In vitro, animal, and human characterization of OPTISON infusions for myocardial contrast echocardiography.

UNLABELLED: Traditionally, performing myocardial contrast echocardiography with OPTISON required maximal bolus dosing. However, sustained and consistent opacification of the myocardium would be preferable for perfusion imaging. METHODS: Images of 5 anesthetized dogs and 6 human volunteers were obtained with a second harmonic ultrasound system during bolus administration of OPTISON and 2 infusion techniques. One infusion technique used diluted OPTISON, and the other used the buoyant properties of OPTISON microspheres by placing the contrast agent between an infusion source and the intravenous site in a vertically oriented extension line (ELT). Myocardial intensities and in vitro microsphere characteristics were analyzed to assess the consistency of microsphere delivery over time. RESULTS: In addition to providing higher myocardial opacification intensity than diluted infusions, ELT infusions provided consistent microsphere concentration, phantom enhancement, and near-peak bolus-level myocardial opacification for 7 to 15 minutes. The myocardial intensity at 3 and 5 minutes in human subjects during ELT infusions (30 mL/h; 2.5 mL) was lower (220 arbitrary units [au] and 165 au, respectively) but not significantly different (P =.3 and.1, respectively) than the peak myocardial intensity (265 au) after bolus administration. CONCLUSION: This new ELT infusion method provides an acceptable alternative to bolus administration of OPTISON for prolonged myocardial opacification.

Contrast-enhanced ultrasound for guidance of local tumor ablation.

The objective was to assess the efficacy of sonography with and without contrast medium enhancement in guiding and monitoring percutaneous ethanol ablation of tumors in an animal model. VX-2 carcinoma was implanted into the thighs of New Zealand white rabbits and examined by grey-scale ultrasound, color, power, and pulse Doppler, before and after injection of 95% ethanol into the tumors. Injections of ethanol were guided by ultrasound to sites of tumor vascularity, until all tumor vascularity had been obliterated. Microbubble contrast medium or saline was injected i.v. prior to each of the ultrasonic interrogations. Arteriography was performed before and after ablation. Selected tumor samples were submitted for histologic examination. Contrast enhanced tumor vascularity over saline controls in all cases. In some, incompletely ablated foci of tumor could only be identified with contrast medium enhancement. Arteriography showed complete ablation of all but 1 tumor. We conclude that ultrasound enhanced by contrast better shows the presence or absence of tumor vascularity. Ultrasound enhanced by contrast might offer an accurate means of guiding and monitoring percutaneous ethanol injection for tumor ablation.

Contrast-enhanced sonography of tumor neovascularity in a rabbit model.

This project was designed to detect the development of tumor neovascularity and determine if intravenous microbubble contrast improves visualization of otherwise undetectable tumors in an animal model. VX-2 carcinoma was implanted into one thigh of 10 New Zealand white rabbits. Tumors were assessed without and with contrast at 1- to 4-day intervals from day 3-19 postimplantation, using gray scale, color flow, pulse Doppler and power Doppler imaging. Tumor vascularity was compared with the contralateral thigh muscle, so each animal was its own control. Contrast injection improved visualization of tumor neovascularity. Early tumors had homogeneous vasculature but, with time, the centers became less vascular, while the periphery increased. Following contrast injection, color gain was decreased by 40% without compromising color intensity. Neovascularity was detected by contrast injection before the tumor could be palpated or visualized by gray scale. Based on these data, we conclude that enhancement of neovascularity by intravenous contrast permits earlier detection and improved visualization of soft tissue tumors in rabbits.

Lack of bioeffects of ultrasound energy after intravenous administration of FS069 (Optison) in the anesthetized rabbit.

The current study was designed to provide a sensitive in vivo model to maximize the potential bioeffects (measured by hemolysis) of B-mode ultrasound energy in combination with FS069 (Optison). B-mode ultrasound energy was delivered to anesthetized male New Zealand white rabbits with a phased array 5 MHz transducer on a Hewlett-Packard Sonos 1500 ultrasonograph, with transmit level set to maximum (40 dB, approx 135 W/cm2). FS069 (Optison), latex particles in human albumin, or human albumin alone (vehicle) was infused via an ear vein at 0.6 mL/kg. No statistically significant changes were noted in serum free hemoglobin or lactate dehydrogenase either over time or between groups.

Contrast enhanced sonography of visceral perfusion defects in dogs.

The purpose of this study was to assess the utility of an intravenous contrast agent (FS069) for visualization of normal visceral perfusion compared with perfusion after segmental infarction in a canine model. Six mongrel dogs were used as subjects. Splenic, renal, hepatic, and small bowel perfusion was assessed without and with intravenous microbubble contrast material using gray scale, color Doppler, pulsed Doppler, and color power Doppler sonography. Each organ was then reassessed after ligation of a segmental vessel. Imaging was again performed without and with contrast material using all four ultrasonographic modalities. In all organs color and spectral Doppler signals were significantly enhanced from normally perfused tissue after intravenous contrast agent injection. Ischemic areas were more conspicuous after contrast medium injection except in the liver. Hepatic perfusion was maintained by portal flow in the liver despite arterial ligation. Ligation of collateral arcades was required to produce bowel ischemia. Intravenous injection of FS069 improves evaluation of visceral perfusion and identification of focal visceral ischemia in dogs. These results suggest that this agent may increase sensitivity for detection of blood flow in small and deep vessels.

Contrast-enhanced ultrasonographic visualization of gonadal torsion.

The purpose of this study was to investigate the effect of use of intravenous ultrasonographic contrast agent in the diagnosis of gonadal torsion in an animal model. After examination of perfusion of normal gonads in male and female dogs, torsion was produced and maintained mechanically. Presence and pattern of blood flow was then reassessed before and after administration of varying doses of perfluorocarbon-filled microsphere intravenous contrast agent. Modes of examination included gray scale, color flow, color power, and spectral analysis using a transducer placed directly on the surgically exposed gonads. Although no enhancement was perceptible on gray scale images, color and spectral Doppler signals were significantly stronger after injection of contrast agent in both normal and rotated gonads. Perfusion asymmetry was more obvious. Some residual flow was seen in partially rotated testes, and absence of flow was documented in both partially and fully rotated ovaries. Use of intravenous contrast medium improves demonstration of altered flow patterns in ischemic gonads, allowing more confident diagnosis.

Myocardial contrast echocardiography: reliable, safe, and efficacious myocardial perfusion assessment after intravenous injections of a new echocardiographic contrast agent.

Reliable and reproducible myocardial opacification after intravenous administration of echocardiographic contrast agents has remained elusive. This study was performed to determine whether a new agent, FS069, a suspension of perfluoropropane-filled albumin microspheres (3.6 microns average microbubble size, concentration 8 x 8(8)/ml), could achieve safe and successful myocardial opacification in open-chest dogs. Seventeen dogs (group 1, n = 7, group 2, n = 10) underwent two-dimensional echocardiography before, during, and after the administration of intravenous FS069. Safety was evaluated by measuring arterial and pulmonary artery pressures, heart rate, blood gases, systolic function, myocardial blood flow, and postmortem analysis of myocardial viability by triphenyl-tetrazolium chloride staining. Efficacy to detect changes in regional myocardial perfusion was assessed by injecting FS069 at baseline, after sequential coronary occlusions and reperfusion, and during intravenous vasodilators with and without coronary occlusions. Results were compared with radiolabeled microspheres. FS069 was found to be safe and effective. In the absence of coronary occlusions, uniform myocardial opacification was observed in all dogs. A perfusion defect was observed in all dogs during coronary occlusions. Background-subtracted peak contrast intensity in the myocardium correctly identified regional myocardial blood flow changes and showed a significant correlation with radiolabeled microspheres (r = 0.65, p = 0.0001).

Myocardial contrast echocardiography in experimental coronary artery occlusion with a new intravenously administered contrast agent.

A new intravenously administered ultrasound contrast agent was studied in eight dogs during intermittent coronary artery occlusion. The area of the myocardial contrast defect was compared with that of the acute wall motion abnormality induced by coronary occlusion. A close correlation was found between these two independent measures of acute myocardial ischemia. The peak change in myocardial intensity during coronary occlusion was significantly less than for the same segment before ischemia and for a remote nonischemic segment. This new, intravenously administered ultrasound contrast agent can be used to evaluate the spatial distribution of hypoperfused myocardium and should therefore prove valuable in the clinical evaluation of ischemic syndromes.

Reduction of animal use with improved procedure for acute tolerance assessment.

A retrospective evaluation of previously conducted acute tolerance tests in rats and mice, following oral and intravenous routes of administration, and a review of the literature indicated that a minimum of three animals per group was adequate to characterize acute tolerance. Limited exploratory testing, using six compounds for which classical LD50 tests had been performed previously, indicated that fewer numbers of rats gave comparable results. A method is proposed by which the acute tolerance of a compound may be adequately characterized in both sexes using approximately 30 animals instead of the 70 to 100 used in the classical LD50 test. The objective of the testing procedure is identification of the maximum nonlethal dose or the minimum lethal dose, rather than determination of the LD50.

Nephrotoxicity of pravadoline maleate (WIN 48098-6) in dogs: evidence of maleic acid-induced acute tubular necrosis.

Single oral administration of pravadoline maleate (WIN 48098-6), the maleic acid salt of WIN 48098, induced acute tubular necrosis (ATN) in male and female beagle dogs at dosages > or = 40 mg/kg (WIN 48098 base (31 mg/kg) and maleic acid (9 mg/kg)). Subsequent oral studies were conducted with equimolar dosages of maleic acid and WIN 48098-7, the ethanesulfonate salt of WIN 48098, to determine the nephrotoxic moiety of WIN 48098-6. ATN was observed for dogs given only maleic acid at single oral dosages > or = 9 mg/kg. This result provided evidence that maleic acid was responsible for the nephrotoxicity observed in dogs given single oral dosages of WIN 48098-6. The induction of maleic acid-related nephrotoxicity in dogs may confound the interpretation of toxicologic studies of maleic acid salts of basic pharmaceutics, if the dosage of test article results in the delivery of dosages of maleic acid > or = 9 mg/kg. Furthermore, the results of these studies underscore the importance of establishing maximum no-observed-effect dosages and target organ toxicity profiles for acids and bases that are commonly used in the development of salts of pharmaceutics.

Chronic (1-year) safety evaluation of ipazilide fumarate, an antiarrhythmic agent, administered orally to rats.

Ipazilide fumarate (WIN 54177-4) is a chemically novel antiarrhythmic agent that prolongs ventricular refractoriness and possesses antiectopic activity. The compound is being developed as oral and iv therapy for ventricular and supraventricular arrhythmias. Since ipazilide therapy may require long-term use, a 1-year oral gavage study (daily dosages of 20, 80, or 160 mg/kg) was conducted in rats. Controls received the purified water vehicle. Treatment-related clinical signs were limited to post-dosing salivation. Increased relative liver weight (females, at 80 and 160 mg/kg) was correlated with centrilobular hypertrophy, but was not associated with significant increased serum liver enzymes activities. These liver weight changes were interpreted as an adaptive metabolic response and were not considered toxicologically significant. An increased incidence of centrilobular hepatocellular vacuolation representing lipid accumulation over that observed for male controls occurred for males in all ipazilide-treated groups. This observation, however, was not correlated with elevated hepatic enzyme activities. Hepatocellular basophilic foci were observed for females only (80 and 160 mg/kg groups); however, the significance of this lesion is unclear. Transient dosage-related duodenal villous atrophy/sloughing was observed for males from the 80 and 160 mg/kg groups. Mild increases in hemoglobin, hematocrit, urea, and creatinine (160 mg/kg), attributed to treatment, were considered of minor toxicologic importance. Likewise, no clinical or anatomical pathologic observations that may indicate cardiac toxicity were determined. It is concluded that a dosage of 20 mg/kg (two to three times the clinical efficacious dosage) was considered a no-effect dosage level since it did not produce any effects of toxicological significance.

Subchronic oral and intravenous (i.v.) safety evaluation and pharmacokinetics in rats and dogs of ipazilide fumarate (Win 54, 177-4), an antiarrhythmic agent.

Ipazilide fumarate (Win 54, 177-4) is a chemically novel antiarrhythmic agent that prolongs ventricular refractoriness and possesses antiectopic activity. Subchronic (29 days) nonclinical safety evaluation of ipazilide was conducted following oral and iv administration in Sprague-Dawley rats (20-320 mg/kg oral and 1.25-10 mg/kg iv) and 14 and 28 days in beagle dogs (3-30 mg/kg oral and 2.5-20 mg/kg iv). The pharmacokinetic parameters of ipazilide indicate that ipazilide is absorbed (tmax less than or equal to 1 hr) in fasted rats and dogs following single and repeated oral administrations. The apparent elimination half-life in the two species is approximately 1 hr (except in rats at a dosage of 320 mg/kg), suggesting rapid clearance. Increases in liver weights (rats 320 mg/kg) accompanied by the observation of centrilobular hypertrophy of hepatocytes were considered an expression of an adaptive metabolic response of the liver to ipazilide and may be associated with the induction of microsomal enzymes. Duodenal villous atrophy and epithelial hyperplasia (rats, 80 and 320 mg/kg) were interpreted to represent an irritant response to the drug. Local irritation was also observed at the injection site in rats and dogs. Dogs tolerated the oral and the iv administration of ipazilide at dosages of up to 30 and 20 mg/kg, respectively. Despite emesis (oral dogs), which was reduced in frequency following repeated treatment over several weeks, plasma levels in treated dogs (i.e., Cmax 4-5 micrograms/ml) were approximately twice that required to convert spontaneous arrhythmias in the conscious dog model 24 hr after myocardial infarction.(ABSTRACT TRUNCATED AT 250 WORDS)

Assessment of the safety of chemicals administered intravenously in the neonatal rat.

A 3-day-old neonatal rat model for the safety assessment of various drugs, chemicals, and nutrients administered intravenously is described. This model was used to assess the safety of di(2-ethylhexyl) phthalate (DEHP), cyclohexanone, and a 3:1 mixture of medium and long-chain triglyceride emulsions following subchronic administration using the intravenous route. The administration of DEHP at dosage of 164.8 mg/kg for 18 consecutive days resulted in a small but statistically significant increase in liver weight and SGOT activity. However, no conclusive histopathological alternations could be discerned between livers from DEHP and normal saline (control) animals. No effects were observed among neonates treated with cyclohexanone at dosages up to 25 mg/kg for 18 consecutive days. Because of technical difficulties emanating from high dosage volumes, the administration of lipid emulsion mixture lasted 9 consecutive days only. Except for transient ataxia and sedation, no adverse effects were observed among neonates that received 3:1 medium- and long-chain triglyceride emulsion for 9 consecutive days beginning on day 3 postpartum. No adverse effects were observed among neonates receiving the lipid emulsions for 7 consecutive days beginning on day 12 postpartum.

Assessment of potential toxic effects of treated Hemofil injection in rats and mice and on the systemic hemodynamics in dogs.

The toxic effects of treated Hemofil (T-AHF) injection were evaluated by acute and subchronic intravenous administration to rats, mice, and dogs. Acute iv administration of T-AHF to rats and mice at dosages of 1320 units (U) of Factor VIII/kg did not produce toxic signs. Both species were found to have an LD50 greater than 1320 U. Seven-day iv administration to rats of T-AHF at dosages of 20, 40, and 60 U/kg and 3-mo administration (3 times/wk for 13 wk) of T-AHF at dosages of 100 and 200 U/kg did not produce any signs of toxicity. There were no treatment-related effects on body weights, hematology, clinical chemistry, urinalysis, ocular tissues, or histopathology. Intravenous administration to dogs at 0.5, 1.0, and 5.0 ml/min . kg (28 U/ml, 100 U/kg at each rate) produced no significant adverse effects on mean arterial pressure, cardiac output, or heart rate. No adverse changes in pulmonary function, as reflected by arterial blood-gas profiles, were observed. It is concluded that animals tolerated well T-AHF administered at dosages and rates similar to or greater than dosages used clinically. The results obtained from these studies establish a reasonable margin of safety and support the acceptability of the T-AHF for clinical use.

Assessment of the cataractogenic potential of cyclohexanone in guinea pigs and rabbits.

The cataractogenic potential of cyclohexanone administered intravenously (0.5 and 5.0 mg/kg) and percutaneously (0.5 ml) was assessed in guinea pigs and rabbits. The negative control article was 0.9% sodium chloride. Positive control articles for guinea pigs were iv galactose and percutaneous dimethyl sulfoxide (DMSO), and for rabbits, iv poly(I) X poly(C) and percutaneous DMSO. An untreated group of guinea pigs was used as a sham control. Animals were treated three times a week for 3 consecutive weeks. Ophthalmic examinations performed monthly for 6 months for treated animals and 7 months for untreated animals revealed the presence of anterior subcapsular vacuoles in guinea pigs in all groups. These lesions consisted of swollen lens fibers that progressed to fiber disruption and protein liquefaction. There was no statistical difference in the incidence and severity of the lesions among treatment groups. No lenticular alterations were noted in any of the rabbits treated with cyclohexanone or any other chemical. It was concluded that these alterations are, apparently, an inherent characteristic of the guinea pig, making that animal an unsuitable model for the assessment of cataractogenic potential of xenobiotics.

In vitro studies on methyl mercury distribution in human blood.

Studies comparing the methyl mercury (mHg) content of maternal and newborn blood have shown increased levels in the newborn. This has been attributed to facilitated transplacental diffusion because of high fetal hematocrit (Hct). This study shows the converse, that the diffusion of mHg diminishes progressively with increasing Hct. The diffusion of m203Hg across a Millipore membrane (0.45 microns) separating compartments A and B of a diffusion cell was studied. When both compartments contained saline or plasma alone, equilibration from A to B occurred in 5 h. Introduction of human red blood cells (RBC) in saline (Hct 20%) into B resulted in a twofold increase in diffusion of mHg when compared to saline alone. Increasing Hct in saline in compartment B resulted in a progressive decrease in diffusion (r = -0.95, P less than 0.001). The presence of RBC in plasma (Hct 20%) in B resulted in a 70% decrease in diffusion; with increasing Hct, diffusion was further reduced (r = -0.95, P less than 0.001). Direct addition of mHg to RBC in saline resulted in 98% RBC uptake. Increasing concentrations of plasma (at a constant Hct) resulted in a progressive decrease in RBC uptake. In undiluted plasma at Hct 14%, RBC uptake of mHg was 35%. Plasma electrophoresis showed that much of the mHg was associated with a high-molecular-weight lipoprotein fraction. Plasma components appear to be important in the distribution of mHg in blood, and may be a factor in the relatively higher blood levels in the fetus.

Methyl mercury toxicity in the chick embryo.

The toxicity of methyl mercury (mHg) in the developing chick embryo was investigated. The relationship of dose, time of administration (i.e., days 4-9 of development), and body levels of mercury was examined. The LD50 for mHg injected into the yolk sac on day 5 of incubation was 40-50 micrograms. Embryos dying within 24 hours showed increased total body mHg levels when compared to survivors (219 +/- 67 vs. 105 +/- 41 micrograms/gm, mean +/- SD). Absorption was dose-related, with a good correlation between mortality and body, blood, and brain levels. Daily analysis of body mHg levels after injection on day 5 showed continued mHg accumulation (0.88 +/- 0.35 micrograms/embryo/day). However, the rate of embryo growth exceeded the rate of mHg absorption, resulting in a progressive decrease in mHg in concentration in tissues (from 94.5 +/- 34.2 micrograms/gm on day 6 to 45.3 +/- 13.4 on day 9). Administration after day 5 resulted in a significant reduction in levels of mHg in the brain on day 18 (from 11.4 +/- 2.1 micrograms/gm when given on day 5 to 8.4 +/- 2.3 when given on day 9) and in mortality (from 64% to 33%). Because blood mHg levels remained unchanged, the increased brain levels and higher mortality early in embryogenesis may reflect facilitated transfer of mHg across a poorly developed blood-brain barrier. Later in development, the reduced mortality and lower brain mHg levels correspond to the formation of specialized interendothelial junctions and a more effective blood-brain barrier.

Chronic intraperitoneal administration of fluids in rats.

A simple method for continuous long-term intraperitoneal administration in rats was devised. Polyethylene tubing was inserted into the peritoneal cavity through an incision in the abdominal wall just below the rib cage. The other end of the catheter was then guided subcutaneously toward an incision located in the back of the neck. The external opening of the catheter was blocked with a small piece of stainless steel wire. The indwelling intraperitoneal catheter permitted instillation of various solutions in rats four times daily for up to 94 consecutive days without apparent stress or pain on the treated animals. The catheter remained patent for the duration of the study in 39 of 60 (65%) rats.

Assessment of the toxicity of cyclohexanone administered intravenously to Wistar and Gunn rats.

The toxicity of cyclohexanone, used as a solvent cement in polyvinyl chloride medical devices, was assessed in Wistar and Gunn rats. The Gunn rat was used because it has a negligible activity of UDP glucuronosyltransferase toward bilirubin and certain other aglycones. Cyclohexanone was administered iv for 28 consecutive days to Wistar and Gunn rats in two doses (50 and 100 mg/kg), using solutions containing 0.25 and 0.50 g per 100 ml, respectively, at a constant volume of 20 mg/kg. Saline (0.9% NaCl) was used as the control. Daily observations for signs of toxicity showed no adverse effects in Wistar or Gunn rats injected with either dose. Daily weight changes of control and test animals were similar. Ophthalmologic examinations revealed no treatment-related structural lesions. No adverse effects were noted when the data from the hemogram or clinical chemistry parameters were evaluated. Gross pathological and histopathologic assessment showed no alterations due to cyclohexanone treatment. Urinary excretions of total and glucuronide conjugates of cyclohexanol were similar for Wistar and Gunn rats; less than 1% was excreted as free cyclohexanone and cyclohexanol. It is concluded that the Gunn rat is capable of forming glucuronides of cyclohexanol and that cyclohexanone at these doses has a negligible toxic potential.