Lower mean weight after 14 days intravenous administration peptide YY3-36 (PYY3-36) in rabbits.

OBJECTIVE: Endogenous peptide YY(3-36) (PYY(3-36)) is associated with postprandial regulation of appetite. We investigated the safety and effectiveness of peripherally administered synthetic human PYY(3-36) for 14 days in New Zealand white rabbits. Weight gain and food consumption were assessed and pharmacokinetics and toxicity characterized. RESEARCH METHODS AND PROCEDURES: In all, 24 animals were randomized to one of four intravenous treatment groups - control (0.9% saline) or PYY(3-36) bolus at 4.1, 41.0, or 205 microg/kg/day. Body weight and consumption of fixed food allotment were measured daily. Hematology and serum chemistries were profiled at baseline and Day 15, and pharmacokinetics measured following dose 14. Histopathologic examination of designated tissues and organs in control and PYY(3-36) 205 mug/kg animals was conducted. All animals were subject to clinical and macroscopic observation. RESULTS: The trend effect of higher dose PYY(3-36) on lower average weight was significant (P = 0.01; Day 14 compared to baseline) and its effect on reduced food consumption was suggested (P = 0.065; number of days < or =75% food eaten, compared with control). Hematology and clinical chemistries were within normal limits pretest and at Day 15. No clinical, macroscopic, histologic, or microscopic changes related to the test article were observed over the course of study. DISCUSSION: Lower average weight occurs in rabbits treated once daily with intravenous injection PYY(3-36) (205 microg/kg/day) over 14 days. No clinical or histologic signs of toxicity were observed. Further research is warranted to describe alternate routes of peripheral administration for optimizing weight control.

Human pharmacokinetics of a perfluorocarbon ultrasound contrast agent evaluated with gas chromatography.

The purpose of this study was to prospectively study the human pharmacokinetics of an ultrasound (US) contrast agent through its active ingredient, dodecafluoropentane (DDFP). Expired air and blood samples were collected from 24 volunteers after IV administration from 0.01 to 0.1 mL/kg. They were analyzed by a gas chromatographic method specially adapted to the study of DDFP. Blood data fitted to an open one-compartment model. Elimination half-life range was 1.8 to 2.5 min. The area under the curve was correlated to the dose (r(2) = 0.99). Mean blood clearance ranged from 30 to 49 mL/min kg. Blood apparent distribution volume ranged from 0.09 to 0.15 L/kg. In expired air, DDFP concentration exhibited a biexponential decay. The percentage of recovery was 98 +/- 19% at 2 h. No extraneous peaks were observed, indicating no detectable DDFP metabolites. It was concluded that DDFP pharmacokinetics in blood fitted to an open one-compartment model with a fast elimination half-life. Recovery in expired air was almost complete 2 h after administration.

Formulation development and antitumor activity of a filter-sterilizable emulsion of paclitaxel.

PURPOSE: Paclitaxel is currently administered i.v. as a slow infusion of a solution of the drug in an ethanol:surfactant:saline admixture. However, poor solubilization and toxicity are associated with this drug therapy. Alternative drug delivery systems, including parenteral emulsions, are under development in recent years to reduce drug toxicity, improve efficacy and eliminate premedication. METHODS: Paclitaxel emulsions were prepared by high-shear homogenization. The particle size of the emulsions was measured by dynamic light scattering. Drug concentration was quantified by HPLC and in vitro drug release was monitored by membrane dialysis. The physical stability of emulsions was monitored by particle size changes in both the mean droplet diameter and 99% cumulative distribution. Paclitaxel potency and changes in the concentration of known degradants were used as chemical stability indicators. Single dose acute toxicity studies were conducted in healthy mice and efficacy studies in B 16 melanoma tumor-bearing mice. RESULTS: QW8184, a physically and chemically stable sub-micron oil-in-water (o/w) emulsion of paclitaxel, can be prepared at high drug loading (8-10 mg/mL) having a mean droplet diameter of <100 nm and 99% cumulative particle size distribution of <200 nm. In vitro release studies demonstrated low and sustained drug release both in the presence and absence of human serum albumin. Based on single dose acute toxicity studies, QW8184 is well tolerated both in mice and rats with about a 3-fold increase in the maximum-tolerated-dose (MTD) over the current marketed drug formulation. Using the B16 mouse melanoma model, a significant improvement in drug efficacy was observed with QW8184 over Taxol. CONCLUSIONS: QW8184, a stable sub-micron o/w emulsion of paclitaxel has been developed that can be filter-sterilized and administered i.v. as a bolus dose. When compared to Taxol, this emulsion exhibited reduced toxicity and improved efficacy most likely due to the composition and dependent physicochemical characteristics of the emulsion.

Safety assessment of the use of perflenapent emulsion for contrast enhancement of echocardiography and diagnostic radiology ultrasound studies.

PURPOSE: The studies were undertaken to assess the safety of the use of perflenapent emulsion (EchoGen, SONUS Pharmaceuticals, Bothell, Wash.) for contrast enhancement of echocardiography and radiology ultrasound studies. MATERIALS AND METHODS: In all, 1,001 patients or subjects were enrolled in 21 clinical studies. Clinical laboratory test, pulse oximetry, vital signs, and electrocardiograms (ECGs) were obtained in 818 patients before and after administration of perflenapent emulsion and active control [5% sonicated human albumin, (Albunex, Molecular Biosystems, Inc., San Diego, Calif.)] or placebo (saline) to determine mean changes from baseline. Adverse event rates were monitored following administration of perflenapent emulsion in 743 patients and placebo in 151 patients. RESULTS: No clinically significant abnormalities in clinical laboratory, pulse oximetry, vital signs, or ECG evaluations were observed. Values following administration of perflenapent emulsion were comparable with those following placebo or active control. Perflenapent emulsion (50/743; 6.7%) was comparable with placebo (4/151; 2.6%) in the overall incidence of adverse events considered related to the test article. Adverse events that occurred with a frequency of > or = 1% within 30 min after perflenapent emulsion administration were vasodilation and taste aberration. Adverse events which were mostly mild to moderate in intensity, began within 10 to 20 min after administration, and resolved spontaneously within 10 to 20 min. CONCLUSION: Perflenapent emulsion is generally well tolerated in patients undergoing echocardiography and ultrasound of other target organs.

A phase I clinical trial with gadodiamide injection, a nonionic magnetic resonance imaging enhancement agent.

Twenty adult male volunteers were studied in an unblinded, ascending-dose study to evaluate the safety, tolerance, and pharmacokinetics of intravenously administered nonionic gadodiamide injection. Dosages administered were 0.05, 0.1, 0.2, and 0.3 mmol/kg. Subjects were monitored from 36 hours before, through 72 hours after administration. There were no clinically relevant changes in vital signs or electrocardiograms. No clinically significant changes occurred in blood or urine laboratory parameters, although a tendency for minor, transient elevations in serum iron levels 8 to 48 hours after administration was noted. These changes were not dose-related. Nine of 20 subjects reported at least one adverse event; all events were transient and of mild intensity, the most common being dizziness/lightheadedness and perversion of taste or smell. One subject reported discomfort consisting of mild stinging at the injection site during administration. Gadodiamide was excreted unmetabolized in the urine with greater than 95% recovery at 72 hours after administration. The serum elimination half-life was approximately 70 minutes.

Preclinical evaluation of MnDPDP: new paramagnetic hepatobiliary contrast agent for MR imaging.

Manganese(II)-N,N'-dipyridoxylethylenediamine-N,N'-diacetate-5,5'-bis (phosphate) (MnDPDP) is a paramagnetic complex designed for use as a hepatobiliary agent. The T1 relaxivity of MnDPDP (2.8 [mmol/L]-1.sec-1 in aqueous solution) was similar to that of gadolinium diethylenetriaminepentaacetic acid (DTPA) (4.5 [mmol/L]-1.sec-1) and gadolinium tetraazocyclodecanetetraacetic acid (DOTA) (3.8 [mmol/L]-1.sec-1). However, in liver tissue the T1 relaxivity of MnDPDP (21.7 [mmol/L]-1.sec-1) was threefold higher than that reported for Gd-DOTA (6.7 [mmol/L]-1.sec-1). Maximum liver T1 relaxation enhancement occurred 30 minutes after injection of MnDPDP, at which time 54MnDPDP biodistribution studies indicated that 13% of total body activity was in the liver. Enhanced (MnDPDP, 50 mumol/kg) MR images showed a fivefold increase in tumor-liver contrast-to-noise ratio over baseline unenhanced images. Results of the authors' acute and subchronic toxicity studies suggest that MnDPDP will be safe at the doses necessary for clinical imaging; at 10 mumol/kg, the safety factor (LD50/effective dose) for MnDPDP is 540, significantly greater than the safety factor of Gd-DTPA (ie, 60-100).

Hepatobiliary MR imaging: first human experience with MnDPDP.

The first human MR imaging results for the hepatobiliary contrast agent manganese(II)N,N'-dipyridoxylethylenediamine-N,N'-diacetate 5,5'-bis(phosphate) (MnDPDP) are reported. MnDPDP is a paramagnetic contrast agent specific for hepatobiliary imaging. An imaging study was performed to investigate the presence of contrast enhancement or facilitated visualization of normal structures. Twelve healthy subjects receiving MnDPDP at doses of 3, 10, or 15 mumol/kg were imaged after injection for approximately 30 minutes at 1-5-minute intervals. Transaxial abdominal images were obtained at 1.5 T in a single breath-hold interval of 21 seconds with use of a spin-echo pulse sequence (repetition time = 150 msec, echo time = 20 msec). Liver parenchyma enhancement was observed 1 minute after injection and persisted for at least 30 minutes. Clearance into the gallbladder was visualized within 15 minutes. Enhancement was dose-dependent; a dose of 10 mumol/kg produced a 75%-100% signal enhancement of the liver at 10 minutes after injection.

Effect of manganese (II) bis(glycinate)dichloride on Ca2+ channel function in cultured chick atrial cells.

Manganese (II) bis(glycinate)dichloride (Mn(glycinate)2) is a coordination complex of manganese with application as a contrast enhancement agent for magnetic resonance imaging in the heart. To determine the cardioactivity of the manganese ion in this chelation cage, the effects of Mn(glycinate)2 on Ca channel function in the cultured chick atrial cell was studied. Mn(glycinate)2 decreased amplitude of contraction in chick atrial cells from embryos 14 days in ovo with complete inhibition of beating at 1 mM and half-maximal effect at 0.1 mM. Under control conditions, Bay K 8644, a Ca channel activator increased amplitude of contraction by 86% with a half maximal effect at 3.2 x 10(-7) M. In the presence of 0.025 mM Mn(glycinate)2, a concentration which had no effect on the amplitude of contraction, the maximum response to Bay K 8644 was decreased to 31%. Mn(glycinate)2 had no effect on the EC50 for the response to Bay K 8644, 1.7 +/- 0.1 x 10(-9) M (S.E.M., n = 4) in control cells compared to 2.2 +/- 0.4 x 10(-9) M (S.E.M., n = 4) in cells incubated with Mn(glycinate)2. 45Ca2+ uptake over 5 min in cultured chick atrial cells decreased from 2.0 nmol/mg protein in control cells to 1.5 nmol/mg protein in the presence of 10(-5) M PN200-110, a Ca2+ channel blocker, a decrease of 28%. 45Ca2+ uptake decreased to 0.94 nmol/mg protein (53%) in the presence of 1 nmol Mn(glycinate)2. Effects of Mn(glycinate)2 and PN200 were not additive. These data demonstrate that Mn(glycinate)2 exerts its negative inotropic effect, at least partially, by interfering with the function of the L-type Ca channels at high concentrations.

Gadodiamide injection: nonionic gadolinium chelate for MR imaging of the brain and spine--phase II-III clinical trial.

Seventy-three patients with clinically suspected central nervous system abnormalities (44 intracranial, 29 medullospinal) were studied with magnetic resonance (MR) imaging before and after administration of nonionic gadodiamide injection. MR imaging showed intracranial lesions in 37 patients. Eight patients had spinal tumors, and 21 had disk disease. Structural abnormalities were shown in 37 of 44 head studies and in all 29 spine studies. Lesions enhancement was seen in 31 head studies and 28 spine studies, and distinction of lesion(s) from associated edema was possible in 10 head studies and in one study of intrinsic cord tumor. Administration of gadodiamide injection provided improved definition of lesion borders in 19 of 44 head studies and 26 of 29 spine studies. The use of the contrast agent changed the diagnosis that was based on the unenhanced images in nine head studies and 13 spine studies. Early postcontrast, T1-weighted spin-echo images of postoperative spines were adequate in distinguishing epidural scar (enhancing) from herniated disk (nonenhancing). The contrast agent was well tolerated, and no drug-related adverse events occurred.

The relationship between thermodynamics and the toxicity of gadolinium complexes.

The suitability of gadolinium complexes as magnetic resonance imaging contrast agents depends on a number of factors. A thermodynamic relationship to toxicity exists if one assumes that the chemotoxicity of the intact complex is minimal but that the toxicity of the components of the complex (free metal and uncomplexed ligands) is substantial. Release of Gd3+ from the complex is responsible for the toxicity associated with gadolinium complexes; this release appears to be a consequence of Zn2+, Cu2+, and Ca2+ transmetallation in vivo. This hypothesis is supported by acute toxicity experiments, which demonstrate that despite a 50-fold range of LD50 values for four Gd complexes, all become lethally toxic when they release precisely the same quantity of Gd3+, and by subchronic rodent toxicity experiments, which demonstrate a set of gross and microscopic findings similar to those known to be caused by Zn2+ deficiency. Finally, this hypothesis predicts that subtle changes in formulation can further enhance the intrinsic safety of these complexes.

Demarcation of myocardial ischemia: magnetic susceptibility effect of contrast medium in MR imaging.

Dysprosium diethylenetriamine-pentaacetic acid-bis (methylamide) (DTPA-BMA), a new nonionic contrast medium for magnetic resonance (MR) imaging, produces signal loss on T2-weighted images because of induced magnetic field gradients. The potential of this agent to delineate myocardial ischemia was investigated in 10 rats with acute (30 minutes) occlusion of the left coronary artery. T2-weighted MR images were acquired before and for 1 hour after intravenous administration of 1 mmol/kg of Dy-DTPA-BMA. Before administration of the contrast medium, signal intensity (SI) in the ischemic region was significantly greater than that in the normal myocardium; however, the borders of the ischemic region were not consistently distinct. The contrast medium caused marked decrease in SI of normal myocardium (18% +/- 3% of the control value), only slight decrease in the jeopardized region (76% +/- 6% of the control value), and no discernible effects on heart rate or blood pressure. Substantial contrast between normal and ischemic myocardium persisted for 1 hour. Moderate signal loss was observed in skeletal muscle. Dy-DTPA-BMA has the potential to demarcate the myocardial area in jeopardy as a region of high signal intensity because it erases signal preferentially in the normal myocardium.

Acute myocardial ischemia: MR imaging with Mn-TP.

Cardiac-gated magnetic resonance (MR) imaging was performed in rats to determine the effects of manganese ethylenediaminetetraphosphonate (TP). Ten normal rats received Mn-TP in a dose of 50 mumol/kg through a tail-vein injection. Spin-echo MR images were obtained before and every 10 minutes after Mn-TP injection for 1 hour. Cardiac signal intensity (SI) increased more than 70% after Mn-TP injection and remained nearly unchanged 1 hour after injection. Myocardial T1 was 517 +/- 49 msec in eight control rats and 282 +/- 61 msec (P less than .001) in six rats 81 +/- 0 minutes after injection. Nine rats underwent occlusion of the left anterior descending coronary artery prior to MR imaging. Images were obtained before and 15, 30, and 60 minutes after Mn-TP injection. In normal myocardium, SI increased up to 82% and remained elevated for 1 hour. In ischemic myocardium, SI rose 11%, leading to a marked contrast between the two tissue zones. T1 was also different in the two regions: In normal tissue, it was 206 msec +/- 54; in ischemic tissue, 338 +/- 82 (P less than .001). With T1-weighted MR imaging, Mn-TP showed a potential for delineating the jeopardized area after acute myocardial ischemia.

The interaction between excimer laser energy and vascular tissue.

The effects of XeF1 excimer laser on isolated normal and atherosclerotic aorta were studied. Experiments were performed in flowing water at constant temperature, flow rate, water depth, pulse width (10 nsec), wavelength (351 nm), beam size (1 mm2) and focal length (50 cm). The number of pulses, the pulse energy, and the pulse frequency were varied, and the vascular tissue was studied histologically. The following observations were made: tissue ablation required a minimum threshold pulse energy and was nonlinearly proportional to the number of pulses and the pulse energy delivered; precise tissue ablation occurred at low pulse frequencies, but changes resembling a thermal process were seen as pulse frequency increased; calcified plaque was more photoresistant than atheroma or normal vessel; excimer laser energy was markedly attenuated by blood; and the time interval between pulses and high peak power are related to the precision of ablation by pulsed excimer laser. It is concluded that excimer laser can rapidly and precisely ablate vascular tissue by a photothermal process.

Probing the renin active site by collisional quenching of endogenous fluorescence.

The structural and enzymatic aspects of renin are of great interest in hypertension research. In this paper, we examine the solution accessibility of the three tryptophan (Trp) residues of mouse submaxillary gland renin by solute collisional fluorescence quenching. Our studies indicate that there are two "classes" of Trp residues in renin: class I, a class of Trp residues which are at or near the surface of renin and fully accessible to the fluorescence quencher iodide; and class II, a class of Trp residues which are, for practical experimental conditions, totally inaccessible to the aqueous solution. The former class contains 2 Trp residues, while only a single Trp is identified in the latter class. The presence of a tetradecapeptide substrate or a nonhydrolyzable substrate analogue (peptide H-77) lowers the accessibility of iodide to the class I Trp residues. These data indicate that the class I Trp residues are at or near the peptide-binding site of renin. In addition, the finding that the class I Trp residues are quantitatively quenched more efficiently than the Trp model compound indole suggests that the environment of the class I tryptophans may be positively charged, and thus have a higher "local" concentration of iodide. These data, taken together with the available sequence and computer-generated three-dimensional structure of renin, permit us to speculate that the class I Trp residues are Trp-39 and Trp-300. This solution study of renin structure is discussed in light of the known information about renin catalysis and physiology.

Bicyclic ring formation is not necessary for the (auto)oxidation of ascorbic acid.

The oxidation rates of ascorbic acid and several of its derivatives have been examined in order to delineate the role of bicyclic ring formation in the autooxidation of ascorbic acid. The compounds evaluated and their respective oxidation rates at pH 7.4 are in uM X min-1: ascorbate, 0.70; 5 methyl 3,4 dihydroxytetrone, 0.65; D-iso ascorbate, 0.73; and ascorbyl palmitate, 0.64. These data do not support the contention that bicyclic ring formation is required for the oxidation of ascorbic acid because both 5 methyl 3,4 dihydroxytetrone and ascorbyl palmitate, neither of which can form the bicyclic intermediate, have oxidation rates similar to that of ascorbate. Furthermore, evidence is presented which suggests that ionization of the oxygen on C3 of ascorbic acid is an obligatory initial step in ascorbic acid autooxidation.

Conformational studies of aqueous melittin. Characteristics of a fluorescent probe binding site.

The structural basis of the interaction of melittin with amphipathic molecular assemblies, i.e. membranes, was investigated by studying the binding of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) to melittin by ultraviolet and fluorescence spectroscopy. Monomeric melittin did not significantly bind TNS as judged by UV and fluorescent spectroscopy. Tetrameric melittin bound two TNS molecules per protomer with dissociation constants (Kd) of 4.2 X 10(-6) M. TNS binding to tetrameric melittin led to an increase in fluorescence quantum yield of 180-fold over the value for TNS alone in aqueous buffer (phi H2O = 0.004). Five independent experimental findings suggest that the arginine residues of melittin provide one portion of the TNS binding site (presumably by formation of an especially stable "ringed-structure" salt bridge between the tetrahedral sulfonyl anion of TNS and the argininyl residues of melittin): 1) the Kd for binding is independent of pH from 6.0 to 10.8, the range in which the alpha-aminoglycine and epsilon-aminolysines titrate; 2) TNS binding fails to perturb the kinetics of the reaction of 2,4,6-trinitrobenzenesulfonate with Lys-21 or Lys-23; 3) 1,7,21,23-acetyl-melittin, in which the NH2 terminus and all lysines are acetylated, binds TNS with a Kd similar to that for normal melittin; 4) guanidination of the NH2-terminal glycines and lysines of melittin (forming N-guanidoglycine and homoargininyl residues, respectively) increases the number of TNS molecules bound per protomer to approximately 5; 5) conversion of Arg-22 and Arg-24 to the anionic N7, N8-(2,3-dihydro(7,7-dimethyl))bicyclo[2.2.1] heptane - 1 - methanesulfonylborate complex abolishes TNS binding, as judged by fluorescence.

Biochemical studies of immune complexes. I. Isolation of circulating immune complexes using a bifunctional exclusion chromatography matrix.

A simple and rapid method of isolating immune complexes from serum is described. The method utilizes the binding properties of a bifunctional chromatography matrix consisting of diethylaminoethyl and Cibacron blue F3GA functional groups linked to agarose beads. This column matrix binds all normal serum proteins except IgG and transferrin, excluding these proteins, and permitting them to be collected in the wash-through, void volume. Immune complexes prepared in vitro with human serum albumin were rapidly isolated from starting serum by this method. This method may have general application for the purification of immune complexes from serum in pathological conditions and should foster subsequent biochemical and immunological studies, including identification and characterization of immune complex antigens.

Biochemical studies of immune complexes. II. Purification of immune complexes from sera of patients with malignant melanoma.

Circulating immune complexes (CICs) from serum samples of patients with malignant melanoma were isolated in high yield utilizing a bifunctional chromatography matrix that binds all normal serum proteins except IgG-containing immune complexes (ICs), IgG, and transferrin. Protein fractions were subsequently analyzed by polyacrylamide gel electrophoresis (PAGE) and characterized by molecular weight. By this method, one common and several unique component nonimmunoglobulin proteins of the ICs were identified. These proteins possess similar molecular weights to several previously described human melanoma antigens. This technique should permit biochemical and immunological characterization of the component antigens of CICs in melanoma and other malignancies.