Long-term stability study of Prussian blue - a quality assessment of water content and thallium binding.

The purpose of this study is to assess the long-term stability of Prussian blue (PB) drug product (DP) and active pharmaceutical ingredient (API) under laboratory storage conditions by monitoring the loss in water content and the corresponding change of the in vitro thallium binding capacity that represents product performance. The bound water content and the in vitro thallium binding capacity of PB DPs and APIs were measured in 2003 and 2013, respectively. Water content, a critical quality attribute that directly correlates to the thallium (Tl) binding capacity was measured by thermal gravimetric analysis (TGA). The thallium binding study was conducted by testing PB in buffered solutions over the human gastrointestinal pH range with thallium concentrations ranging from 600 to 1,500 ppm. Samples were incubated at physiological temperature of 37°C in a shaking water bath to mimic gastric flux and intestinal transport. The binding equilibrium was reached at 24h. Following incubation, each sample was filtered and the free thallium was analyzed using a validated inductively coupled plasma spectroscopic method (ICP). The Langmuir isotherm was plotted to calculate maximum binding capacity (MBC). Compared with 2003, the water content of DP-1 decreased by about 14.1% (from 15.6 to 13.4 mol), and the MBC of DP-1 decreased by about 12.5% (from 714 to 625 mg/g) at pH 7.5. When low concentration of thallium (600 ppm) was used at pH 7.5, the Tl binding remained comparable for both API-1 (286 vs 276 mg/g) and DP-1 (286 vs 268 mg/g). Similarly, the Tl binding remained unchanged for both API-1 (237 vs 255 mg/g) and DP-1 (234 vs 236 mg/g) at pH 5.0. However, at pH 1.0 the binding was reduced 32.3% and 25.9% for API-1 and DP-1, respectively. Since the majority of binding takes place in the upper GI tract where pH around 5 can be expected, and therefore, the Tl binding capacity of PB should be comparable for new and aged samples. The findings that Tl binding changes with the water loss of PB and pH conditions are consistent with our previously published data. The study also represents the first quantitative assessment of the long-term stability of PB. Over last 10 years, PB DPs and APIs have lost about 20% water under ambient laboratory storage conditions which are consistent with a controlled warehouse environment. While the maximum binding capacity of PB to thallium was decreased after about 10 years of long-term storage, it is still very effective, suggesting that the shelf life of PB should be much longer than the manufacturer ascribed expiration date of 2008 under proper storage conditions.

Tl+-induced micros gating of current indicates instability of the MaxiK selectivity filter as caused by ion/pore interaction.

Patch clamp experiments on single MaxiK channels expressed in HEK293 cells were performed at high temporal resolution (50-kHz filter) in asymmetrical solutions containing 0, 25, 50, or 150 mM Tl+ on the luminal or cytosolic side with [K+] + [Tl+] = 150 mM and 150 mM K+ on the other side. Outward current in the presence of cytosolic Tl+ did not show fast gating behavior that was significantly different from that in the absence of Tl+. With luminal Tl+ and at membrane potentials more negative than -40 mV, the single-channel current showed a negative slope resistance concomitantly with a flickery block, resulting in an artificially reduced apparent single-channel current I(app). The analysis of the amplitude histograms by beta distributions enabled the estimation of the true single-channel current and the determination of the rate constants of a simple two-state O-C Markov model for the gating in the bursts. The voltage dependence of the gating ratio R = I(true)/I(app) = (k(CO) + k(OC))/k(CO) could be described by exponential functions with different characteristic voltages above or below 50 mM Tl(+). The true single-channel current I(true) decreased with Tl+ concentrations up to 50 mM and stayed constant thereafter. Different models were considered. The most likely ones related the exponential increase of the gating ratio to ion depletion at the luminal side of the selectivity filter, whereas the influence of [Tl+] on the characteristic voltage of these exponential functions and of the value of I(true) were determined by [Tl+] at the inner side of the selectivity filter or in the cavity.

Assessment of myocardial viability in patients with myocardial infarction using twenty-four hour thallium-201 late redistribution imaging.

BACKGROUND: Rest thallium-201 (201Tl) myocardial perfusion imaging has been widely used for evaluation of myocardial ischemia/viability after myocardial infarction, but the ideal timing for imaging after injection to maximally estimate viability is not well established. METHODS: Thirty-six patients with myocardial infarction underwent the initial, 3 h, and 24 h redistribution imaging after intravenous injection of 148-185 MBq 201Tl. The initial and 3 h images, the initial and 24 h images, and the 3 and 24 h images were compared double-blinded. RESULTS: Out of the 184 abnormal segments based on the initial imaging, 56 (30%) segments improved by at least 1 grade on the 3 h imaging while 78 (42%) segments improved by at least 1 grade on the 24 h imaging. The 24 h late imaging detected more viable myocardium than the 3 h imaging did, with a significant difference (chi2= 5.680, p = 0.017). There were 158 abnormal segments on the 3 h imaging, with average 28% (44) segments improved by at least 1 grade on the 24 h imaging. There were 128 initial abnormal segments with no improvement on the 3 h imaging. Out of these segments, the 24 h late redistribution imaging detected additional redistribution in 26 segments, taking up 20%. CONCLUSIONS: Twenty-four hour late 201Tl imaging will demonstrated additional redistribution in patients who have incompletely reversible defects on early redistribution imaging at 3h.

Gating models of the anomalous mole-fraction effect of single-channel current in Chara.

The dependence of single-channel current on the Tl+/K+ mole fraction exhibiting a minimum at [Tl+]/[K+] of about 1:15 is proportional to open probability in bursts. Five models are suggested to explain modulation of gating by the Tl+/K+ ratio. Three models start from a channel with 4 identical subunits, each with an allosteric binding site for K+ or Tl+. In the first model, ion binding is directly observable as a transition from one Markov state to another. This model can explain the dependence of the apparent single-channel current on Tl+ concentrations. However, the predicted linear dependence on ion concentrations of the apparent rate constants was not observed in measurements in 25 or 250 mM KNO3 and 250 mM Tl NO3. The second model can overcome this problem by introducing saturation kinetics for ion binding. In the third model, gating is caused by inherent vibrations of the protein, and the rate constants of the related transitions depend on the occupation of the allosteric sites. The fourth model is based on the foot-in-the-door approach with the essential feature that two K+ ions in the selectivity filter are necessary to keep the pore radius suitable for K+ ions. The fifth model is also a foot-in-the-door model, but non-Markovian because, similar to model 3, it is assumed that the conformation of the protein (and thus the rate constants of the Markov model of the time series) depends on the force exerted by the temporal average over the states of a Markov model of ion occupation. These ions may reside in the pore itself or outside.

Quantitative assessment of thallium myocardial washout rate: importance of peak heart rate and lung thallium uptake in defining normal values.

Traditionally, the results of exercise thallium scintigraphy were interpreted by transient defect analysis using initial and delayed images. Recently, washout rate analysis has been used for the relative quantification of exercise thallium scintigraphy. A diffuse slow washout from all myocardial regions has been defined as the indicator of extensive coronary artery disease. However, slow washout has occasionally been observed in normal cases and in healthy myocardial segments which are not supplied by a stenosed artery in patients with single or double vessel disease. We evaluate the factors influencing washout rate in 100 normal patients and 63 patients with angina pectoris (33 cases of single vessel disease and 30 cases of double vessel disease). The washout rates were calculated using circumferential profile analysis. In normal patients, washout rate was closely related to peak heart rate (r = 0.72) and inversely related to lung thallium uptake (r = -0.56). A diffuse slow washout was observed in seven (7%) of 100 normal patients, six (18%) of 33 cases of single vessel disease and eight (24%) of 30 cases of double vessel disease. The patients with diffuse slow washout showed significantly higher lung thallium uptake values and lower peak heart rates than those without diffuse slow washout (P less than 0.01). Thus, this false positive slow washout should be considered in the interpretation of quantitative exercise thallium scintigraphy.

Thallium circumferential profiles in the detection of coronary artery disease--assessment by receiver operating characteristic curve analysis.

Thallium uptake and redistribution in the myocardium was quantitated using circumferential activity profiles. The profiles were determined from images in the LAO 35, LAO 70 and anterior views, obtained after peak exercise and after 3-4 h redistribution. A retention curve was constructed from these profiles, displaying the redistribution profile as a fraction of the stress profile. Normal values were obtained from 21 normal patients and compared to the images from 68 patients suspected of having coronary artery disease (CAD). Of the latter, 20 had normal/non-significant CAD and 48 had 70% or greater stenosis of one or more vessels on coronary angiography. Several methodological variations were examined: calculation of the profiles using the peak or average counts around the ventricle, uniform or interpolative background subtraction, normalisation of the profiles to their peak or mean counts. Receiver operating characteristic (ROC) curves were generated for profiles calculated using these variations and compared with the ROC curves obtained by visual interpretation of both analogue and computer-enhanced images by two experienced observers. The uniform background subtraction method was found to be better than the interpolative method, and normalisation to the mean of each curve was preferable to normalisation to the peak. ROC curves using profiles calculated using the peak myocardial counts were identical to the ROC curves from profiles using the average counts, but the operating points on the two curves differed. Computer enhancement of the digital images resulted in increased sensitivity for CAD without loss of specificity when compared to the interpretation of the analogue images. The circumferential profiles were found to provide a further increase in sensitivity and were highly reproducible.(ABSTRACT TRUNCATED AT 250 WORDS)

Myocardial imaging with thallium-201 for assessment of regional myocardial perfusion and viability after intracoronary thrombolytic therapy.

Whereas coronary arteriography demonstrates success of reflow or recanalization after intracoronary thrombolysis, myocardial perfusion imaging with thallium-201 (201Tl) permits assessment of viability of reperfused myocytes. The initial distribution of 201Tl in the myocardium immediately after intravenous injection is the result of both blood flow delivery of the radionuclide to the heart and the extraction fraction for 201Tl. If 201Tl is administered during a period of coronary occlusion, a scintigraphic defect will be observed in the territory of the occluded vessel. If, subsequently, blood flow is restored, as with reperfusion, and cellular kinetics transport of 201Tl returns to normal, myocardial regions initially deprived of 201Tl will demonstrate delayed redistribution and the defect will tend to normalize within several hours. In a sustained coronary occlusion, the defect will persist after 201Tl injection. Specific imaging protocols that have been or can be used to assess thrombolytic therapy are reviewed. If intracoronary streptokinase infusion is associated with a substantial enhancement of perfusion and preservation of cellular integrity in the ischemic zone, delayed 201Tl images obtained several hours after intravenous injection of the radionuclide will demonstrate significantly more 201Tl activity than the images before reflow. Direct intracoronary injections of 201Tl down the infarct vessel have also been used to evaluate the benefit of thrombolytic therapy by the demonstration of increased 201Tl uptake after reperfusion in the ischemic zone. Finally, computer methods for subtracting serial images obtained after two separate intravenous 201Tl injections administered before and after streptokinase infusion might be used to derive a "functional image" of the ischemic region that would quantitatively be proportional to the amount of myocardial salvage achieved.

Assessment of drugs by functional imaging of thallium-201 distribution in the dog.

The effect of drugs on thallium-201 biodistribution in the body has been studied in dogs using a new functional imaging method. The calculation is based upon the comparison of the activities of the two scintigraphic images obtained after two successive injections of thallium-201 separated by an interval of at least 10 min, during which some drug of interest can be administered. This imaging technique was applied in control dogs (n = 6) and in animals treated with dipyridamole (n = 7) or grisorixin (n = 7). As expected, these two vasoactive drugs increased mainly the myocardial uptake, whereas smaller variations were noted in the other organs studied: liver, kidney, lungs, and skeletal muscles. Thus this method should allow a rapid and reliable noninvasive assessment of cardiovascular drugs with thallium-201.

[201Thalium scintimetry of the heart as a new method for functional assessment in coronary heart disease (author's transl)]. / Die 201-Thallium-Szintimetrie des Herzens als neues Verfahren zur funktionellen Differenzierung der koronaren Herzkrankheit

201Tl scintimetry of the heart was used for a statistical comparison between normal people (10 individuals) and those with acute anterior wall infarcts (5 patients), anterior wall hypokinesia (5 patients) and dyskinesia (8 patients). Numerical results were obtained by a computer and depended on the relative 201tl storage in the left ventricular myocardium (64 mm2 area) related to mediastinal background uptake. It was found that maximal myocardial uptake, compared with mediastinal activity (about 290%) did not differ between normals and patients with coronary heart disease. Aneurysms of the anterior ventricular wall showed a reduction (with the ventricle perpendicular to the collimator) of 45.8%; in hypokinesia and acute myocardial infarcts, it was 64.2% and 64.6% respectively (normal 82.6%). 201Tl uptake in aneurysms (42.4%) approached background activity (36%) if a projection parallel to the collimator was used. 201Tl scintimetry provides a means of defining functional ability of the myocardium (depdending on myocardial perfusion and mass). Aneurysms and hypokinetic portions of the myocardium can be differentiated statistically from normal 201Tl uptake.