Evaluation of KCB-328, a new IKr blocking antiarrhythmic agent in pacing induced canine atrial fibrillation.

HYPOTHESIS: KCB-328 is a new potassium channel blocker, which prolongs action potential duration with exhibition of minimal reverse use dependence. We tested the efficacy and proarrhythmic potential of KCB-328, dofetilide and propafenone in the pacing induced canine model of atrial fibrillation (AF). METHODS: Mongrel dogs in complete heart block were paced for 1-6 weeks to produce AF, and given KCB-328 or dofetilide. A subset then received propafenone 14+/-3 days after testing the first drug. RESULTS: KCB-328 prolonged right and left atrial (RA and LA) activation times and AF cycle length (CL), terminating AF in 3 of 6 dogs. RA effective refractory period (ERP) and ventricular ERP and QT interval were prolonged. Dofetilide terminated AF in 1/6 dogs, and increased AF CL and ventricular ERP and QT interval. Dofetilide's reverse use dependency on the QT interval was greater than KCB-328. Propafenone prolonged RA and LA activation times and AF CL and terminated AF in 8 of 9 dogs. One death occurred with dofetilide, none with KCB-328 or propafenone. CONCLUSION: The spectrum of effect of the three drugs differed significantly: propafenone showed the greatest success in AF termination, and both propafenone and KCB-328 appeared less proarrhythmic than dofetilide in this model.     

Preliminary Studies on the Absorption,Distribution, Metabolism,and Excretion of THIP in Animal and Man using 14C-labelled Compound

Abstract: Distribution of radioactivity in rats, serum levels in human volunteers and rats and elimination of radioactivity in volunteers, rats, and mice following oral administration of ¹⁴C-labelled THIP have been investigated. Peak values of radioactivity in the organs and in serum were seen half an hour after administration, indicating a rapid absorption. Highest concentrations of radioactivity were found in the kidneys, but radioactivity was seen in all investigated tissues including the brain. The radioactivity was mainly excreted with urine (84–93%). Thin-layer chromatography of urine from volunteers, rats, and mice showed that most of the excreted radioactivity corresponds to unchanged THIP. Three metabolites were found in urine from rats each in amounts of 2–7% of the total dose given. Two of these metabolites were also found in urine from the volunteers in amounts of 30–35% and <2%, respectively, and in urine from mice in amounts of 21% and 6% of the total dose, respectively. No radioactivity corresponding to unchanged THIP was found in faeces indicating complete absorption of THIP following oral administration. One of the metabolites, the main one in man and mouse, seemed to be a glucuronic acid conjugate of THIP, but the chemical structure of the metabolites has not yet been established.     

Influence of dietary fat on factors in serum that regulate thyroid cell metabolism

The type of dietary fat affected the production of cAMP by cultured thyroid cells incubated with mouse and rat sera. Greater amounts of cAMP were produced with serum from mice fed 30% rac-1(3)-palmitoyl glycerol and 4% safflower oil (PG + SO) than with serum from mice fed 30% rac-1(3)-palmitoyl glycerol (PG). The serum from mice fed PG + SO gave a response similar to that with calf serum. Sera were separated into lipoprotein and aqueous fractions by centrifugation. A combination of both the lipoprotein and aqueous fractions of serum from mice fed PG + SO was required for the increased response. Proteolysis of the aqueous fraction of the serum from either mice fed PG or rats fed 30% hydrogenated coconut oil (HCO) reduced the amount of cAMP produced by the thyroid cells. However, the same effect was not seen with aqueous fractions of calf serum or serum from mice fed PG + SO or rats fed HCO plus 5% corn oil. These findings suggest that there are at least three factors in serum capable of regulating thyroid cell metabolism that are controlled by the type of fat fed the animal.     

Selection of Filters and Evaluation In Vitro of the Selective Dual Filtration Artificial Kidney, SEDUFARK

Transport properties of different artificial kidney membranes have been studied employing substances with molecular weights ranging from 60 daltons (urea) to 62,000 daltons (dextran). The results from these studies were used to select filters for the SElective DUal Filtration ARtificial Kidney, SEDUFARK.
Selection criteria for filters to be used in long-term clinical trials with SEDUFARK were based on biological tests showing toxicity of large molecular weight substances in uremic blood plasma and clinical advantages of returning middle molecular weight substances to the patient. The filters selected were used for evaluation in vitro of the complete SEDUFARK system. The results confirmed a theoretical analysis of this system, demonstrating that selective removal of substances within given molecular weight ranges from uremic blood plasma is feasible with a combination of commercial filters. The efficiency of the system, however, is limited due to the "cut-off' characteristics and hydraulic permeabilities of membrane types currently available.     

Iso-Immune Neonatal Purpura Caused by Anti-PlGrLyB1

A case of iso-immune neonatal thrombocytopenic purpura caused by a maternal, complement fixing, platelet iso-antibody, anti-PlGrLy^B1, is reported. The problems concerning the diagnosis of this disease are discussed. Although the PlGrLy^B1-antigen is known to be present on both platelets and leukocytes, and though accordingly the mother's blood contained strong leukocyte agglutinins no leukocytopenia was observed in the patient. The reasons for this observation are discussed. No platelet antibody could be demonstrated in the patient's blood, but the maternal antibody belonged to the γG globulins and was thus placenta-permeable. Evidence is presented of a gene dosage effect of the PlGrLy^B1-gene among family members.     

Age-associated changes in electrophysiologic remodeling: a potential contributor to initiation of atrial fibrillation

OBJECTIVE: Although the incidence of atrial fibrillation (AF) increases with age, the cellular electrophysiological changes that render the atria of aged individuals more susceptible to AF remain poorly understood. We hypothesized that dispersion of atrial repolarization increases with aging, creating a substrate for initiation of AF. METHODS: Four groups of dogs were studied: adult and old dogs in normal sinus rhythm (SR) and adult and old dogs with chronic AF (CAF) induced by rapid atrial pacing. In each dog, action potentials (AP) were recorded with microelectrodes from isolated endocardial preparations of four regions of right atrium and three regions of left atrium. Two indices of AP duration (APD) heterogeneity were obtained in each dog by calculating standard deviation (SD) and the coefficient of variation (COV=[SD/mean] x 100%). RESULTS: In SR groups, APD averaged across all regions was significantly longer in old than in adult tissues. Both indices of APD heterogeneity were higher in old dogs in comparison to adult. At both ages, CAF was associated with significant APD shortening and a decrease in APD adaptation to rate. While CAF significantly increased both indices of APD heterogeneity in adult dogs, it significantly decreased them in old dogs. CONCLUSIONS: The increase of spatial variability in repolarization in old atria may contribute to the initiation of AF in the aged. CAF-induced APD shortening and a decrease in APD adaptation appear to be important for the maintenance of sustained AF in both adult and old atria. The CAF-induced increase in dispersion of repolarization may be important for AF stabilization in adults, while previously reported fibrosis and slowed conduction of premature beats may be important in the old for both AF initiation during SR and subsequent stabilization of AF.     

Development of the "Cell Chip": a new in vitro alternative technique for immunotoxicity testing

Predictive testing of immunotoxicity associated with chemical compounds is complicated and cannot be accomplished with a single test. As most of the existing tests for immunotoxicity employ experimental animals, there is an increasing need for alternative tests in vitro. We have developed a new system for in vitro immunotoxicity testing, which employs changes in cytokine expression observed in vitro as an endpoint indicating potential for perturbation of the immune system in vivo. This system named "fluorescent cell chip" (FCC) is based on a number of genetically modified cell lines that regulate the expression of a transgene coding for fluorescent protein enhanced green fluorescent protein (EGFP) in a similar way as they regulate expression of IL-1beta, IL-2, IL-4, IFN-gamma, IL-10, TNF-alpha, and beta-actin. Morphological and functional features of selected cell lines expressing EGFP under the control of cytokine promotors were compared with maternal cell lines and this comparison showed that critical functional features of the maternal cell lines were preserved in EGFP expressing cells. Two chemicals with known immunotoxic activities, cyclosporine A and potassium tetrachloro-platinate(II), mediated compound-specific pattern of inhibition and activation of reporter gene expression. Thus, the "fluorescent cell chip" has demonstrated potential for application as a predictive screening test for immunomodulatory activities of chemicals. The major advantage of this approach is the possibility to apply this test in high throughput screening of high number of compounds for their well defined biological activity.     

Depressed adolescents in a case-series were low in vitamin D and depression was ameliorated by vitamin D supplementation

Aim: The relationship between depression in adolescents and vitamin D was studied in a case‐series that included effects of vitamin D supplementation. Methods: Serum 25OH vitamin D (25OHD) levels in 54 Swedish depressed adolescents were investigated. Subjects with vitamin D deficiency were given vitamin D₃ over 3 months (n = 48). To evaluate well‐being and symptoms related to depression and vitamin D status, the WHO‐5 well‐being scale, the Mood and Feelings Questionnaire (MFQ‐S) and a vitamin D deficiency scale were used. Results: Mean serum 25OHD in the depressed adolescents was 41 at baseline and 91 nmol/L (p < 0.001) after supplementation. Basal 25OHD levels correlated positively with well‐being (p < 0.05). After vitamin D supplementation, well‐being increased (p < 0.001) and there was a significant improvement in eight of the nine items in the vitamin D deficiency scale: depressed feeling (p < 0.001), irritability (p < 0.05), tiredness (p < 0.001), mood swings (p < 0.01), sleep difficulties (p < 0.01), weakness (p < 0.01), ability to concentrate (p < 0.05) and pain (p < 0.05). There was a significant amelioration of depression according to the MFQ‐S (p < 0.05). Conclusion: This study showed low levels of vitamin D in 54 depressed adolescents, positive correlation between vitamin D and well‐being, and improved symptoms related to depression and vitamin D deficiency after vitamin D supplementation.     

Staging of women with breast cancer after introduction of sentinel node guided axillary dissection

Today, sentinel lymph node dissection (SLND) has replaced axillary lymph node dissection (ALND) as standard procedure for staging of the axilla in the treatment of breast cancer. SLND can accurately stage the axilla by removing on average only two lymph nodes. Only in case of metastatic spread to sentinel nodes an ALND is offered. Removing fewer nodes has made more extensive histopathological examinations of the lymph nodes possible and as a consequence more metastases are found. This has resulted in stage migration. Based on data from the nationwide Danish Breast Cancer Cooperative Group (DBCG) database we have estimated the magnitude and therapeutic consequences of this stage migration in Denmark by comparing the distribution of lymph node metastases in breast cancer patients operated in 1993-1996 and 2005-2008; before and after introducing SLND. The proportion of patients having macrometastases was not significantly different in the two periods, whereas the proportion of patients with micrometastases increased significantly from 5.1% to 9.0%. However, the proportion of patients offered adjuvant systemic treatment due to positive nodal status as the only high-risk criterion did only increase from 7.8% to 8.8%, when estimated using today′s criteria for risk-allocation, because nodal status is now less important in risk-allocation. In general, only 15-20% of patients with micrometastases and 10-15% of patients with isolated tumor cells (ITC) in sentinel node have further metastatic spread to non-sentinel nodes (NSN). Thus, the majority of these patients does not benefit from additional ALND but still run the risk of arm morbidity. Based on data from the DBCG database, we have developed two models to predict NSN metastases in breast cancer patients with micrometastases or ITC in the sentinel node. A total number of 304 breast cancer patients with ITC and 1577 patients with micrometastases in sentinel node operated in 2001-2008 with SLND and subsequent ALND were identified in the database. In patients with ITC in sentinel node the risk of NSN metastases was significantly associated with younger age at diagnosis, increasing tumor size and increasing proportion of positive sentinel nodes in a multivariate analysis. If patients were ≥ 40 years at diagnosis with tumor size ≤ 2 cm as well as one or more negative sentinel nodes, NSN metastases were found in only 2%. Omission of ALND in this group would spare 1/3 of patients with ITC in sentinel node for an ALND. In patients with micrometastases in sentinel node the risk of NSN metastases was significantly associated with increasing tumor size, lymphovascular invasion, negative hormone receptor status, location of tumor in the upper lateral quadrant of the breast and increasing proportion of positive sentinel nodes in a multivariate analysis. However, a model based on these traditional prognostic markers could not identify a subgroup of patients with a risk of NSN metastases less than 10%. We then investigated whether the biochemical prognostic markers TIMP-1, Ki67 and HER2 could support the model. In a matched case-control study 25 cases with micrometastases in sentinel node and additional metastatic spread to NSN were compared to 50 matched controls with micrometastases in sentinel node but without NSN metastases. Despite being prognostic markers in breast cancer, we found no significant differences in the expression of these three biochemical markers between patients with and without NSN metastases. Not all NSN metastases will become clinically relevant, making ALND redundant in many breast cancer patients. Accordingly, there is a trend towards omission of ALND in breast cancer patients with minimal metastatic disease in sentinel node. As a result, a tool is needed to identify a group of patients with high risk of recurrence, where ALND should still be offered. In our model a small group of patients with micrometastases had a high risk of NSN metastases on nearly 40%, comparable to patients with macrometastases, indicating that ALND may still be recommended in this subgroup in the future.     

Protein targeting constructs in alpha therapy

The progress in the field of targeted α-particle therapy (TAT) has to a great extent been enhanced by developments in both recombinant DNA technology and radionuclide labeling chemistry. Advances in genomics and proteomics have promoted an increase in the identification of novel targets and molecules that can define different diseases, such as cancer. In radioimmunotherapy (RIT), the primary goal is to improve delivery to and therapeutic efficacy of the cancer cells, whilst minimizing toxicity. Different approaches have been investigated to achieve this, such as reducing the size of the carrier, pretargeting, multidosing, locoregional administration and using a cocktail of radiolabeled monoclonal antibodies for targeting multiple antigens simultaneously. Some of these approaches have been encouraging, but translation of TAT into the clinic has been slow, in part because of the limited availability and the short physical half-lives of some of the available α-particle emitters. The clinical studies carried out to date have been promising, although many challenges remain in order to make TAT safe and economically feasible. In this paper a number of different targeting constructs used hitherto that may be promising carriers for TAT in the future are presented and discussed. The constructs include enzymatic cleaved antibody fragments (Fab and F(ab˙)2 fragments); genetically engineered antibody fragments (scFv monomer, dimer (i.e. diabody) and tetramer, CH2 domain deleted antibody fragments); other protein targeting constructs such as affibodies and peptides as well as liposomal delivery.