Accelerated Caco-2 cell permeability model for drug discovery.

INTRODUCTION: By culturing Caco-2 cells according to a new and optimized protocol, it has been possible to accelerate the cell culture process in such a way that the cells can be used for experiments after only 6 days. The accelerated Caco-2 model has been compared to the traditional model (requiring 21-25 days of culture) in terms of tightness of the junctions, ability to rank chemical compounds for apparent permeability, active efflux and to discriminate P-gp substrates. METHODS AND RESULTS: In the new protocol, Caco-2 cells were cultured with the classical Caco-2 medium supplemented with puromycin. The initial cell seeding density was increased two times compared to the traditional procedure and the presence of a low concentration of puromycin in the culture medium reduced the Caco-2 permeability of mannitol. Bi-directional studies were performed with known P-gp substrates (rhodamine 123, digoxin and saquinavir) and with a total of 20 marketed drugs covering a wide range of physicochemical characteristics and therapeutic indications. Strong correlations were obtained between the apparent permeability in absorptive (Papp A→B) or secretory (Papp B→A) of the drugs in the accelerated model and in the traditional models and comparable efflux ratios were observed in the two studied models. DISCUSSION: The new protocol reduces costs for screening and leads to higher throughput compared to traditional Caco-2 cell models. This accelerated model provides short time-feedback to the drug design during the early stage of drug discovery.

Transport screening of drug cocktails through an in vitro blood-brain barrier: is it a good strategy for increasing the throughput of the discovery pipeline?

PURPOSE: The objective of the current study was to investigate whether blood-brain barrier (BBB) permeability studies in vitro could be accelerated by running several compounds together in the same experiment. METHODS: To address this question, we compared the transport of six compounds run separately with the results of the same compounds run together (cocktails). RESULTS: The study clearly demonstrated that the outcome of the experiments were totally different depending on the strategy used. Furthermore, the study highlights the importance of having the resistance to drug transport offered by filters without cells under control, as the filter membrane itself can be the rate-limiting step for some compounds; in addition, there is always a potential risk of interactions between molecules in cocktails as well as drug-drug interaction at the level of BBB transporters. In this study, the presence of several P-glycoprotein substrates in the drug cocktail was found to cause breakdown of the BBB. CONCLUSIONS: The results demonstrate that unless a strategy that involves running several compounds in the same experiment is properly validated, the results are of little predictive value.

Diseases of the foreskin, penis, and urethra.

Diseases of the foreskin, penis, and urethra are uncommon conditions that constitute a small percentage of emergency department visits. However, it is important that the emergency physician recognize and treat these conditions and understand which conditions require urologic consultation. Rapid and proper treatment in the emergency department can alleviate patient discomfort, increase patient satisfaction, and prevent future morbidity.

Physicochemical characterisation of a drug-containing phospholipid-stabilised o/w emulsion for intravenous administration.

Clomethiazole (CMZ) was used as a model drug to be incorporated into an emulsion vehicle. The effects of drug concentration and number of homogenisation steps were evaluated using multiple linear regression. The droplet size, measured as a z-average diameter by photon correlation spectroscopy (PCS), was found to be between 60 and 260 nm in the investigated range of CMZ concentrations, highly dependent on the concentration, but more weakly so on the number of homogenisation steps. Slow-scanning high-sensitivity differential scanning calorimetry (DSC) measurements showed that CMZ depresses the phospholipid chain melting temperature in the emulsion system, whereas (13)C nuclear magnetic resonance (NMR) experiments suggested that the CMZ molecules are to a large extent located in the surface region of the emulsion droplets. This interpretation is compatible with results from NMR self-diffusion measurements, which showed that most of the CMZ molecules are rapidly exchanged between emulsion droplets and the aqueous surrounding. It can be concluded that the surface-active drug CMZ has a significant influence on the characteristics of phospholipid-stabilised emulsions through its ability to interact with the phospholipid interface. Thus, the results underline the importance of characterising drug-lipid interactions for the development of lipid-based formulations.

A comparison of elevated blood lead levels among children living in foster care, their siblings, and the general population.

OBJECTIVES: To assess the prevalence of elevated blood lead levels (EBLLs) among children before and after foster care placement, and to compare the prevalence of EBLLs among children in foster care with that of their siblings and the general population. METHODS: We conducted a retrospective cohort study using administrative databases from the Philadelphia Department of Human Services and the Birth Certificate Registry and the Childhood Lead Poisoning Prevention Program at the Philadelphia Department of Public Health. Logistic regression analyses were performed to control for confounding variables, including age, race, gender, and the year, seasonal timing, and source (capillary vs venous) of test. RESULTS: From June 1992 to May 1997, there were 1824 children in foster care with available blood lead results in the Childhood Lead Poisoning Prevention Program database. Of these, 519 (28%) had initial lead screening before foster care placement and 654 (36%) after placement. There were 821 siblings and 73 608 children in the general population with available blood lead results. Before entering foster care, children were nearly twice as likely to have EBLLs as their siblings (adjusted odds ratio [OR] = 1.7; 95% confidence interval [CI] = 1.4, 2.0), those in placement (adjusted OR = 1.9; 95% CI = 1.6, 2.2), and the general population (adjusted OR = 1.7; 95% CI = 1.5, 2.0). At the highest point prevalence, 50% of children before placement had lead levels >/=20 microg/dL, and nearly 90% had levels >/=10 microg/dL. For all age categories, siblings of children in foster care placement had a higher prevalence of EBLLs than did the general population. After placement, children in foster care were nearly half as likely as the other groups to have EBLLs. CONCLUSIONS: Our findings suggest that children are at high risk for lead poisoning before entering foster care and that placement in foster care may have a beneficial effect on lead exposure. Children before foster care placement are nearly twice as likely to have EBLLs compared with children in foster care placement, the general population, and their siblings. Furthermore, siblings of children in foster care are at high risk for lead poisoning. Children receiving social services in their own homes and children suffering from abuse and neglect should be actively screened for lead poisoning. Greater efforts at preventing lead poisoning among these children must be made.

In vitro model for evaluating drug transport across the blood-brain barrier.

The passage of substances across the blood-brain barrier (BBB) is regulated in the cerebral capillaries, which possess certain distinct different morphological and enzymatic properties compared with the capillaries of other organs. Investigations of the functional characteristics of brain capillaries have been facilitated by the use of cultured brain endothelial cells, but in most studies some characteristics of the in vivo BBB are lost. To provide an in vitro system for studying brain capillary functions, we have developed a process of coculture that closely mimics the in vivo situation by culturing brain capillary endothelial cells on one side of a filter and astrocytes on the other. In order to assess the drug transport across the blood-brain barrier, we compared the extraction ratios in vivo to the permeability of the in vitro model. The in vivo and the in vitro values showed a strong correlation. The relative ease with which such cocultures can be produced in large quantities facilitates the screening of new centrally active drugs. This model provides an easier, reproducible and mass-production method to study the blood-brain barrier in vitro.

A comparative profile of clinical pharmacy activities for pharmacotherapists and residents in a university hospital.

Due to increasing financial pressure on maintaining and improving pharmacy services, there is a need for productivity data and time distribution among different activities for pharmacy practice residents and their faculty preceptors (pharmacotherapists). This study measured the clinical productivity of 13 residents and 25 pharmacotherapists for a 14-day period. The study identified the average time (minutes) and frequency spent each day on categories of activities, which included direct patient care, chart use, rounds, professional encounter, teaching, research, and administration. Results showed that the productivity profiles for residents and pharmacotherapists were comparable. Findings were utilized to defend the educational programs for the department and as a baseline for periodic monitoring of the productivity of these programs.

Systemic and pulmonary haemodynamic effects of intravenous infusion of non-ionic isoosmolar dimeric contrast media. An investigation in the pig of two ratio 6 contrast media.

The systemic and pulmonary haemodynamic effects of i.v. infusion (1 ml/s) of high doses (4 ml/kg) of 2 non-ionic, isoosmolar dimeric contrast media (CM) were investigated in 17 female pigs. The 2 CM were iodixanol and iotrolan. Both CM induced a significant increase of the following parameters: mean arterial, mean right atrial, mean pulmonary arterial, mean pulmonary arterial occlusion pressure, cardiac output, stroke volume, and diuresis. The plasma concentration of atrial natriuretic peptide was significantly increased following infusion of the 2 CM. A significant decrease was seen in the systemic and pulmonary vascular resistance.

Analysis of a recombination hotspot for gene conversion occurring at the HIS2 gene of Saccharomyces cerevisiae.

The properties of gene conversion as measured in fungi that generate asci containing all the products of meiosis imply that meiotic recombination initiates at specific sites. The HIS2 gene of Saccharomyces cerevisiae displays a high frequency of gene conversion, indicating that it is a recombination hotspot. The HIS2 gene was cloned and sequenced, and the cloned DNA was used to make several different types of alterations in the yeast chromosome by transformation; these alterations were used to determine the location of the sequences necessary for the high levels of meiotic conversion observed at HIS2. Previous work indicated that the gene conversion polarity gradient is high at the 3' end of the gene, and that the promoter of the gene is not necessary for the high frequency of conversion observed. Data presented here suggest that at least some of the sequences necessary for high levels of conversion at HIS2 are located over 700 bp downstream of the end of the coding region, extend over (at least) several hundred base pairs, and may be quite complex, perhaps involving chromatin structure. Additional data indicate that multiple single base heterologies within a 1-kb interval contribute little to the frequency of gene conversion. This contrasts with other reports about the role of heterologies at the MAT locus.

A meiotic gene conversion gradient opposite to the direction of transcription.

Genetic recombination involves classical crossing-over and gene conversion (aberrant segregation). In fungi that produce an ascus containing four spores, a gene conversion event is manifested as 3:1 or 1:3 (or more rarely 4:0 or 0:4) segregations, in contrast to the normal mendelian 2:2 segregation. Polarity is one of the properties of gene conversion; in almost all cases the frequency of conversion exhibits a gradient across the gene monitored. The frequency of conversion is usually independent of the specific allele used as a marker, but dependent on its location. An interpretation of conversion polarity is that it is caused by the existence of specific initiation sites for meiotic recombination, located at the high end of the polarity gradient. Here we show that the polarity gradient for the HIS2 gene of Saccharomyces cerevisiae is high at the 3' end of the gene, implying that the promoter of HIS2 is not the initiation site.

Primary care referral management: a marketing strategy for hospitals.

With increasing competition among hospitals, primary care referral development and management programs offer an opportunity for hospitals to increase their admissions. Such programs require careful development, the commitment of the hospital staff to the strategy, an integration of hospital activities, and an understanding of medical practice management.

Studies on renal damage from percutaneous nephrolitholapaxy.

To study in detail the effects of percutaneous nephrolitholapaxy on renal function, a consecutive series of 11 patients were investigated preoperatively by excretory urography, gamma camera renography for determination of individual renal function and computerized tomography of the kidneys. Postoperatively, gamma camera examination, computerized tomography, antegrade nephrostography, renal angiography and excretory urography were performed. With 2 exceptions, percutaneous nephrostomy, dilation of the tract and stone removal were done in 1 stage with the patient under continuous epidural anesthesia. Nephrostomy tract dilation was done with an Olbert type balloon catheter or Alken metallic dilators. Thickening of Gerota's fascia was demonstrated by computerized tomography in most cases, and small to moderate perirenal hematomas were found in several. At gamma camera examination decrease of renal function was noted regularly on postoperative day 1 and returned to near initial levels 2 weeks postoperatively in most cases. Angiography in 10 patients showed discrete parenchymal scarring in some and a peripheral arteriovenous fistula in 1. We conclude that percutaneous renal stone surgery usually is tolerated well by the kidney.

Dilatation of experimental renal artery stenosis by balloon catheter.

An experimental model has been developed in the pig in order to evaluate the effect of percutaneous transluminal angioplasty on fibrous renal artery stenosis. Stenoses were induced by bilateral subtotal ligation with chromic catgut. Six weeks following ligation unilateral transluminal dilatation was performed in 9 pigs using Grüntzig balloon catheters. In only one case a serious complication occurred with rupture of the renal artery. Seven pigs with dilated stenoses were re-examined by angiography 8 to 10 weeks after dilatation. In 6 cases the stenoses were practically completely eliminated while in one cases a re-stenosis developed. Microscopy of the dilated arteries revealed several small and large ruptures of th internal elastic membrane.