Evaluation of various static in vitro-in vivo extrapolation models for risk assessment of the CYP3A inhibition potential of an investigational drug.

Nine static models (seven basic and two mechanistic) and their respective cutoff values used for predicting cytochrome P450 3A (CYP3A) inhibition, as recommended by the US Food and Drug Administration and the European Medicines Agency, were evaluated using data from 119 clinical studies with orally administered midazolam as a substrate. Positive predictive error (PPE) and negative predictive error (NPE) rates were used to assess model performance, based on a cutoff of 1.25-fold change in midazolam area under the curve (AUC) by inhibitor. For reversible inhibition, basic models using total or unbound systemic inhibitor concentration [I] had high NPE rates (46-47%), whereas those using intestinal luminal ([I]gut) values had no NPE but a higher PPE. All basic models for time-dependent inhibition had no NPE and reasonable PPE rates (15-18%). Mechanistic static models that incorporate all interaction mechanisms and organ specific [I] values (enterocyte and hepatic inlet) provided a higher predictive precision, a slightly increased NPE, and a reasonable PPE. Various cutoffs for predicting the likelihood of CYP3A inhibition were evaluated for mechanistic models, and a cutoff of 1.25-fold change in midazolam AUC appears appropriate.

A method for the estimation of hydration state during hemodialysis using a calf bioimpedance technique.

Although many methods have been utilized to measure degrees of body hydration, and in particular to estimate normal hydration states (dry weight, DW) in hemodialysis (HD) patients, no accurate methods are currently available for clinical use. Biochemcial measurements are not sufficiently precise and vena cava diameter estimation is impractical. Several bioimpedance methods have been suggested to provide information to estimate clinical hydration and nutritional status, such as phase angle measurement and ratio of body fluid compartment volumes to body weight. In this study, we present a calf bioimpedance spectroscopy (cBIS) technique to monitor calf resistance and resistivity continuously during HD. Attainment of DW is defined by two criteria: (1) the primary criterion is flattening of the change in the resistance curve during dialysis so that at DW little further change is observed and (2) normalized resistivity is in the range of observation of healthy subjects. Twenty maintenance HD patients (12 M/8 F) were studied on 220 occasions. After three baseline (BL) measurements, with patients at their DW prescribed on clinical grounds (DW(Clin)), the target post-dialysis weight was gradually decreased in the course of several treatments until the two dry weight criteria outlined above were met (DW(cBIS)). Post-dialysis weight was reduced from 78.3 +/- 28 to 77.1 +/- 27 kg (p < 0.01), normalized resistivity increased from 17.9 +/- 3 to 19.1 +/- 2.3 x 10(-2) Omega m(3) kg(-1) (p < 0.01). The average coefficient of variation (CV) in three repeat measurements of DW(cBIS) was 0.3 +/- 0.2%. The results indicate that cBIS utilizing a dynamic technique continuously during dialysis is an accurate and precise approach to specific end points for the estimation of body hydration status. Since no current techniques have been developed to detect DW as precisely, it is suggested as a standard to be evaluated clinically.

CD14(++)CD16+ monocytes but not total monocyte numbers predict cardiovascular events in dialysis patients.

Migration of monocytes into the vessel wall contributes to the onset and progression of atherosclerosis. Because monocytes are a heterogeneous population, we determined potential associations between monocyte subsets and cardiovascular events in a prospective cohort of 94 dialysis patients followed for 35 months. The incidence of cardiovascular events and death measured by Kaplan-Meier plots and flow cytometric analysis of monocyte subsets showed that total leukocyte and monocyte numbers failed to predict event-free survival. Among monocyte subsets, a high CD14(++)CD16(+) monocyte number was associated with higher rates of cardiovascular events and death. In a multivariate proportional hazards model adjusted for classical cardiovascular risk factors, patients with CD14(++)CD16(+) monocyte numbers in the top quartile were at higher risk of cardiovascular events and death compared to patients in the lowest quartile. Our study suggests that the number of CD14(++)CD16(+) monocytes was independently associated with cardiovascular events and death in a high-risk population of dialysis patients.

Bioimpedance, dry weight and blood pressure control: new methods and consequences.

PURPOSE OF REVIEW: Chronic overhydration contributes to the development of left ventricular hypertrophy and a high cardiovascular mortality in end-stage renal disease. Assessment of dry weight is highly dependent on clinical assessment. Bioimpedance technology offers the potential to quantify body fluid compartments and to facilitate dry weight prescription. This review covers recent innovative approaches to dry weight assessment using bioimpedance technology. RECENT FINDINGS: Three different bioimpedance approaches to determine dry weight have been published. The normovolemic/hypervolemic slope method applies whole body multifrequency bioimpedance to assess predialysis total body extracellular fluid volume and compares the extracellular fluid volume/body weight relation at hypervolemia with the standard value in normovolemic individuals. The resistance-reactance graph method uses whole body single frequency bioimpedance for assessment of hydration state and nutritional status from height-adjusted resistance and reactance. The resulting resistance-reactance vector is set in relation to a distribution range in a normovolemic population. An alternative method uses segmental bioimpedance in the form of continuous intradialytic calf bioimpedance to record changes in calf extracellular volume during dialysis. Dry weight by this method is defined as the weight at which calf extracellular volume is not further reduced despite ongoing ultrafiltration. SUMMARY: Although promising, none of these methods has gained much popularity, probably due to the difficulties in understanding bioimpedance and the lack of gold standard methods for dry weight determination. Bioimpedance will improve dry weight assessment, but further refinement of the methods as well as large-scale clinical studies to demonstrate the accuracy and the clinical value of objective dry weight determination are needed.

Conformation and dynamics of abasic sites in DNA investigated by time-resolved fluorescence of 2-aminopurine.

Abasic sites are highly mutagenic lesions in DNA that arise as intermediates in the excision repair of modified bases. These sites are generated by the action of damage-specific DNA glycosylases and are converted into downstream intermediates by the specific activity of apurinic/apyrimidinic endonucleases. Enzymes in both families have been observed in crystal structures to impose deformations on the abasic-site DNA, including DNA kinking and base flipping. On the basis of these apparent protein-induced deformations, we propose that altered conformation and dynamics of abasic sites may contribute to the specificity of these repair enzymes. Previously, measurements of the steady-state fluorescence of the adenine analogue 2-aminopurine (2AP) opposite an abasic site demonstrated that binding of divalent cations could induce a conformational change that increased the accessibility of 2AP to solute quenching [Stivers, J. T. (1998) Nucleic Acids Res. 26, 3837-44]. We have performed time-resolved fluorescence experiments to characterize the states involved in this conformational change. Interpretation of these studies is based on a recently developed model attributing the static and dynamic fluorescence quenching of 2AP in DNA to aromatic stacking and collisional interactions with neighboring bases, respectively (see the preceding paper in this issue). The time-resolved fluorescence results indicate that divalent cation binding shifts the equilibrium of the abasic site between two conformations: a "closed" state, characterized by short average fluorescence lifetime and complex decay kinetics, and an "open" state, characterized by monoexponential decay with lifetime approximately that of the free nucleoside. Because the lifetime and intensity decay kinetics of 2AP incorporated into DNA are sensitive primarily to collisional interactions with the neighboring bases, the absence of dynamic quenching in the open state strongly suggests that the fluorescent base is extrahelical in this conformation. Consistent with this interpretation, time-resolved quenching studies reveal that the open state is accessible to solute quenching by potassium iodide, but the closed state is not. Greater static quenching is observed in the abasic site when the fluorescent base is flanked by 5'- and 3'-thymines than in the context of 5'- and 3'-adenines, indicating that 2AP is more stacked with the neighboring bases in the former sequence. These results imply that the conformation of the abasic site varies in a sequence-dependent manner. Undamaged sequences in which the abasic site is replaced by thymine do not exhibit an open state and have different levels of both static and dynamic quenching than their damaged homologues. These differences in structure and dynamics may be significant determinants of the high specific affinity of repair enzymes for the abasic site.

Membrane associated antigens of human malignant melanoma. III. Specificity of human sera reacting with cultured melanoma cells.

Sera from melanoma patients, healthy donors, pregnant women and patients with types of tumors other than melanoma were tested on various melanoma lines as well as on a cultured brain tumor and adult skin fibroblasts, using a microimmune adherence test. Positive reactions against all cell lines were found in serum from each group of donors. The degree of reactivity was dependent on the cell line used. Sequential absorption with AB Rh+ and pooled platelets of more than 200 donors either reduced the titer of sera or rendered a great part of the sera negative, demonstrating that antibodies against HL-A antigens and other tissue antigens were involved. The remaining positive sera were further absorbed with pooled cells from 6- to 8-week-old fetuses. This step abolished the reactivity of most sera, indicating the relatively high frequency of antibodies in males and females against fetal antigens expressed also on melanoma and other cells. In order to determine the specificity of the few remaining positive sera, absorptions with three different melanoma cell lines, a brain tumor and fibroblasts were carried out. The results showed only partial cross-reactivity between different cell lines. No evidence was obtained from this study for the existence of a common cross-reacting membrane-associated antigen on human malignant melanoma. Antigens that could be readily detected seemed mostly to be tumor-associated fetal antigens.

[Immunology of malignant melanoma (author's transl)]. / 75. Immunologie des malignen Melanoms

Melanoma cells carry membrane-bound antigens that induced both antibody production and cellular immunity. However, these antigens appear not to be tumor-specific, as the activity of human antisera can be absorbed out by fetal antigens. Nonetheless, the phenomenon of spontaneous regression, though mostly affecting only parts of a lesion, indicates that effective attack mechanisms do exist. Simultaneous tumor progression is due to heterogeneity of tumor cells, which vary widely in antigen expression. Cells that are not recognizable sneak through defense mechanisms and produce metastases.

[The importance of radio-allergo-sorbent-test in comparison with intracutaneous test in diagnosis of allergic diseases].

A comparison of clinical skin-tesing using the prick-test and the intracutaneous test with the radio-allergo-sorbent-test (RAST) in 167 patients with different immediate-type allergies showed concordant results in 75.7 per cent of the cases. In 20 per cent of the cases, the skin-test was positive while the simultaneous RAST was negative. Results in male and female patients did not show any important difference. The two test methods agreed well with selected antigens, e.g. grass-pollen, animal-epithelium, fungi and egg white; whereas, the two methods showed considerably different results using tree pollen and housedust as antigens.