larynx.

In 24 men aged 32 to 58 years with precancerous states of the larynx, i.e., leukoplakia, papillomas and pachydermia the peripheral blood lymphocytes were cytochemically stained for N-acetyl-beta-glucosaminidase, beta-glucuronidase, acid phosphatase and glycogen; and the neutrophils were stained for alkaline phosphatase, myeloperoxidase and lipids. The results were expressed in terms of the absolute counts of reaction-positive cells and of the activity index score. The serum immunoglobulins IgG, IgA and IgM were also determined by Mancini's method. The results obtained were compared with those in 20 healthy men aged 20 to 30 years. The patients exhibited elevated numbers of N-acetyl-beta-glucosaminidase and beta-glucuronidase-positive lymphocytes. A characteristic feature was an increase in the absolute counts of lymphocytes with diffuse and granular-diffuse types of cytochemical reaction for all enzymes studied. The number of cells with the granular type of enzymatic reaction (intact enzyme-positive lysosomes) was significantly diminished. These cytochemical alterations were accompanied by a significant increase in the serum IgA level. These results are discussed with reference to the lymphoid system response to tissues of precancerous lesions of the larynx. So far as the neutrophils are concerned the patients exhibited significant intracellular deficiency of beta-glucuronidase and decrease in the lipid content as well as an elevated alkaline phosphatase activity. The possible significance of the beta-glucuronidase deficiency in neutrophils for the diminished cytotoxic response of these cells against the tumor and precancerous lesion cells is discussed.

Agenesis of corpus callosum: clinical description and etiology.

In 135 children (aged 3 months to 15 years) with structural defects of the central nervous system found on magnetic resonance imaging, agenesis of the corpus callosum was evident in 7. The etiology of agenesis of the corpus callosum has been established in four children: partial trisomy of chromosome 13, partial duplication of the long arm of chromosome 10, Aicardi's syndrome, and intracranial bleeding during the fetal period as a result of injury. Agenesis of the corpus callosum coexisted with a Dandy-Walker malformation in one other patient, which suggests a genetic etiology. In spite of these variable etiologies, dysmorphic features were identified in all seven patients, as was psychomotor retardation. Epileptic seizures had occurred in six patients, and all manifested abnormalities on neurologic examination.

N-acetyltransferase phenotype of patients with cancer of the larynx.

In the present paper N-acetyltransferase phenotype has been evaluated in 128 patients with cancer of the larynx. Out of 128 patients, 86.7% were smokers. The corresponding control group consisted of 106 men who did not suffer from neoplastic diseases. 83.6% of the patients were slow acetylators. This is significantly different from the frequency of 60.3% of slow acetylators observed in the control group. The slow acetylators exhibited a greater susceptibility to the cancer of the larynx.

Zinc, vitamins A and E, and retinol-binding protein in sera of patients with cancer of the larynx.

Serum vitamin A concentrations were measured in 22 patients with laryngeal cancer and found to be significantly lower than those of patients of similar age with either nonmalignant laryngeal or other than laryngeal diseases. The levels of vitamin A in the patients with laryngeal cancer, but not in the controls, significantly correlated with serum concentrations of retinol-binding protein (RBP) and zinc. It is suggested that low levels of zinc might reduce the synthesis of RBP and thus reduce the mobilization of vitamin A from the liver stores.

Sperm chromatin structure assay of bulls qualified for artificial insemination.

The goal of our study was to find the relationship between fertility of bulls qualified for AI and the percentage of spermatozoa with abnormal chromatin structure as an independent parameter. We used the frozen semen of 8 mature bulls from one AI center. Each bull was represented by 3 ejaculates collected with at least 2-week intervals. Bull fertility was calculated on the basis of non-return ratio and was expressed as a scale where 100 points represented the average fertility of all the AI center's bulls. Bulls with lower or higher fertility received a lower or higher score respectively. Fertility scores of bulls used in the study ranged from 83 to 104 . Semen was processed according to the SCSA (sperm chromatin structure assay) method and was analyzed by flow cytometry. "Artificial" alpha(t) (alpha(t)=red/green+red fluorescence) and red fluorescence histograms were used for calculation of COMPalpha(t), SDalpha(t), %Red, %PeakR and MeanR parameters. The percentage of spermatozoa with abnormal chromatin ranged from 1.2% to 23.8%. A large variation among ejaculates was found for bulls with lower fertility. Fertility correlated significantly with COMPalpha(t) (-0.50, P < 0.05), SDalpha(t) (-0.55, P < 0.01), %Red (-0.53, P < 0.01), %PeakR (-0.58, P < 0.01) and MeanR (-0.45, P < 0.05). The SCSA method has a practical application in analyzing spermatogenesis disorders in bulls. If regularly applied, it allows us to identify and eliminate ejaculates with a high level of sperm chromatin abnormalities.

N-acetyl-beta-glucosaminidase of peripheral blood lymphocytes in patients with cancer of the larynx.

In 20 men, age 35 to 55 years, with cancer of the larynx of N-acetyl-beta-glucosaminidase in peripheral blood lymphocytes was determined cytochemically according to Hayashi's method. Numbers of enzyme-positive cells were counted with regard to the type of cytochemical reaction (granular, granular-diffuse, or diffuse), and to the number of enzyme-positive lysosomal granules within a single cell. In comparison to 20 healthy men, age 20 to 30 years, the following changes were noted in the patients: 1) the decrease of the total count of enzyme-negative lymphocytes; 2) the decrease of the total count of lymphocytes with granular type of reaction; 3) the increase of the counts of lymphocytes with granular-diffuse and diffuse type of reaction. The authors suggest that these changes could be related to the immune reaction of specific tumor antigens.

The trial of quantitative determination of the lead, chlorine, and sulfur content in the peripheral blood lymphocytes from patients with cancer of the larynx using the X-ray microanalysis method.

Using own modification of X-ray microanalysis method the authors evaluated the lead, chlorine, and sulfur content in the peripheral blood lymphocytes in 20 patients with laryngeal carcinoma in comparison with 20 healthy subjects of the control group. In patients with advanced clinical stages of that carcinoma the elevated lead and chlorine content has been stated in above cells whereas that of sulfur was decreased. According to the authors' opinion above changes reflect functional disturbances of lymphocytes in the patient with laryngeal carcinoma related to the decreased detoxicative properties, the intracellular electrolyte imbalance, and the enzymatic machinery alterations within that cells.

The surface structure of peripheral blood lymphocytes from patients with laryngeal carcinoma evaluated by scanning electron microscopy.

Using the scanning electron microscopic technique the surface structure of the peripheral blood lymphocytes has been studied in 20 men aged 46 to 62 years with the laryngeal carcinoma diagnosed clinically and histopathologically. The control group consisted of 20 healthy men aged 39 to 58 years. According to the international classification 15 of 20 patients studied showed the T3N0M0 stage of the disease and other five the T4N0M0 stage respectively. The peripheral blood lymphocytes of the patients showed significantly greater variability of the surface structure as compared with the controls. The characteristic pattern of the lymphocytes in patients with cancer of the larynx was quantitative increase in numbers of lymphocytes having smooth surface deprived of microvilli and significant decrease of cells exhibiting numerous surface microvilli; these cells may correspond to the T and the B cell series, respectively. Moreover, the patients showed significant, approximately threefold increase in number of lymphocytes having the intermediate surface morphology between above mentioned two basic lymphocyte topographical types. According to the authors opinion above differences between the lymphocytes of patients with cancer of the larynx and those from healthy subjects are associated with the antitumor response of the lymphocytes.

Enzymatic deficiencies of the immune system cells in patients with cancer of the larynx and other malignancies.

Activity of N-acetyl-beta-D-glucosaminidase, beta-glucuronidase, and acid phosphatase and myeloperoxidase was determined in neutrophils and lymphocytes of patients with cancer of the larynx and precancerous states of the larynx as well as--for comparative reasons--in patients with malignant tumors of female generation organs, breast carcinoma, cancer of the stomach and endometriosis. The main result of investigations performed was in fact that intracellular deficiency of beta-glucuronidase within the neutrophils characterizes patients with cancer and precancerous states of the larynx. Patients with cancer of the larynx show additionally a deficiency of neutrophil myeloperoxidase. Deficiency of N-acetyl-beta-D-glucosaminidase occurs, in contrast, in patients with malignancies of female generation organ. Activity of myeloperoxidase in neutrophils from patients with gastric carcinoma is slightly elevated.