Synthesis and evaluation of several new (2-chloroethyl)nitrosocarbamates as potential anticancer agents.

Seven new (2-chloroethyl)nitrosocarbamates have been synthesized as potential anticancer alkylating agents. These compounds were designed with carrier moieties that would either act as prodrugs or confer water solubility. All compounds were screened in an in vitro panel of five human tumor cell lines: CAKI-1 (renal), DLD-1 (colon), NCI-H23 (lung), SK-MEL-28 (melanoma), and SNB-7 (CNS). Several agents showed good activity with IC(50) values in the range of 1-10 microg/mL against at least two of the cell lines. One compound, carbamic acid, (2-chloroethyl)nitroso-4-acetoxybenzyl ester (3), was selected for further study in vivo against intraperitoneally implanted P388 murine leukemia. In addition to the aforementioned compound, both carbamic acid, (2-chloroethyl)nitroso-4-nitrobenzyl ester (9) and carbamic acid, (2-chloroethyl)nitroso-2,3,4, 6-tetra-O-acetyl-1-alpha,beta-D-glucopyranose ester (24) were evaluated against subcutaneously implanted M5076 murine sarcoma in mice. None of these compounds were active in vivo.

Improved retroviral transduction efficiency of vascular cells in vitro and in vivo during clinically relevant incubation periods using centrifugation to increase viral titers.

Vascular cells are an important target for gene transfer because of their potential to deliver gene products both locally and systemically. Direct retroviral gene transfer to vascular cells in vivo has been limited by inefficient rates of transduction. We hypothesized that vascular cell transduction efficiency (TE), during short retroviral incubation periods, is significantly improved in vitro and in vivo using centrifugation to increase viral titer. Furthermore, we hypothesized a linear relationship between concentration of viable viral particles (measured as colony-forming units (CFUs)/cell) and retroviral TE during short incubation periods. Cultured rat pulmonary artery endothelial cells (RPAECs), rat aortic smooth muscle cells (RSMCs), and human iliac artery endothelial cells (HIAECs) demonstrated a strong correlation between TE and high concentrations of virus (> 100 CFU/cell) during retroviral incubation periods of 10 to 60 minutes. High titers, and thereby high concentrations, were achieved by centrifugation and resuspension in a fraction of the original volume. Titers was consistently increased tenfold, for a twentyfold increase in concentration by volume. A 20-minute incubation with a Moloney murine leukemia-derived retroviral vector coding for human placental alkaline phosphatase, pLJhpAP, at a concentration of 1150 CFU/cell yielded TEs of 10.6% +/- 0.7%, 40.4% +/- 1.6%, and 15.1% +/- 2.0% for RPAECs, RSMCs, and HIAECs, respectively. A similar effect was shown using the Moloney murine leukemia-derived MFGlacZ retroviral vector, coding for Escherichia coli beta-galactosidase. Increased titer and concentration had no effect on target cell viability, as shown by trypan blue exclusion. Although RSMCs had the most cells transduced in a given incubation period (p < 0.05), RPAECs had the highest replication rate (p < 0.05), suggesting the importance of factors other than cell cycle on retroviral TEs during short, clinically relevant incubation periods. In subsequent in vivo experiments, gene transfer was achieved in the rat carotid artery during a 20-minute incubation period infusing the concentrated pLJhpAP retrovirus after carotid balloon injury. Rats infused with virus 2 days after balloon injury exhibited hpAP activity (0 to 10 cells/section/rat) in the neointima of five out of six rats. Rats infused 4 days after balloon injury exhibited hpAP activity (0 to 25 cells/section/rat) in the media and adventitia of five out of five rats. Control rats that received the balloon injury alone or the balloon injury and unconcentrated retrovirus exhibited zero hpAP activity. We conclude that the TE of retroviral-mediated gene transfer to vascular cells in vitro and in vivo can be improved during short, clinically relevant incubation periods using centrifugation to increase retroviral titer, and thereby concentration of viable viral particles.

Interaction between ionizing radiation and supercoiled DNA within human tumor cells.

We have analyzed DNA supercoiling within histone-free nuclei (nucleoids) using four human squamous cell carcinoma cell lines that express varying degrees of radiosensitivity. The entire DNA, arranged as negative supercoiled loops attached to the nuclear matrix, was extracted from single cells, stained with ethidium bromide, and passed through a flow cytometer recording both light scatter and red (DNA) fluorescence. Supercoiled loops of DNA from all cells were unwound with a low concentration of ethidium bromide, as seen by increased light scatter. Nucleoids from radiosensitive but not radioresistant cells resisted the transition from zero to positive supercoiling at higher concentrations of ethidium bromide. The profile of red DNA fluorescence from ethidium bromide-stained nucleoids showed that the radiosensitive cells expressed a greater variation in the total amount of ethidium bromide bound. After 12 Gy of gamma-radiation, radiosensitive cell lines produced nucleoids that contained a greater proportion of relaxed supercoiled DNA, making them larger than those from radioresistant cell lines. We suggest these observations are secondary effects resulting from an altered affinity between supercoiled looped DNA and the nuclear matrix. Combined with radiation damage, these structural alterations may lead to a more complex type of damage to repair within the radiosensitive cell lines.

Pyrroxamide, a nonionic nitroxyl spin label contrast agent for magnetic resonance imaging. Mutagenesis and cell survival.

Pyrroxamide [N-(1-hydroxymethyl-2,3-dihydroxypropyl)-2,2,5,5-tetramethyl pyrrolidine-1-oxyl-3-carboxyamide] is a newly tested nonionic monomeric nitroxyl compound with demonstrated effectiveness for MRI contrast enhancement at doses as low as 10(-3) M. Pyrroxamide and its hydroxylamine metabolic derivative were tested in concentrations from 10(-9) to 10(-2) M with a battery of cytotoxic and mutagenic assays using mammalian Chinese hamster ovary cells. Loci-specific mutation induction was examined at the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) and the Na+/K+ ATPase loci, both in the presence and absence of a liver microsomal metabolic activating mixture (S-9 mix). Cell survival and induction of sister chromatid exchanges also were studied. All tests yielded negative results indicating that pyrroxamide and and hydroxylamine derivative were both noncytotoxic and nonmutagenic at the doses tested.

Evaluation of SR-2508 induced neurotoxicity and myotoxicity in rats using brainstem auditory evoked potentials and the posterior auricular muscle response.

Brainstem auditory evoked potential (BAEPs) and a middle latency component attributed to the posterior auricular muscle response (PAMR) to an intense auditory stimulus were used to measure the onset of neurotoxic and myotoxic effects in rats after chronic exposure to the radiosensitizer SR-2508. The rats received intraperitoneal injections of SR-2508, 500 mg/kg, 5 days/week for 6 weeks. BAEP and PAMR were measured after 10, 20, and 30 injections and 4 weeks after the drug treatment was stopped. A significant neurotoxic effect was observed: After 10 injections of SR-2508, latency of the fourth positive (P4) component of the BAEP, which is thought to represent activity from the superior olivary nuclei, increased from baseline levels, and a further increase was measured after 30 injections. Four weeks after drug treatment was stopped, P4 latency had not returned to baseline levels, indicating permanent injury. PAMR latency was also increased after 10 injections of SR-2508, but increased no further during the drug treatment period. Four weeks after the last injection, PAMR latencies had returned to pretreatment levels, indicating that the myotoxic effects of SR-2508 were reversible.

Evaluation of desmethylmisonidazole-induced neurotoxicity in the rat using brainstem auditory evoked potentials.

Brainstem auditory evoked responses (BAEPs) and a scale that evaluates clinical signs of neurotoxicity were used to measure the onset of neurotoxic effects seen in rats after chronic injection of 400 mg/kg/day of the radiosensitizer desmethylmisonidazole (DMM) for 5 days/week for 7 weeks. A significant neurotoxic effect was indicated by increases in the latencies of peaks 4 and 5 of the BAEP after the 24th injection of DMM; clinical signs of neurotoxicity were observed after the 30th injection. Histologic examination of brainstems from rats sacrificed after selected number of injections during treatment showed that the onset of lesions in the brainstem was gradual but not extensive. Pentobarbital used as an anesthetic agent had no effect on the induction of neurotoxicity.

Dynamical relations for left ventricular ejection: flow rate, momentum, force and impulse.

An investigation was carried out to quantitatively evaluate left ventricular volume flow rate, momentum, force and impulse derived from application of conservation principles for mass and momentum of blood within the ventricle during the ejection phase. An automated digital image processing system was developed and applied to left ventricular angiograms which are computer processed and analyzed frame by frame to determine the dynamical relations by numerical methods. Our initial experience with force and impulse has indicated that neither quantity seemed to be a sensitive indicator of coronary artery disease as evaluated by qualitative angiography for the particular patient group studied. Utilization of the dynamical relations in evaluating human left ventricular performance requires improved means of measurement and interpretation of clinical studies.

Misonidazole neurotoxicity in rats: Part I. Evaluation of misonidazole neurotoxicity in rats by analysis of brain stem auditory and cortical evoked potentials.

The effects of misonidazole (MISO) on brain stem evoked potentials (BAEPs) and cortical evoked potentials (CEPs) were evaluated in 16 Sprague-Dawley rats treated with the agent. As found in previous studies, serial BAEP values were diagnostic of the onset of MISO toxicity before clinical signs and symptoms appeared. However, MISO had no effects on CEPs, which remained essentially unchanged through the course of the experiment. At histologic examination, significant changes were found in the area of the brain stem, but there was no histologic evidence of damage to cortical or subcortical structures caused by MISO administration. The results of this study suggest that the neurotoxic effects of MISO are species-specific, and that while the rat model may be useful for comparison of the relative toxic effects of nitroimidazole radiosensitizers, it is not a model suited for measurement of neurotoxicity caused by MISO in humans and nonhuman primates.

Misonidazole neurotoxicity in rats: Part II. Effect of pre- and intermittent treatment with pentobarbital on misonidazole neurotoxicity in the rat.

Rats pretreated for 5 days with 50 mg/kg of pentobarbital tolerated statistically significant higher doses of misonidazole before the onset of misonidazole-induced neurotoxicity than rats treated intermittently with the same dose of pentobarbital. Presumably in pretreated rats, pentobarbital induced an increase in the activities of hepatic microsomal enzymes that led to a more rapid metabolism of misonidazole than in rats treated only intermittently.

Comparison of rest/exercise ECG, thallium-201 scans and radionuclide angiography in patients with suspected coronary artery disease.

The results of rest and exercise ECG, 201Tl myocardial perfusion imaging and equilibrium radionuclide angiography were analyzed in 71 consecutive patients referred for diagnosis or evaluation of coronary artery disease (CAD). In 45 patients the diagnosis was established either by catheterization or typical history. In this group the overall sensitivity for rest/exercise ECG was 66%, for 201Tl scans 74%, for both combined 79% and for the ejection fraction response to exercise determined by radionuclide angiography 97%. If only the exercise response was considered, the corresponding sensitivity values were 58% (ECG), 50% (201Tl scans), 71% (ECG + 201Tl) and 97% (radionuclide angiography). The specificity for coronary artery disease was determined to be 71% for ECG, 86% for 201Tl scans and 42% for radionuclide angiography. All patients with false-positive results by radionuclide angiography had cardiomyopathies, thus this test has a high specificity for left ventricular dysfunction rather than for CAD alone. Criteria developed from the analysis of the test results in the 45 patients with definite diagnoses were then applied to the evaluation of 26 additional patients with atypical chest pain. A diagnosis could be made in all but 5 of them and radionuclide angiography was again the single most reliable test. Based on this study a new approach for the noninvasive evaluation of patients with suspected coronary artery disease is proposed.

Validity of left-ventricular ejection fractions measured at rest and peak exercise by equilibrium radionuclide angiography using short acquisition times.

To validate ejection fraction (EF) calculations from 5 and 2 minutes of multiple-gated equilibrium radionuclide angiographic data and to establish its utility during alterations in cardiac performance, we studied 38 patients with chest pain suggestive of coronary artery disease. Twenty-four patients underwent contrast ventriculography (CV) as well as first-pass (FP) and equilibrium (EQ) radionuclide angiography at rest, and 14 additional patients had both radionuclide tests performed at rest as well as during peak supine bicycle exercise. The resting 5-min acquisition ejection fractions were compared between each method and the following correlations were generated: r = .92, n = 24 (CV-EQ), r = .92, n = 24 (CV-FP), and r = .95, n = 38 (FP-EQ). The variability of EQ-EF calculations between two independent observers was less than 2%; the mean absolute difference between two sequential 2-min acquisitions and the 5-min recordings was -.1 +/- 1.6%, and the reproducibility of sequential 2-min ejection fractions was excellent (r = .98). EQ and FP ejection fractions at symptom-limited exercise correlated well (r = .96, n = 14). We conclude that equilibrium radionuclide angiography is a valid method to measure EF both at rest as well as during peak exercise even when 2-min acquisition periods are used.

The effect of diaphragmatic attenuation on 201Tl images.

The effect of diaphragmatic attenuation on 231Ti images of the heart was studied by intermittent recording with patients in inspiration and expiration. These studies suggest that the commonly observed posteroinferior defects in the left lateral projection can be explained by this mechanism. Possible effects on the anterior view are also suggested.

Profiles of radionuclide left ventricular ejection fraction changes induced by supine bicycle exercise in normals and patients with coronary heart disease.

This paper presents the profiles of left ventricular ejection fraction (EF) during and following supine bicycle exercise in normal subjects and in patients with coronary heart disease, as well as the relationship of the described patterns to clinical parameters. Twenty normal men and 40 patients with coronary artery disease were studied using gated equilibrium radionuclide angiography (EQ-EF). In the normals, during exercise, EF increased by a mean of 25% of the resting value, with an increase of no less than 11%. The exercise-limiting symptom in patients with coronary artery disease was angina pectoris in 20 and fatique in the other 20 patients. In the angina patients, there was a mean decrease in EF of 20%, and in the other coronary artery disease patients ejection fraction change little. Only two patients with coronary artery disease increased from a normal resting value to peak exercise by more than 11%, and they had isolated right coronary lesions. An "overshoot" elevation of ejection fraction above resting levels was demonstrated following termination of exercise in most patients. The patients with a significant fall in exercise ejection fraction more frequently had abnormal exercise-induced ECG changes as well as abnormal left ventriculograms and more severe coronary artery disease at cardiac catheterization than the patients with little change in ejection fraction. We conclude that 1) normals could be separated from most patients with significant coronary artery disease in this study population; 2) ejection fraction must be measured at maximal exercise for it to have diagnostic value, since there could be normal rise before and after peak exercise and an abnormal response missed; and 3) the ejection fraction response to exercise reflects the severity of the underlying coronary artery disease. The described patterns of exercise-induced changes in left ventricular ejection fraction are important to consider when using this new technique to diagnose and evaluate patients with coronary artery disease.

Extrusion of endodontically treated teeth.

Extrusion in a vertical direction was used in efforts to retain 14 teeth that had subcrestal fractures, perforations, or caries below the gingival tissue. Indications for the procedure, techniques, and complications are discussed.

The physics of left ventricular ejection and its implications for muscle mechanics.

The ejection stage of the left ventricle of the heart is analyzed using the proper form of Newton's Second Law of Motion and a simple cylindrical ventricular model. The Laplace relation is then used to calculate the dynamic relationship between force, velocity and acceleration in an average, circumferential, equitorial myocardial wall fiber, yielding: Fm=Fa + Cacf + Dvcf2 where Fm is the force in the fiber, Fa the fiber force that would be required to match aortic pressure, acf the acceleration and vcf the velocity of shortening of the fiber. C is a constant, and D is a geometrical parameter that varies smoothly with time. The significance of the above equation is discussed both in terms of muscle function (total muscular force) and pump function afterload Fa and the differential force, Fm - Fa): The afterload component Fa provides the pressure head needed to force the blood through the capillaries (Poiseuille flow) and varies with time in accordance with the impedance of the vascular system (auxotonic loading). The differential component force Fm - Fa provides the ventricular impulse needed to overcome the inertia of the system due to (1) the mass of the blood, (2) the geometrical constriction of the outflow tract as one moves downstream (Bernoulli effect), and (2) the moving ventricular walls. Analysis of this sort should help in attempting to separate the intrinsic properties of cardiac muscle and the pump function of the intact heart. The important role that isolated muscle experiments can play in this effort is discussed.