Characterization and evaluation of solid self-microemulsifying drug delivery systems with porous carriers as systems for improved carbamazepine release

The purpose of this study was to investigate solid self-microemulsifying drug delivery system (SSMEDDS), as potential delivery system for poorly water soluble drug carbamazepine (CBZ). Self-microemulsifying drug delivery system (SMEDDS) was formulated using the surfactant polyoxyethylene 20 sorbitan monooleate [Polysorbate 80] (S), the cosurfactant PEG-40 hydrogenated castor oil [Cremophor(?) RH40] (C) and the oil caprylic/capric triglycerides [Mygliol(?) 812] (O). Four different adsorbents with high specific surface area were used: Neusilin(?) UFL2, Neusilin(?) FL2 (magnesium aluminometasilicate), Sylysia(?) 320 and Sylysia(?) 350 (porous silica). Microemulsion area at the surfactant to cosurfactant ratio (K(m)) 1:1 was evaluated and for further investigation SMEDDS with SC/O ratio 8:2 was selected. Solubilization capacity of selected SMEDDS for CBZ was 33.771±0.041mg/ml. Rheological measurements of unloaded and CBZ-loaded SMEDDS at water content varied from 10 to 60% (w/w) were conducted. It has been found that CBZ has great influence on rheological behaviour of investigated system upon water dilution. Photon correlation spectroscopy has shown the ability of CBZ-loaded SMEDDS to produce microemulsion droplet size. SSMEDDS improved release rate of CBZ, but the type of adsorbent significantly affects release rate of CBZ. For SSMEDDS with different magnesium aluminometasilicate adsorbents, release rate of CBZ decreased with increasing specific surface area due to entrapment of liquid SMEDDS inside the pores and its gradual exposure to dissolution medium. With porous silica adsorbents no difference in release rate was found in comparison to physical mixtures. In physical mixtures at 12.5% (w/w) CBZ content, presence of amorphous CBZ led to high dissolution rate.     

Absolute quantitation of DNA methylation of 28 candidate genes in prostate cancer using pyrosequencing

Aberrant DNA methylation plays a pivotal role in carcinogenesis and its mapping is likely to provide biomarkers for improved diagnostic and risk assessment in prostate cancer (PCa). We quantified and compared absolute methylation levels among 28 candidate genes in 48 PCa and 29 benign prostate hyperplasia (BPH) samples using the pyrosequencing (PSQ) method to identify genes with diagnostic and prognostic potential. RARB, HIN1, BCL2, GSTP1, CCND2, EGFR5, APC, RASSF1A, MDR1, NKX2-5, CDH13, DPYS, PTGS2, EDNRB, MAL, PDLIM4, HLAa, ESR1 and TIG1 were highly methylated in PCa compared to BPH (p < 0.001), while SERPINB5, CDH1, TWIST1, DAPK1, THRB, MCAM, SLIT2, CDKN2a and SFN were not. RARB methylation above 21% completely distinguished PCa Separation based on methylation level of SFN, SLIT2 and SERPINB5 distinguished low and high Gleason score cancers, e.g. SFN and SERPINB5 together correctly classified 81% and 77% of high and low Gleason score cancers respectively. Several genes including CDH1 previously reported as methylation markers in PCa were not confirmed in our study. Increasing age was positively associated with gene methylation (p < 0.0001).Accurate quantitative measurement of gene methylation in PCa appears promising and further validation of genes like RARB, HIN1, BCL2, APC and GSTP1 is warranted for diagnostic potential and SFN, SLIT2 and SERPINB5 for prognostic potential.     

Tolerance liability of diazepam is dependent on the dose used for protracted treatment

BackgroundBehavioral effects of benzodiazepines following repeated exposure vary according to the intrinsic efficacy of the benzodiazepine studied, treatment schedule and the behavioral parameters evaluated.MethodsWe applied the behavioral paradigms of spontaneous locomotor activity, elevated plus maze and grip strength to investigate the sedative, anxiolytic and myorelaxant effect of acute challenge with 2 mg/kg diazepam administered after 14 days of protracted treatment with 0.5, 2 or 10 mg/kg of diazepam. In addition, we studied the effects of everyday handling and intraperito-neal (ip) administration on animal behavior.ResultsTolerance to the sedative effect of 2 mg/kg diazepam ensued after 14 days of protracted treatment with 2 and 10 mg/kg of diazepam. In contrast, treatment with the lowest dose (0.5 mg/kg) of diazepam resulted in potentiation of the sedative effect of acute challenge with 2 mg/kg diazepam thus confounding the detection of the anxiolytic effect of diazepam. A sensitization-like response to the anxiolytic action of 2 mg/kg diazepam was seen after protracted treatment with the intermediate dose (2 mg/kg); however, anxiolytic effect was absent after protracted administration of the highest dose. Partial tolerance to the muscle relaxant effect of 2 mg/kg diazepam ensued after protracted treatment with diazepam regardless of the dose. Daily handling or ip administration did not alter the behavioral response to acute challenge with 2 mg/kg diazepam in all the three behavioral paradigms studied.ConclusionThe presented results showed that behavioral effects of acute challenge with diazepam were differently affected by the dose administered during protracted treatment.     

Osteopontin expression correlates with nuclear factor-κB activation and apoptosis downregulation in clear cell renal cell carcinoma

Osteopontin (OPN) is a phosphoglycoprotein implicated in tumorigenesis and tumor cell metastasis. Apoptosis inhibition is one of the mechanisms that contribute to development and progression of cancer, and might be initiated by OPN interaction with tumor cells. The aim of this study was to analyze the relation between OPN and nuclear factor-kappa B (NF-κB) expression in clear cell renal cell carcinoma (CCRCC), as well as their relation to apoptotic activity of tumor cells.Expression of OPN protein and p65 NF-κB subunit was analyzed immunohistochemically in 87 CCRCC samples, and compared mutually and with apoptotic index. Expression of OPN mRNA was analyzed using quantitative real-time PCR and compared with OPN and NF-κB protein expression in 22 CCRCC samples.Statistical analysis showed an association of p65 NF-κB with OPN mRNA (p = 0.015) and protein (p < 0.001). Also, we found an inverse relationship of OPN with NF-κB protein expression and apoptotic activity of tumor cells (p = 0.006 and p = 0.022, respectively). Our results indicate that p65 NF-κB signaling pathway may be involved in OPN-mediated CCRCC progression, partly by protecting tumor cells from apoptosis. Therefore, both molecules can constitute potential targets for therapeutic intervention in CCRCC.     

Stress among Croatian physicians: comparison between physicians working in emergency medical service and health centers: pilot study

Aim

To determine the sources of stress, its intensity, frequency, and psychophysical and behavioral reactions in physicians working in emergency medical service and those working in health centers.

Methods

To a convenience sample of primary care physicians employed in emergency medical service (n = 79) and health centers (n = 81), we administered the list of demographic questions, Scale of Sources of Stress, Scale of Intentions of Leaving the Job, and Scale of Psychosomatic Symptoms.

Results

Emergency medicine physicians experienced significantly more intense and more frequent uncontrollable working situations, conflict between work and family roles, and unfavorable relationships with coworkers than physicians working in health centers. They were also more likely to leave the job during the next few years and/or change jobs within the profession (scores 2.2 ± 0.9 vs 1.7 ± 0.9 out of maximum 5.0, F = 12.2, P = 0.001) and they had a poorer physical health status (scores 1.8 ± 0.5 vs 1.7 ± 0.5 out of maximum 4.0, F = 5.3, P = 0.023).

Conclusion

Physicians working in emergency medical service experience more stress in almost all aspects of their work than physicians working in health centers. They also have a stronger intention of leaving the job, which decreases with years of experience.It has been shown that health workers are highly susceptible to stress at work and experience more negative outcomes of stress than other professions (1-8). For example, a survey conducted in Irish hospitals indicated that work stress caused dissatisfaction in 79% of physicians, 56% evaluated their job as stressful or extremely stressful, and as many as 68% considered leaving the profession, half of them very seriously (9). Work stress in physicians is associated with an array of other negative outcomes: adverse psychological well-being (6), job burnout (10), significantly larger number of suicide attempts (11,12), alcohol dependence, and other psychosocial problems (11). Stress in physicians affects not only their private lives and health but also the quality of medical care that they provide, patient’s satisfaction with the physician, and patient’s adherence to treatment (13).Some of the most important sources of stress for general practitioners and hospital physicians of different specializations were identified as intrinsic factors of work, administration, stressors related to financial opportunities, contact with patients, relationships with coworkers, organizational structure and climate, and interference of private and work life (4-6). One of the most stressful areas of medicine is emergency medicine, in which physicians and other medical personnel are frequently exposed to unexpected and serious medical conditions, trauma, and life or death situations. Important sources of stress specific to work in emergency medicine were found to be time pressure and the need to make critical decisions (8), with numerous negative effects of stress, particularly the intention of leaving the job and deteriorated psychological well-being (11,14-16).The activities of emergency medical service in Croatia include the treatment of severe acute and urgent conditions at the site of the incident, at the dispensary in which the patient arrived, and during transport to the nearest hospital or other health care institution (17,18).The aim of this pilot case study was to identify specific sources of stress in emergency medicine physicians in Croatia, since we assumed that, due to the unpredictable nature of their job, they have different perception of sources of stress, health status, and intent of leaving the job than physicians working in health care centers.     

Muscle strength and bone density in patients with different rheumatic conditions: cross-sectional study

Aim

To explore the relationship between muscle strength and bone density in patients with different rheumatic diseases and to examine whether inflammatory arthritis was more harmful for muscle strength and bone loss than degenerative joint diseases.

Methods

The study included 361 men and women with a mean ± standard deviation age of 60.5 ± 11.4 years and different rheumatic conditions: regional syndromes, osteoarthritis of the hands, shoulders, knees, and hips, and inflammatory arthritis. Maximum voluntary back strength was measured by isometric dynamometry. Bone mineral density (BMD; g/cm²) of the lumbar spine, femoral neck, and distal radius was measured by dual-energy x-ray absorptiometry. Anthropometry and lifestyle characteristics were also assessed.

Results

Back strength was lowest in patients with hand and shoulder osteoarthritis (20.0 ± 17.9 kg), followed by patients with inflammatory arthritis (24.8 ± 19.2 kg). Patients with inflammatory arthritis had the lowest BMD at the mid-radius (0.650 ± 0.115 g/cm²) and femoral neck (0.873 ± 0.137 g/cm²), while patients with hand and shoulder osteoarthritis had the lowest BMD at the mid-radius (0.660 ± 0.101). In both sexes, muscle strength was significantly lower in patients who had lower BMD (T score<-1.0). Multiple regression analysis identified significant predictors of back strength to be spine BMD (P = 0.024) and body mass index (P = 0.004) in men and femoral neck BMD in women (P = 0.004).

Conclusion

Muscle strength decline may be connected to bone loss in patients with rheumatic conditions, especially those with inflammatory joint diseases.There is a concomitant decline in muscle strength of the upper and lower limbs and bone density after the fifth decade of the life (1,2). Impaired muscle function is a common consequence in patients with rheumatic diseases, especially those with inflammatory joint diseases. Muscle strength may also be significantly reduced around joints affected with osteoarthritis. Several studies showed greatly reduced isokinetic strength in patients with rheumatoid arthritis (3-5) and patients with knee osteoarthritis (6).It is also known that muscle strengthening can yield a bone-building effect (7). Exercises with greater loading and higher impact activities produce the greatest skeletal benefit (8). Increased muscle weakness can also compound the problem of low bone density by increasing the risk of falls and fracture. A positive correlation between muscle strength and bone density has been shown in several studies (9-17). Some of them demonstrated the association only in postmenopausal women (12,17) but not in men (9,13), while other found a site-specific correlation between muscle strength and bone mineral density (BMD) (4,12). However, several studies did not find a correlation between any measures of muscle strength and BMD (18,19). With such contradictory reports, it is difficult to make clinically relevant conclusions about the relationship between muscle strength and bone mass, although this may be one of the key factors that affect the rehabilitation outcome.The aim of the study was to assess the differences in muscle strength and bone density between patients with different rheumatic conditions. Since muscle strength is an important determinant of bone density, we explored whether the age-related decline in bone density and muscle strength was more pronounced in patients with inflammatory arthritis than in those with degenerative joint diseases.     

Application of microsatellite loci on the chromosome X for rapid prenatal detection of the chromosome X numerical abnormalities

Aim

To determine the value of short-tandem repeat markers on the chromosome X (X-STR) for prenatal diagnostics of the chromosome X numerical disorders.

Methods

We investigated the genetic variability of 5 X-markers (DXS9895, DXS6810, DXS6803, GATA172D05, and HPRTB) in 183 healthy Croatian individuals (90 men and 93 women). We also tested 13 patients with X chromosome disorders (Turner syndrome – 6 cases; Klinefelter syndrome – 5 cases, and Triple X syndrome – 2 cases). The analysis was performed using polymerase chain reaction amplification with specific primers and electrophoresis on a polyacrylamide gel. The study was performed in 2010.

Results

Our sample showed no significant differences in allelic frequencies of the investigated X-markers from other European populations. A set of 5 X-STR markers was sufficiently informative for a successful determination of the chromosome X numerical abnormalities.

Conclusion

Since no false positive or negative results were observed, diagnostic value of the investigated X-STR loci for prenatal detection of chromosome X numerical disorders was confirmed. Our study represents an important step toward an improved prenatal diagnostics in Croatia.The analysis of short tandem repeat (STR) markers using polymerase chain reaction (PCR) method has become a widely applied technique in forensic individual identification, rapid detection of chromosome aneuploidies in prenatal and postnatal diagnosis, as well as paternity testing (1-5). Until now, a large number of autosomal and Y-chromosomal markers has been forensically evaluated and used for various purposes. Although X-chromosomal markers have been increasingly applied in both forensic and medical field, their role has not been as extensively investigated as that of autosomal and Y-chromosomal markers. Several investigations have documented the accuracy of fluorescent PCR using STR loci for the rapid prenatal diagnoses of numerical disorders affecting the chromosomes 21, 18, and 13 (6-8). However, the low polymorphism of the most chromosome X and Y markers has hampered the use of the PCR-STR approach for the detection of numerical disorders of sex chromosomes, such as the Turner (45, X) or Klinefelter (47, XXY) syndromes (1,7). A few years ago, a group of authors reported on the application of PCR-STR method in the detection of X-chromosome abnormalities (9,10). One of the biggest challenges is the Turner syndrome, in which a sufficient number of STR loci has to be included to be sure that the individual has only a single X chromosome. The aims of the present study were to investigate the diagnostic informativeness of 5 X-linked STR markers: DXS9895 (Xpter-Xp22.1), GATA172D05 (Xq26.1), DXS6810 (Xq12-Xq21.33), DXS6803 (Xq24-Xq27), and HPRTB (Xq27.3) in the Croatian population and to evaluate the diagnostic value of these 5 loci.     

Urban-rural differences in a population-based breast cancer screening program in Croatia

Aim

To investigate urban-rural differences in the distribution of risk factors for breast cancer.

Methods

We analyzed the data from the first round of the “Mamma” population based-screening program conducted in Croatia between 2007 and 2009 and self-reported questionnaire results for 924 patients with histologically verified breast cancer. Reproductive and anthropometric characteristics, family history of breast cancer, history of breast disease, and prior breast screening history were compared between participants from the city of Zagreb (n = 270) and participants from 13 counties with more than 50% of rural inhabitants (n = 654).

Results

The screen-detected breast cancer rate was 4.5 per 1000 mammographies in rural counties and 4.6 in the city of Zagreb, while the participation rate was 61% in rural counties and 59% in Zagreb. Women from Zagreb had significantly more characteristics associated with an increased risk of breast cancer (P < 0.001 in all cases): no pregnancies (15% vs 7%), late age of first pregnancy (≥30 years) (10% vs 4%), and the most recent mammogram conducted 2-3 years ago (32% vs 14%). Women from rural counties were more often obese (41% vs 28%) and had early age of first live birth (<20 years) (20% vs 7%, P < 0.001 for both).

Conclusion

Identification of rural-urban differences in mammography use and their causes at the population level can be useful in designing and implementing interventions targeted at the reduction of inequalities and modifiable risk factors.Significant differences in breast cancer frequency have been identified in different socioeconomic groups, ethnic groups, and between urban and rural populations (1,2). Living in rural areas may be associated with lower access to health care and mammography screening (3), as well as with late-stage diagnosis (4). This often means that patients need to travel great distances to receive care (5). Blair et al found that people in rural and urban areas were diagnosed with breast cancer at similar stages of the disease, although those from rural communities lacked basic cancer information because they did not have access to cancer education programs offered in urban areas (6). Robbins et al explained the higher breast cancer incidence in the San Francisco Bay Area than in other regions by known risk factors: parity, age at first full-term pregnancy, breast-feeding, age at menarche, and age at menopause (7). In Croatia, Polašek et al found that in a period without a national cancer screening program access to health care was the strongest cancer screening utilization predictor in adult rural population (8).Risk factors for breast cancer are mostly those related to the reproductive life of women (9,10): menarche, nulliparity or late age at first birth, late menopause, as well as hormonal factors, be they endogenous or exogenous (eg, term use of oral contraceptives or menopausal hormonal replacement). Other risk factors related to hormonal status include obesity and a diet characterized by a high caloric intake, low intake of fruits and vegetables, and lack of physical activity (11). Radiation, in particular during breast development, was also found to be a risk factor (12), while the role of contaminants, such as xenoestrogens and certain pesticides, remains controversial. Four- to 5-fold risk of developing breast cancer was associated with epithelial proliferative lesions, particularly atypical ductal or lobular hyperplasia (11).In Croatia, breast cancer is the leading cancer among women, amounting to 27% of new female cancer cases; moreover, the incidence rate in 2007 was 17% higher than in the previous year (13). In 2007, cancer incidence by county and age-standardized rates per 100 000 women varied considerably: from 273.1 (Šibensko-kninska county) to 437.7 (the city of Zagreb), but the prevalence of breast cancer risk factors remains unknown. A government-funded mammography screening program was established in October 2006 and has since been implemented in 21 counties, including the city of Zagreb (14).Population-based screening for breast cancer is conducted through mammographic examination of all women of a specified age at prescribed time intervals. The implementation of population-based screening requires technical resources and trained personnel for double reading of mammograms, as well as a major media campaign (15).Within a more extensive study of breast cancer risk factors, this study investigated urban-rural differences in reproductive, anthropometric, and family history of breast cancer and personal history of breast disease among women aged 50-69 from 13 rural counties and the city of Zagreb who participated in the first round of population-based mammography screening in Croatia.     

Forensic DNA databases in Western Balkan region: retrospectives, perspectives, and initiatives

The European Network of Forensic Science Institutes (ENFSI) recommended the establishment of forensic DNA databases and specific implementation and management legislations for all EU/ENFSI members. Therefore, forensic institutions from Bosnia and Herzegovina, Serbia, Montenegro, and Macedonia launched a wide set of activities to support these recommendations. To assess the current state, a regional expert team completed detailed screening and investigation of the existing forensic DNA data repositories and associated legislation in these countries. The scope also included relevant concurrent projects and a wide spectrum of different activities in relation to forensics DNA use. The state of forensic DNA analysis was also determined in the neighboring Slovenia and Croatia, which already have functional national DNA databases. There is a need for a 'regional supplement' to the current documentation and standards pertaining to forensic application of DNA databases, which should include regional-specific preliminary aims and recommendations.