Intestinal and hepatic lesions in mice, rats, and other laboratory animals after intravenous administration of gas-carrier contrast agents used in ultrasound imaging.

Single intravenous administration of three different gas-carrier contrast agents used in ultrasound imaging to mice caused inflammation, necrosis, and ulceration of cecum and proximal colon (cecocolonic area) and focal necrosis in the liver. Similar intestinal lesions were also found in rats after treatment with a single iv administration of a gas-carrier contrast agent. Strain differences in the incidences of these lesions were found in both rats and mice. HsdHan:NMRI mice were among the most sensitive of the strains of mice studied. Even at the lowest dose of Sonazoid technically possible to inject in HsdHan:NMRI mice, lesions were found and a no-effect dose could not be determined. In a time-course experiment in HsdHan:NMRI mice, it was found that the lesions began to develop in the cecum and colon within 15 to 30 min after dosing. Lesions in the liver were first observed 120-240 min after dosing. Diet played a role in the etiology of the lesions, as HsdHan:NMRI mice given a diet with reduced amounts of cellulose and starch had reduced incidences of lesions, and when glucose was the only carbohydrate source, no lesions were observed. No intestinal or hepatic lesions were found in guinea pigs or rabbits after repeated intravenous administrations of Sonazoid. In dogs, minimal to mild granulocytic inflammation of the cecum and/or colon was found after daily repeated intravenous injections for 28 days, but not after daily repeated administration for 14 days nor after a single administration. It is proposed that the intestinal and hepatic lesions in rats and mice after a single intravenous injection of gas-carrier contrast agents are caused by a common mechanism: intravascular growth of gas-carrier agents in tissues with gas supersaturation, as occurs in the cecal wall of rats and mice. In this particular environment the growing gas bubbles cause ischemia and necrosis in the cecal and colonic wall and liver. This proposed mechanism of action is consistent with the absence of clinical reports indicative of intestinal and/or hepatic lesions in humans after administration of gas-carrier contrast agents.

Air-filled ultrasound contrast agents do not damage the cerebral microvasculature or brain tissue in rats.

RATIONALE AND OBJECTIVES: Air microemboli may damage the cerebral microvasculature. The aim of this study was to evaluate the safety of ultrasound contrast agents composed of air microspheres with regard to cerebral damage when administered into the arterial system (ie, when not filtered by the capillary system of the lungs). METHODS: Three experimental methods were used in 75 rats after injection of either Albunex, Echovist, or Levovist into the left heart ventricle. The alkaline phosphatase (ALP) method to demonstrate small segmental brain capillary and arteriolar dilatations (SCADs), intravenous injections of Evans blue and fluorescence microscopy to detect increased vascular permeability (blood-brain barrier damage), and histologic examination of the brain to detect microinfarction. Intracardiac injections of saline, air, and corn oil were used as controls. RESULTS: Brain microinfarcts and SCADs formation of the brain microvasculature occurred only after control injections with corn oil. None of the brains from animals that received ultrasound contrast agent showed gross discoloration, as an indication of increased vascular permeability, with the Evans blue/fluorescence microscopy method. Definite leakage of Evans blue occurred only after large doses (150 microL) of air. CONCLUSIONS: This study indicates that ultrasound contrast media composed of air microspheres do not cause lesions of the brain microvasculature or parenchyma.

Effect of microsphere size distribution on the ultrasonographic contrast efficacy of air-filled albumin microspheres in the left ventricle of dog hearts.

RATIONALE AND OBJECTIVES: The in vitro ultrasonographic contrast efficacy of air-filled albumin microspheres has been found to depend on the size distribution of microspheres. The objective of the current study was to empirically describe the relationship between the size distribution of injected air-filled albumin microspheres and the in vivo contrast efficacy after lung capillary filtration in a dog model. METHODS: Twenty different air-filled microspheres with large and well-defined differences in size distribution were prepared from nine different batches of Albunex (Molecular Biosystems Inc.) and subsequently characterized by Coulter counting. The in vivo ultrasonographic contrast enhancement of these preparations was investigated with a VingMed CFM750 in closed chest model in six mongrel dogs. The observed contrast efficacy, measured as gray-level enhancement in the left ventricle (LV), was correlated to the microsphere size distribution, using both univariate and multivariate approaches. RESULTS: The results demonstrated a significant contribution to LV contrast efficacy from microspheres larger than approximately 7 microm, and a lack of contribution from microspheres smaller than approximately 7 microm. Linear relationships were found between LV contrast efficacy, and both the number concentration of microspheres between 8 to 12 microm and the total microsphere volume concentration. No significant covariance between in vivo contrast efficacy and the number concentration between 1 to 38 microm or 4 to 10 microm was observed. The multivariate model showed a significant contribution to the in vivo gray-level enhancement from microspheres in the size range 7 to 15 microm, with optimal efficacy per microsphere at approximately 13 microm. CONCLUSIONS: Large microspheres (> 7 microm), which had been expected to be trapped in the lung capillary bed, contribute most of the observed ultrasound contrast in the LV of the heart.

Neural tolerance of the non-ionic dimers iodixanol and iotrolan and the non-ionic monomer iopamidol during myelography in non-anaesthetised rabbits.

PURPOSE: The neural tolerance of the recently introduced dimer iodixanol (320 g I/l) was compared with that of the dimer iotrolan (300 g I/l) and of the monomer iopamidol (300 g I/l), both used in clinical myelography. MATERIAL AND METHODS: Non-anaesthetised rabbits were injected into the cisterna magna with Ringer's solution (control) or contrast media (CM) at doses of 1.0 or 0.5 ml/kg b.w. The behaviour of the animals (10 in each of 7 groups) was evaluated for signs of excitation and depression during the first 3 hours after injection. RESULTS: At the dose level of 1.0 ml/kg b.w., iodixanol produced no seizures but did cause focal twitching in 4/10 rabbits. Iopamidol produced grand mal seizures in 2/10 and hyperexcitability in 4/10 rabbits. Iotrolan produced generalised grand mal seizures in 8/10 rabbits, an incidence of excitation significantly greater than that of iodixanol (p < 0.01) and iopamidol (p < 0.05). The excitative effects of iodixanol were not significantly different from those of iopamidol and Ringer's solution. All 3 CM produced similar depressive effects on rabbit behaviour. Ringer's solution caused no depressive effects (p < 0.01). A clear dose response was produced with all 3 CM after treatment with a lower dose of 0.5 ml/kg b.w. CONCLUSION: The results indicate that the neural tolerance in the rabbit of iodixanol is higher than that of iotrolan, and is at least equal to that of iopamidol.

Renal effects of iodixanol in experimental animals.

The effects of the new nonionic dimeric hexa-iodinated contrast media (CM) iodixanol on renal function and morphology were investigated in 7 independent studies in rats, rabbits and monkeys and compared with other iodinated CM. No significant effect on serum creatinine levels was seen at doses up to and including 5 g I/kg in rats and 10.5 g I/kg in rabbits. An immediate and transient increase in proteinuria was found in rabbits when 10.5 g I/kg was administered as a bolus, and when 12.5 g I/kg was administered as a slow infusion in a comparative study with several CM. Increased serum elimination half-life was shown by measuring serum iodine concentrations after the infusion of 12.5 g I/kg. The effect of a high dose of iodixanol on proteinuria and elimination half-life were in this study in the same range as those of the monomeric nonionic CM, but less pronounced than those of the monomeric ionic CM. Reduced renal capacity was induced in male rats by performing unilateral nephrectomy 4 weeks before i.v. injection of iodixanol or iopamidol (2g I/kg). The administration of CM did not affect renal function monitored as serum concentrations of creatinine and urea. The vacuolation of renal proximal tubular cells and kidney iodine retention were investigated in rats 48 hours after administration of different doses of iodixanol or iotrolan. The no-effect level for vacuolation was 0.5 g I/kg for both CM. Iodine retention was higher in male than females rats, and was higher for iodixanol than iotrolan at the 2 highest dose levels (3 and 5 g I/kg). No difference in iodine retention was found at the other dose levels (0.25-1g I/kg). The reversibility of renal proximal tubular vacuolation after administration of iodixanol was studied in male rats (1.2 g I/kg) and monkeys (1.2 and 3.6 g I/kg). The vacuolation was more pronounced in rats than in monkeys. Vacuolation was completely reversed in all rats 3 weeks after dosing, and 2 of 3 monkeys 3 days after a dose of 1.2 g I/kg. The degree of vacuolation evident in renal percutaneous biopsy specimens from monkeys 14 days after i.v. administration of iodixanol at a dose of 3.5 g I/kg was not significantly different to that in control animals. In conclusion, iodixanol affected renal function to the same degree as did the nonionic monomeric and dimeric comparative media, but to a lesser degree than the ionic monomers. The degree of renal proximal tubular cell vacuolation induced by iodixanol seems to be species-dependent, being less pronounced and more quickly reversed in monkeys than rats.

Neurotoxicity of water-soluble contrast media.

A review is given of the development of the water-soluble contrast media (CM) with particular attention to the frquency of neurological complications. A remarkable improvement was achieved following the introduction of the nonionic agent metrizamide in 1974, and a further decrease in neurotoxicity was obtained with the newer nonionic monomers, which have multlple hydroxyl groups included at different sites of the molecule. Theoretical considerations and experimental studies suggest that the neurotoxicity of the new nonionic dimeric agents shuold be at least within the low range seen with the monomeric ones, but further experience is needed before definite conclusions can be drawn in this respect. The mechanisms responsible for the neurological complications seen with CM are unknown but certain critical groups on the CM molecules are known. Several animal models have been developd, which may help predict the degree of neurotoxicity.

Adverse reactions in myelography.

An attempt was made to assess the usefulness of using animal models to predict the neural tolerability in man of iodinated contrast media (CM) in general, and of the new nonionic dimer iodixanol in particular. For this purpose, the results from 6 animal experiments evaluating excitative and depressive effects of subarachnoidally injected CM in nonanesthetized rabbits were compared with the results from 22 randomized double-blind clinical trials dealing with post-myelographic adverse reactions. Comparisons were made as regards the nonionic monomers metrizamide, iohexol, and iopamidol, and the dimer iotrolan. The results seem to justify the conclusion that the convulsive effects of CM can be reliably predicted from animal experiments. The animal model cannot be used to predict specific types of nonconclusive adverse reactions in man, but reflects well the differences in frequencies of minor reactions following clinical myelography with different nonionic CM. In general, the neural tolerability of iodixanol may be expected to be better than that of the nonionic monomers and approximately equal to that of iotrolan.

Neural tolerability of iodixanol in mice and dogs after single and repeated intracisternal administration.

The neural tolerability of iodixanol has been assessed in studies in mice and dogs. The animals received up to 4 injections in the cisterna cerebellomedullaris while under light anesthesia. Iotrolan was included as a reference study in 1 study. The observations comprised assessment of clinical behavior, cerebrospinal fluid analysis, hematology, clinical chemistry and/or macroscopic and microscopic examination at necropsy. In addition, the repeated-dose dog study, urinalysis and opthalmoscopy were performed, electrocardiograms obtained, and respiratory rate, blood pressure and rectal temperature measured. Clinical signs and minor pathological changes caused by the injection procedures were seen in all studies in some animals treated with iodixanol as well as in control animals. Single (2.0 g I/kg) and repeated (0.960 g I/kg) intracisternal administration of iodixanol to mice caused no significant toxicological effects. Two dogs treated with a high dose of iodixanol (0.256 g I/kg; 0.8 ml/kg) had pathological changes (meningeal inflammation and/or necrosis) that were more severe than those observed in control dogs. Single and repeated intracisternal administration of 0.128 g I/kg (0.4 ml/kg) of iodixanol to dogs, however, caused no significant toxicological effects. Apart from the findings in the 2 dogs, the neurological and neuropathological changes elicited by iodixanol were similar to those induced by control or reference substances. The results from these intracisternal toxcity studies in mice and dogs indicate a significant margin of safety regarding the use of iodixanol in clinical intra-thecal indications.

Tissue reaction follwing intratracheal application of roentgen contrast media in rats.

Contrast media (CM) given orally for roentgen examination of the upper gastrointestinal tract may inadvertently enter the lungs. The present paper describes the local effects on the lungs of rats after a single intratracheal instillation of the nonionic, iso-osmolar, dimer CM iodixanol and iotrolan, and the ionic hyperosmolar, monomeric CM diatrizoate. Hydrochloric acid (HCl) and saline were included as positive and negative controls, respectively. The test compounds were given by intratracheal instillation to anesthetized rats at low dose volumes of 0.5 ml/kg b.w. The animals were killed 6 hours, 24 hours or 7 days after dosing, and the trachea and lungs subjected to histopathological examination. Acute signs of dyspnea were observed in 7 out of 15 animals that received HCl. No clinical signs could be related to treatment with any of the CM. Histomorphological assessment of the respiratory tract did not reveal any CM-related adverse effects, whereas animals treated with HCl showed marked histopathological changes. The results indicate that accidental exposure of the respiratory system to iodixanol, iotrolan or diatrizoate is unlikely to cause any significant tissue damage or lead to respiratory complications.

Absorption and excretion of iodixanol after intragastric administration to rats.

Absorption and excretion of iodixanol 320 mg I/ml were investigated in rats after intragastric administration of 2.5 g I/kg b.w. Animals were observed for up to 96 hours after treatment, and blood, urine and feces taken at several time-points throughout the experiment. Concentrations of iodixanol in serum and urine were measured by means of reversed-phase high-performance liquid chromatography. Fecal concentrations of iodixanol, based on iodine measurements, were determined by X-ray fluorescence spectrometry. Serial radiographs were obtained and histopathological examination was performed on selected tissues. The results indicate that less than 1% of the intragastric dose of iodixanol is absorbed from the intestine into the blood stream. No adverse clinical signs were observed, and there were no treatment-related histomorphological findings.

Biodistributions of air-filled albumin microspheres in rats and pigs.

The air-filled microspheres of the ultrasound-contrast agent Albunex are unique in that the walls consist of human serum albumin molecules which have been made insoluble by sonication of the albumin solution. The microspheres were isolated by flotation, and the washed microspheres were labelled with 125I. The labelled material was cleared from the circulation mainly as particles, not as soluble albumin molecules. In rats, 80% of intravenously injected microspheres were cleared from the blood within 2 min. Nearly 60% of the dose was recovered in the liver, only 5% in the lungs, 9% in the spleen, and negligible quantities in kidneys, heart and brain. Of the radioactivity in the liver, more than 90% was taken up by Kupffer cells (liver macrophages). The protein in the liver was degraded apparently with first-order kinetics (half-life 40 min). In pigs, over 90% of the intravenously injected dose was recovered in the lungs. The vastly increased recovery in pig lungs, compared with that in rats, is probably due to the pulmonary intravascular macrophages of the pig; macrophages are not normally found in this location in rats (or humans). In a separate series of experiments in rats, the biodistribution of shell material from the microspheres was examined. The microspheres were made to collapse by applying external pressure on the suspension, leaving sedimentable protein material consisting of layers of insoluble albumin from the 'shells' surrounding the air bubble. The 'shells' and the microspheres were cleared from the circulation and taken up by the liver with the same kinetics. In the lungs, a higher proportion (15%) of shells than of microspheres was recovered.

Effect of pyridostigmine pretreatment, HI-6 and Toxogonin treatment on rat tracheal smooth muscle response to cholinergic stimulation after organophosphorus inhalation exposure.

The ex vivo contraction response of the rat tracheal smooth muscle was examined after 10 min in vivo inhalation of soman and/or pretreatment with pyridostigmine and/or post-exposure treatment with HI-6 ([[[(4-aminocarbonyl)pyridinio]methoxy]methyl]-2[(hydroxy imino) methyl]pyridinium dichloride) or Toxogonin (1,1'-[oxybis-(methylene)]bis[4-[(hydroxyimino)methyl]-py rid inium] dichloride). In vivo pretreatment with pyridostigmine was achieved by subcutaneous (s.c.) implantation of an osmotic pump that delivered pyridostigmine continuously (0.01 mg/h) in the neck region of the rat 18 h before soman exposure. The ex vivo cholinergic tracheal smooth muscle response increased during the first 60 min after soman exposure in animals pretreated with pyridostigmine. The amplitude of the contraction response in pyridostigmine pretreated animals was about 60% of control, compared to 15% of control without pyridostigmine pretreatment. Pyridostigmine pretreatment also produced significant recovery of the total cholinesterase (ChE) activity in plasma, but not in trachea and lung. Intraperitoneal (i.p.) injection of HI-6 or Toxogonin (50 mg/kg), immediately after 10 min inhalation exposure to soman, also significantly improved the ex vivo cholinergic contraction response of the trachea (decapitation 15 min after oxime administration). The recovery of the physiological response with Toxogonin was, however, not stable. HI-6 was superior to Toxogonin with respect to the initial airway contraction response, and the response increased up to a stable level not significantly different from control. There was no significant reactivation of the ChE activity after treatment with the oximes. Combination of pyridostigmine pretreatment and oxime treatment enhanced the recovery of the tracheal contraction response and the ChE activity in the trachea compared to treatment with oximes alone.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of calcium antagonists (omega-conotoxin GVIA, verapamil, gallopamil, diltiazem) on bronchial smooth muscle contractions induced by soman.

The effect of the calcium antagonists omega-conotoxin GVIA, verapamil, gallopamil and diltiazem was investigated on in vitro bronchial smooth muscle contraction in the rat induced by the nerve agent soman. Soman inhibits the acetylcholinesterase activity irreversibly. The effect of the calcium channel antagonists on contractions induced by electrical field stimulation and carbachol was also investigated, in order to elucidate the mechanism by which calcium antagonists inhibit the soman induced contraction. omega-Conotoxin GVIA reduced the bronchial smooth muscle contraction induced by electrical field stimulation with an almost complete inhibition at approximately 1.0 x 10(-6) M. The soman induced contraction was only inhibited by 15% at a concentration of 3.0 x 10(-6) M omega-conotoxin GVIA. The organic calcium antagonists verapamil, gallopamil and diltiazem reduced both electrically and soman induced smooth muscle contraction. Complete inhibition of the contractions induced by soman was achieved at 1.4 x 10(-4) M for verapamil and gallopamil, while diltiazem inhibited the contraction to 7% of control at 1.4 x 10(-4) M. Verapamil, gallopamil and diltiazem increased the EC50 for carbachol significantly, while omega-conotoxin GVIA had no effect. None of the calcium antagonists had any effect on the maximal contraction induced by carbachol. Verapamil, gallopamil and diltiazem blocked, however, sub-maximal contractions induced by carbachol (10(-7)-10(-5) M) resulting in a right-shift of the dose response curve. The results show that omega-conotoxin GVIA inhibits the calcium-dependent release of acetylcholine which causes contraction of airway smooth muscle, while it has no effect on smooth muscle contraction induced by soman.(ABSTRACT TRUNCATED AT 250 WORDS)

Prejunctional stimulation of cholinergic nerves in rat airway smooth muscle by an adenosine analogue.

The effect of 5'-N-ethylcarboxamidoadenosine (NECA) on rat bronchial smooth muscle was examined in vitro. Both the nerve mediated muscle contraction induced by electrical stimulation and the potassium evoked release of [3H]ACh were enhanced by NECA. The apparent affinity (EC50) of NECA in the contraction experiments was 0.30 +/- 0.06 microM. The adenosine (ADO) receptor antagonist, 8-phenyltheophylline (8-PT), inhibited the NECA induced potentiation of both the electrical induced contraction and the potassium evoked release of [3H]ACh. The EC50 and intrinsic activity of exogenous ACh were not altered in the presence of NECA (1 microM) in experiments where smooth muscle contraction were measured, indicating that NECA has a prejunctional effect and not a postjunctional effect on muscarinic receptors. The new A2 specific ADO receptor agonist 2-p-(2-carboxyethyl)-phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680) and ADO also enhanced the nerve-mediated contraction (EC50 = 35 +/- 8 microM and 69 +/- 20 microM, respectively). 8-PT (10 microM) and enprofylline (ENPF) (10 microM) inhibited the electrically induced contraction by 55 +/- 16% and 45 +/- 5% respectively. The potassium evoked release, however, was stimulated 56 +/- 6% and 39 +/- 7% by 50 microM 8-PT and ENPF respectively. The results provide evidence for a NECA specific ADO receptor in rat bronchi that is most likely prejunctional. Stimulation of this receptor, which may be of an A2 receptor subtype, enhances the nerve mediated release of ACh and thereby induce contraction of the bronchial smooth muscle.

The effect of acetylcholinesterase-inhibition on the tonus of guinea-pig bronchial smooth muscle.

The irreversible acetylcholinesterase inhibitor soman (O-[1,2,2-trimethylpropyl]-methyl-phosphonofluoridate) induced contraction of guinea-pig primary bronchial smooth muscle. The apparent affinity (ED50) of acetylcholine (ACh) was altered from control value of 12 microM to 0.3 microM following exposure of the bronchial smooth muscle to 14 microM soman for 15 min in vitro. The ED50 of the cholinergic agonist carbachol was not changed even when the acetylcholinesterase (AChE) activity was inhibited completely. The intrinsic activity (alpha) of ACh and carbachol was not significantly changed after exposure to soman for 15 min. The results demonstrate that the effect of soman is only due to its anticholinesterase activity. Furthermore, the contraction induced by histamine was not altered by concentrations of soman which increase the cholinergic stimulation. This indicates that histamine does not induce contraction of bronchial smooth muscle in guinea pig through the release of ACh or by modulation of muscarinic receptors. Furthermore, soman also inhibited the carboxylesterase activity in the primary bronchi. In respiratory tissue this group of enzymes may have a major protective function, due to their ability to bind several organophosphorus compounds. Compared to studies performed on other species, this study shows that guinea-pig bronchi are very sensitive to the AChE-inhibitor soman. Therefore, exposure to very low concentrations of AChE-inhibitors may induce contraction of bronchial smooth muscle.