Overcoming drug impurity challenges in amorphous solid dispersion with rational development of biorelevant dissolution-permeation method.

Hot-melt extrusion is often used to prepare amorphous solid dispersion to overcome low drug solubility and enhance bio-performance of the formulation. Due to the uniqueness of each drug - polymer combination and its physico-chemical properties, setting the appropriate HME barrel temperature, feed rate and screw speed ensures drug amorphization, absence of residual crystallinity, absence of water, and a suitable drug release profile. In this research, samples with BCS II/IV model drug and PVP/VA polymer were prepared to evaluate the impact of HME process parameters, incoming drug form (anhydrous vs. hydrate), and drug supplier (i.e., impurity profile), on biorelevant drug release. This study provides a relationship between observed in vitro supersaturation and precipitation behavior of amorphous solid dispersion formulation with in vivo results, on patients, by using the acceptor profile of side-by-side dissolution-permeation apparatus. An in vitro dissolution method, in small volumes, in an apparatus with paddles and dissolution-permeation side-by-side method was developed on the MicroFlux™ apparatus to assess if the differences observed in vitro bears relevance to the bioequivalence outcome in vivo. The former was used to guide the generic drug product development due to high discriminatory strength, while the latter was biorelevant, due to the inclusion of the second compartment assuring absorptive environment to capture the impact of supersaturation and subsequent precipitation on bioavailability. Bio-relevancy of the in vitro method was confirmed with the in vivo dog study and clinical study on patients, and an in vitro - in vivo correlation was established. For the investigated BCS II/IV drug, this research highlights the importance of considering supersaturation and formation of colloidal species during amorphous solid dispersion release testing to assure product quality, safety and efficacy.

Compaction properties of crystalline pharmaceutical ingredients according to the Walker model and nanomechanical attributes.

This study investigates the extent to which single-crystal mechanical properties of selected active ingredients (famotidine, nifedipine, olanzapine, piroxicam) influence their bulk compressibility and compactibility. Nanomechanical attributes of oriented single crystals were determined with instrumented nanoindentation, and bulk deformational properties were assessed with the Walker and Heckel models as well as the elastic relaxation index. Good correlations were established between bulk and single-crystal plasticity parameters: the Walker coefficient and indentation hardness. The Walker model showed more practical value for evaluating bulk deformational properties of the APIs investigated because their properties differed more distinctly compared to the Heckel model. In addition, it was possible to predict the elastic properties of the materials investigated at the bulk level because a correlation between the elastic relaxation index and compliance was established. The value of using indentation hardness for crystalline APIs was also confirmed because their compactibility at the bulk level was able to be predicted. Mechanically interlocked structures were characteristic of most polymorphic forms investigated, resulting in single crystals having isotropic mechanical properties. It was revealed that in such cases good correlations between single and bulk mechanical properties can be expected. The results imply that innate crystal deformational properties define their compressibility and compactibility properties to a great extent.

Consolidation trend design based on Young's modulus of clarithromycin single crystals.

The key aim of this study was to determine single mechanical properties of clarithromycin polymorphic forms in order to select some of them as more suitable for the tableting process. For this purpose, AFM single-point nanoindentation was used. The Young's moduli of clarithromycin polymorphs were substantially different, which was consistent with the structural variations in their packing motifs. The presence of the adjacent layers, which can easily slide over each other due to the low energy barrier (the lowest Young's modulus was 0.25 GPa) resulted in better bulk compressibility (the highest Heckel coefficient) of clarithromycin Form I. We also addressed the importance of tip geometry screening because the stress during the force mode often results in tip apex fracture. Even the initial manufacture of the diamond-coated tips can result in defects such as double-apex tips.

Anemia is independently associated with NT-proBNP levels in asymptomatic predialysis patients with chronic kidney disease.

BACKGROUND: Although anemia and renal dysfunction are related to increased natriuretic peptides levels in heart failure patients, less is known about this relationship in asymptomatic predialysis patients with chronic kidney disease (CKD). The aim of this study was to investigate relationship between hemoglobin (Hb) concentration, N-terminal proBNP (NT-proBNP) levels and echocardiographic findings in these patients. METHODS: The study included 61 patients with CKD stage IV-V (34 male, mean age 62.6 ± 13.6 years) and 22 age- and sex -matched healthy persons as control group. All participants underwent clinical, laboratory and echocardiographic examination, including Tissue Doppler Imaging and colour M-mode Doppler. RESULTS: Patients with CKD had lower Hb levels (p<0.001), and higher levels of NT-proBNP (p<0.001) than healthy controls. Patients were divided into two groups according to their mean Hb levels: group A, Hb<10.3 g/dL and group B, Hb≥10.3 g/dL. Patients from group A was significantly older (p<0.001), left ventricular mass index was significantly higher (LVMI, p<0.001), LV diastolic function was worse (septal and lateral E'/A' ratio: p<0.05 and p<0.01, respectively), and the level NT-proBNP was higher (p<0.001) compared to patients from group B. The natural logarithm of NT-proBNP (lnNT-proBNP) showed highly significant correlation with Hb (p<0.001) and significant correlation with estimated glomerular filtration rate (p=0.035) in CKD patients. Multiple regression analysis revealed Hb levels (p<0.01), cholesterol (p<0.001), LV ejection fraction (p<0.001) and septal E/E' ratio (p<0.01) as the independent variables predicting as much as 54% variability of lnNTpro-BNP. CONCLUSIONS: The increased NT-proBNP levels in asymptomatic patients with advanced CKD were independently associated with echocardiographic parameters of LV function, but anemia may represent one of the important confounder of the relationship between NT-proBNP and cardiovascular abnormalities.

Is there evidence of optimisation for carbon efficiency in plant proteomes?

Flowering plants, angiosperms, can be divided into two major clades, monocots and dicots, and while differences in amino acid composition in different species from the two clades have been reported, a systematic analysis of amino acid content and distribution remains outstanding. Here, we show that monocot and dicot proteins have developed distinct amino acid content. In Arabidopsis thaliana and poplar, as in the ancestral moss Physcomitrella patens, the average mass per amino acid appears to be independent of protein length, while in the monocots rice, maize and sorghum, shorter proteins tend to be made of lighter amino acids. An examination of the elemental content of these proteomes reveals that the difference between monocot and dicot proteins can be largely attributed to their different carbon signatures. In monocots, the shorter proteins, which comprise the majority of all proteins, are made of amino acids with less carbon, while the nitrogen content is unchanged in both monocots and dicots. We hypothesise that this signature could be the result of carbon use and energy optimisation in fast-growing annual Poaceae (grasses).

Novel H1N1 influenza in neonates: from mild to fatal disease.

Analysis of pediatric deaths associated with pandemic A H1N1 influenza shows that fatal outcome is more likely in young children, under the age of 5. Neonates, because of the immaturity of their immune system, could represent a high-risk group for severe disease and fatal outcome. We present a group of five neonates with confirmed novel influenza A H1N1 infection. This report indicates that the full spectrum of influenza A H1N1 infection ranging from mild febrile illness with spontaneous recovery to severe disease with fatal outcome may be expected even in neonates.

Efficacy of hyperphosphatemia control in the progression of chronic renal failure and the prevalence of cardiovascular calcification.

BACKGROUND AND AIMS: Chronic kidney disease mineral- and bone disorder (CKD-MBD) has been studied more often in dialysis than in predialysis CKD patients. The association between efficacy of hyperphosphatemia control and chronic renal failure (CRF) progression, prevalence of bone disease and cardiovascular calcification was the objective of the present investigation. MATERIAL AND METHODS: 42 patients with CKD in Stage 5, regularly monitored for 5 years, were divided into Group 1 of 20 patients with normal serum phosphate (sPO4) levels and Group 2 of 22 patients with hyperphosphatemia registered at the majority of checks. Serum urea, creatinine, calcium (sCa) and sPO4 levels were regularly determined in the retrospective 5-year period. At the end of this period iPTH, bone alkaline phosphatase-BAP and inflammation markers (CRP, fetuin-A) were measured, valvular and arterial calcifications were detected by B mode echocardiogram and soft-tissue native radiograms of the pelvis and the wrist. RESULTS: Progression of CRF (1/sCr over time) was faster in Group 2 than in Group 1 (b = -0.0577 vs. -0.0288, p = 0.003) during the study period. Average BAP and iPTH values were similar in both groups and 23/42 patients had PTH > 300 pg/ml. Arterial and valvular calcifications were found in 5/23 patients from Group 1 and 14/22 patients from Group 2 (p = 0.011). Linear regression analysis revealed sPO4 as a predictor for total calcification number, inflammatory diseases as a predictor for valvular calcifications, while sPO4 and iPTH were predictors for arterial calcifications. CONCLUSIONS: More than half the patients with Stage 5 CKD not yet on dialysis exhibited elevated PTH. Faster CRF progression and frequent arterial and valvular calcifications were seen in patients with poor phosphate control and sPO4 was selected as an independent predictor of total calcification score.

Comparison between pimonidazole binding, oxygen electrode measurements, and expression of endogenous hypoxia markers in cancer of the uterine cervix.

BACKGROUND: Although tumor hypoxia has been associated with a more aggressive phenotype and lower cure rate, there is no consensus as to the method best suited for routine measurement. Binding of the chemical hypoxia marker, pimonidazole, and expression of the endogenous hypoxia markers HIF-1alpha and CAIX were compared for their ability to detect hypoxia in tumor biopsies from 67 patients with advanced carcinoma of the cervix. METHODS: Two biopsies were taken one day after administration of pimonidazole and were analyzed for pimonidazole binding using flow cytometry or immunohistochemistry. CAIX and HIF-1alpha expression and degree of colocalization were measured in sequential antibody-stained sections. Patient subsets were examined for tumor oxygen tension using an Eppendorf electrode, S phase DNA content, or change in HIF-1alpha expression over the course of treatment. RESULTS: Approximately 6% of the tumor area stained positive for pimonidazole, HIF-1alpha, or CAIX. The CAIX positive fraction correlated with the pimonidazole positive fraction (r = 0.60). Weaker but significant correlations were observed between pimonidazole and HIF-1alpha (r = 0.31) and CAIX and HIF-1alpha (r = 0.41). Taking the extent of marker colocalization into consideration increased the confidence that all markers were identifying hypoxic regions. Over 65% of stained areas showed a high degree of colocalization with the other markers. Oxygen microelectrode measurements and S phase fraction were not correlated with the hypoxic fraction measured using the three hypoxia markers. HIF-1alpha levels tended to decrease with time after the start of therapy. CONCLUSIONS: Endogenous hypoxia marker binding shows reasonable agreement, in extent and location, with binding of pimonidazole. CAIX staining pattern is a better match to the pimonidazole staining pattern than is HIF-1alpha, and high CAIX expression in the absence (or low levels) of HIF-1alpha may indicate a different biology.

Suppression of salivary Streptococcus mutans and lactobacilli by topical caries preventive agents.

Reduction of cariogenic bacteria, especially salivary Streptococcus mutans and lactobacilli is a valuable clinical procedure that in many ways alleviates implementation of targeted caries preventive procedures in the entire population. The aim of this study was to investigate the caries preventive values of certain preventive procedures in in vivo conditions. Four groups of subjects, each with 18 children aged from 4-5 and 10-12 years (n = 72) were treated with different caries preventive agent (aminfluoride solution, Proxyt paste, chewing gum containing xylitol and fluoride and chlorhexidine solution). During a period of two months five control measurements for number of salivary Streptococcus mutans (SM) and lactobacilli (LB) were performed. At the end of the study the best result in the reduction of the bacteria was achieved by the application of Proxyt paste and daily use of chewing gum (p < 0.001). In patients treated with this preventive procedure the number of SM was reduced by 1 class and LB to < 10(4) CFU/ml saliva after two months of study. The results obtained indicate that professional teeth cleaning and use of chewing gum with xylitol and fluorides on daily basis can be very effective protocol for cariogenic bacteria reduction and in the individual caries prevention.

Influence of diet on dental caries in diabetics.

Two groups of population consisting of 84 patients suffering from diabetes (60 type I, 24 type II) and 69 nondiabetics of the same age have been examined on: oral hygiene index (OHI), frequency of daily tooth brushing, dietary habits and incidence of dental caries by registration of the decayed, missed and filled dental surfaces (DMFS-index). OHI in type I and type II diabetes was found to bee slightly worse than in nondiabetics, but not significantly (p > 0.05). In the number of daily tooth brushing there is not significant difference between diabetics and nondiabetics. All diabetics have considerably lower daily intake of total as well as simple carbohydrates than nondiabetics. The diabetics have a significantly higher daily intake of dietary fibers, calcium and phosphorus as well as the number of meals with simple carbohydrates and also DMFS-index than the nondiabetics. A significantly higher incidence (p < 0.01) of caries location was found on the buccal and labial cervical areas among patients suffering from diabetes. Explanation for this could be more frequent daily intake of low molecular carbohydrates with an improper calcium phosphorus ratio.

Double tooth.

The form of primary and permanent teeth can differ morphologically from that which is considered normal, completely or in some parts. The changes in tooth form can be hereditary or caused by some disease or trauma. Fusion is a union of one or more teeth during development. Gemination means that two separate morphological units were created by division of the tooth germ. The intention of this study was to state the prevalence of double teeth (fusion and gemination) among the persons tested, as to gender, distribution in the maxilla or mandible, and whether the anomaly occurred bilaterally or unilaterally. The results of this investigation have shown that in a total of examined 3,517 plaster models, a prevalence of double teeth was 0.2%. 57.2% of them were fusioned and 42.9% geminated.

Interspike background activity in extracelullarly recorded Purkinje neurons: spectral analysis.

The aim of this study was to investigate the spectral characteristics of Purkinje cell interspike background activity caused by the occurrence of particular action potentials or by electrically induced enhancement of cerebellar inhibitory and excitatory input drive. Spontaneously active Purkinje neurons were extracellularly recorded in anesthetized rats before and after cessation of stimulation from the inferior olive (10) or locus coeruleus (LC). After A/D conversion (30 kHz), direct spectral analysis of extracted interspike background activity was done. Our results have shown that, in contrast to simple spikes, the occurrence of complex spikes induces changes in the spectra of interspike background activity. The different spectral changes of interspike background activity induced by LC and 10 stimulation also indicated the importance of this extracellularly recorded phenomenon.

Transient neonatal diabetes mellitus in a child with paternal uniparental disomy of chromosome 6.

We report a male infant with transient neonatal diabetes mellitus (TNDM; MIM 601410), macroglossia, hypertelorism, umbilical hernia, inguinoscrotal hernia and onychomycosis. Diabetes mellitus was diagnosed 10 days after birth and resolved after 6.5 months of treatment. Genetic investigation indicated the presence of paternal uniparental disomy of chromosome 6 (UPD 6). The finding of paternal UPD 6 allows prediction of a transient, rather than permanent NDM, and no increased recurrence risk of TNDM in subsequent pregnancies. Therefore, finding of NDM should be a strong indicator for genetic testing.

[C-reactive protein concentrations during initial (empiric) treatment of neonatal sepsis]. / Koncentracije C-reaktivnog proteina tokom pocetnog lecenja sepse kod novorodjenceta.

INTRODUCTION: Neonatal septicaemia is characterized by high mortality so that intravenous antibiotics must be administered on clinical suspicion. Initial antibiotic therapy, before the results of microbiological evaluation, is based on empirical data in regard to sensitivity of prevalent bacterial strains. In the past years, aetiological causes of neonatal sepsis have been changed with an increased bacterial resistance to the usual combination of initial antibiotics. AIM: We compared changes of serum C-reactive protein (CRP) concentrations in neonates with proven neonatal sepsis in response to initial antibiotic therapy (inappropriate or appropriate). Our hypothesis was that changes of CRP levels during the first 48 hours of treatment and before microbiologic results could be useful in evaluation of effectiveness of empiric antibiotics. METHODS: Our prospective study included all neonates with suspected sepsis referred to our Intensive Care Unit from January 1992 to December 1996. Neonates received ampicillin and gentamycin/or amikacin (during the first week of life), while infants older than 7 days of life were given combination of cloxacillin and aminoglycozides. In patients with late neonatal sepsis who also had meninigitis, cloxacillin was substituted with ampicillin. Microbiological identification was performed with routine bacteriological methods. Susceptibility of isolated bacterial strains to antibiotics was performed by Kirby-Bauer disc-diffusion method. Serum concentration of CRP was measured by immunoturbidimetry (Turbox CRP, Orion Diagnostica) and CRP concentration higher than 20 mg/l was regarded as elevated. Blood sampling for CRP measurements were taken before the treatment (CRP0), and during the first (CRP1) and second (CRP2) day of empiric therapy. The interval between sampling was from 12 to 24 hours. RESULTS: A total of 1520 neonates were evaluated during this study period and 47 patients fulfilled criteria for final analysis. In 14 of 47 patients initial antibiotic treatment was inappropriate. The most frequent resistant strains was KI. pneumoniae (6) followed with St. aureus (4), E. coli (2) and Pseudomonas (2). During initial evaluation six patients had concomitant meningitis while two had concomitant septic arthritis and two necrotizing enterocolitis, respectively. Seven (50%) of 14 patients with non-adequate initial treatment died. In 33 cases of adequately treated septicaemia the course was uncomplicated and no lethal outcome was observed. In the first group of 14 patients who received inappropriate treatment serum CRP concentations (mg/L; mean and +/- SD) were: CRP0 = 107.5 +/- 65.6; CRP1 = 155.3 +/- 75.7; CRP2 = 209.1 +/- 67.0, while in 33 repeated samples of the 33 patients in the second group who received adequate treatment the following results were recorded: CRP0 = 124.0 +/- 78.1; CRP1 = 133.8 +/- 63.5; CRP2 = 94.6 +/- 46.4. Increase in serum CRP concentration in the first group during the first 48 hours of initial non-adequate therapy was significantly higher (p = 0.015, two way ANOVA) than in the second group with appropriate treatment. During the first 24 hours of treatment increase in serum concentration of CRP was registered in 12 (85.7%) of 14 measurements in patients with non-adequate therapy and in 19 (56.7%) of 33 measurements in patients with adequate therapy. In the first group during the second day of treatment, in 11 (78.6%) of 14 cases an increase in serum CRP concentration was recorded while in 3 (14.3%) cases CRP concentration decreased. In 31 (91.2%) of 34 measurements in patients with adequate treatment CRP concentration decreased during the second day of treatment and in only 3 (8.8%) cases CRP concentration increased. With an increase in serum concentration of CRP more than 10 mg/L in the second day of antibiotic treatment, probability of non-adequate antibiotic therapy (positive predictive value) was estimated to be 77.0%. Any recorded decrease of serum CRP concentration may confirm appropriate choice of antibiotics during the second day of treatment with probability of 93.3% (negative predictive value). DISCUSSION: Measurement of serum CRP concentration is useful for diagnosis of severe neonatal sepsis. In our study all isolated bacterial strains were comparable in their ability to activate systemic inflammatory response and CRP production. It is known that serial CRP measurements during neonatal sepsis are useful in making decision to cease antibiotic treatment. The highest serum CRP concentrations were detected during the first day of illness but, in some cases even with appropriate treatment, CRP peak levels due to sustained pro-inflammatory action of interleukin-6 production could be detected even 24 hours after treatment was started. Our study showed that in patients with non-adequate initial antibiotic therapy of neonatal sepsis serum CRP concentrations increase further during the second day of treatment. By contrast, the use of appropriate antibiotic therapy in the same time period followed the significant decrease of serum CRP levels in our patients. Therefore, increase of CRP level during initial treatment, especially during the second day of treatment of neonatal sepsis should be taken as indication for replacement of initial antibiotics, even before sensitivity of microbiological causes to given antibiotics is known.

[Results of intensive therapy of meningococcal sepsis in children]. / Rezultati intenzivne terapije meningokokne sepse kod dece.

The authors report on the method, course and results of treatment of patients with meningococcal septicaemia. The two most common forms of meningococcal disease are meningococcal septicaemia (MS) and meningitis. Severe MS is a fulminant form of sepsis characterized by a rapidly spreading purpuric rash, haemodynamic instability and rapid progression to shock or death. The diganosis of MS was confirmed by isolation of NM in blood or cerebrospinal fluid, and/or positive solubile bacterial antigenes. However, in some children whose symptoms were consistent in MS (temperature and extensive purpura), no bacterial or soluble antigens were detected, particularly when they had been previours antibiotic treatment. Several scoring systems have been used to predict morbidity and mortility from MS. We selected the prognostic scor developed by Malley et al. Absolute neutrophil count less than 3 x 10(3)/mm3, platelet count less than 150 x 10(3)/mm3 and poor perfusion are indicators of poor prognosis. The presence of at least two of these indicators was associated with an 82% of risk of death. We reviewed the hospital records of 36 paediatric patients with acute meningococcal infection during a 5-year period. The age of our patients ranged from 2 months to 15 years (mean 4.4 yrs). Twenty seven (70%) of 36 children had MS and 11 (40.7%) had both MS and meningitis. Based on Malley scor, 13 (48.1%) patients had at least two predictors with > 82% of risk of lethal outcome. Four children (30.7%) died. Severe MS was diagnosed in 16 (59.2%) patients, who required mechanical ventilation (16; 59.2%), or continuous inotropic support and invasive measures of circulatory parametars (15; 55.5%). Shock treatment consisted of large volumes of crystalloid or colloid infusions thad ranged from 140 to 500 ml/kg/24 hrs (mean 215 ml/kg) Our results indicate that early controlled mechanical ventilations increase safety of large volume infusion with continuous invasive monitoring and inotropic stimulation, and may contribute to a greater survival of children with severe MS.

Alterations in optic canal following resection of nerve in rats.

The authors have studied relations between bony optic canal and nerve elements resulting from lesion in the optic nerve. Experimental animals were divided in two groups: young adults and old adult rats; the non-operated sides of rat skulls were used as a control group. At the same time in control samples the changes in osseous openings resulting from ageing were studied. Four months after surgery the animals were sacrificed, their skulls macerated and morphometric measurements taken by photometric and planimetric techniques. The obtained results show no significant difference in size of bony openings between the operated and non-operated sides. A significant difference has been found in the size of optic canal between the young and the old rats, regardless of the operated side. The authors have thus come to the conclusion that the size of bony canals in adult animals depends on their age in the first place. Narrowing of bony canaliculi, observed in the course of ageing, is most probably caused by primary osseous apposition, while atrophy of nerve elements may be considered as a secondary event.

Gestational exposure to loud noise alters the development and postnatal responsiveness of humoral and cellular components of the immune system in offspring.

Gestational exposure of the female to environmental toxins can alter immune function in the offspring. We have recently shown that prenatal maternal stress, that is, stress applied to or induced in the female during pregnancy, can also alter the development of humoral immunocompetence in the offspring and their hormonal and immunologic responses to postnatal stress. This report presents data from two experiments on the effects of prenatal exposure to loud noise-prenatal sound stress (PSS)-on the development and responsiveness of in vitro and in vivo humoral and cellular immune function in the offspring. Pregnant rats were exposed daily from Day 15 to Day 21 of gestation to an inescapable loud noise (an 85- to 90-decibel fire alarm bell) delivered randomly for 1 hr. In developing offspring, PSS produced age-dependent and mitogen-specific alterations in lymphoproliferative activity and reduced immunoglobulin G levels at Postnatal Day 21. Antibody titers to herpes simplex virus type 1 were also reduced. Exposure to loud noise before or after infection produced an additional reduction in titers in these offspring. Arthus skin reaction (AR) to old tuberculin was reduced by PSS. Combined prenatal/postnatal sound stress further reduced this response and the AR to bovine serum albumin (BSA). Delayed hypersensitivity reaction to BSA was reduced in PSS offspring; postnatal sound stress enhanced the reaction to both antigens, but only in males. Antibody titers to BSA were increased by PSS; adjuvant-induced inflammation was attenuated by postnatal sound stress. These data suggest that in utero exposure to loud noise, which can occur in the workplace, is toxic to the developing immune system.