Influence of day of pregnancy on rat placental, uterine, and ovarian prostaglandin synthesis and metabolism.

Synthesis and metabolism of prostaglandins in reproductive tissues of the gravid rat were studied from the time of post-implantation to just prior to parturition. Rat placental prostaglandin synthesis is low on day 8 of pregnancy, sharply increases on day 11, falls on day 14, and remains at a low level for the remainder of gestation. In the tissue PGE2 synthesis is 6 times greater than that of PGF2alpha on day 11. Prostaglandin metabolism in the placenta was high on day 11, low on days 8 and 14, and elevated on days 16, 18, and 21 of pregnancy. PGE1 metabolism was 8 times greater than that of PGF2alpha. Uterine prostaglandin synthesis was low until day 16, and then increased until the end of pregnancy. PGE2 synthesis was very low in this tissue in comparison to PGF2alpha synthesis. Prostaglandin metabolism in the uterus was relatively low until day 16 and then sharply increased for the remainder of gestation. This increase in metabolism was not directly proportional to uterine growth. PGE1 metabolism was 5 times higher than PGF2alpha metabolism in this organ. Ovarian prostaglandin synthesis was very low in comparison to that of the other reproductive organs. Prostaglandin metabolism in this tissue decreased from day 8 through day 18 of pregnancy. PGE1 metabolism in the ovary was twice that of PGF2alpha. These studies demonstrate patterns for synthesis and metabolism of prostaglandin in each tissue studied which may indicate inter-relationships with the physiological requirements of pregnancy.

Central benzodiazepine receptors in the spinal cord and other regions of the CNS of the cat.

Central benzodiazepine (BZ) receptors were looked for in the spinal cord, cerebral cortex and cerebellum of the cat. Both [3H]Ro 15-1788 and [3H]flunitrazepam bound to benzodiazepine receptors with apparent dissociation constants (KD) in the nanomolar range and Hill coefficients near unity. The concentration of binding sites was much greater (10-40 times,depending on the ligand used) in the cortex and cerebellum than in the spinal cord. gamma-Aminobutyric acid (GABA) significantly reduced the KD value of the binding of tritiated flunitrazepam in all three areas of the central nervous system (CNS). The displacement of [3H]Ro 15-1788 by different benzodiazepine ligands indicates a relative prevalence of BZ1 (high affinity for beta-carboline esters and the triazolopyridazine, CL 218 872) in the cerebellum, a predominance of BZ2 (low affinity for the same agents) in the spinal cord and a mixture of both types in the cortex. The possibility that there is regional heterogeneity of receptors for benzodiazepines in CNS of the cat is discussed.

Benzodiazepine receptor binding and anticonflict activity in a series of 3,6-disubstituted pyridazino[4,3-c]isoquinolines devoid of anticonvulsant properties.

A series of 3,6-disubstituted pyridazino[4,3-c]isoquinolines were synthesized and tested for their ability to inhibit the binding of [3H]diazepam to rat brain receptors in vitro. Compounds bearing a phenyl, 4-methoxyphenyl, or methyl group at position 3 and a dialkylamino group at position 6 showed the highest affinity in the binding assay and were subsequently evaluated for their anticonflict and anticonvulsant effects. All of these compounds (5a-1 and 5q) were active in the Vogel rat conflict procedure, but none prevented convulsions in mice induced either by metrazol or bicuculline. 3-Phenyl-6-pyrrolidinylpyridazino[4,3-c]isoquinoline (5d) with a Ki = 11.4 nM in the binding assay exhibited the best potency in the anticonflict assay (MED 5 mg/kg ip) and did not produce neuromuscular impairment at the highest dose tested (50 mg/kg ip).

6-(Alkylamino)-3-aryl-1,2,4-triazolo[3,4-a]phthalazines. A new class of benzodiazepine receptor ligands.

Some 6-(alkylamino)-3-aryl-1,2,4-triazolo[3,4-a]phthalazines have been shown to displace diazepam from rat brain specific binding sites, in vitro, with Ki (nM) values comparable to those of reference benzodiazepines and to have anticonvulsant (pentylenetetrazole test, mice) and anticonflict activity (Vogel test, rat) in vivo. Separation between the doses causing anticonflict effects (Vogel test, rat) and those impairing motor coordination (rotarod test, rat) has been shown for N,N-bis(2-methoxyethyl)-3-(4-methoxyphenyl)-1,2,4-triazolo[3,4-a] phthalazin-6-amine (80). This compound, unlike diazepam, was inactive in counteracting the strychnine (mouse) and maximal electroshock (mouse) induced convulsions and in the "aggressive monkey" model. These differences from the classical benzodiazepines in the animal tests indicate that 80 may have some selective anxiolytic activity.

Characterization of spironolactone binding sites distinct from aldosterone receptors in rat kidney homogenates.

The binding of [3H]spironolactone to kidney homogenates from adrenalectomized rats was studied by dextran-charcoal absorption methods. [3H]Spironolactone binds with high affinity and low capacity (KD = 12.9 +/- 0.6 nM; Bmax = 93.4 +/- 3.8 fmoles/mg protein) at low temperatures (0 degrees-2 degrees). Its hormone specificity, as measured by relative binding affinity (RBA) is spironolactone greater than prorenone greater than methyltrienolone greater than testosterone greater than progesterone greater than aldosterone greater than dexamethasone. In the same tissue preparation, specific spironolactone binding sites and classical mineralocorticoid receptor sites labelled with [3H]aldosterone differ in their thermal stability, binding parameters and hormone specificities, whereas their tissue distributions are similar. In conclusion, [3H]spironolactone binds specifically to kidney homogenates from adrenalectomized rats and these binding sites, apparently, are different from the classical mineralocorticoid receptors. The theoretical and practical aspects of this finding are discussed.

Neurotolerability of nonionic X-ray contrast media. The role of chemotoxicity.

RATIONALE AND OBJECTIVES: Because small quantities of x-ray contrast agents can cross the blood-brain barrier, the authors evaluate the properties that contribute to neurotoxicity. METHODS: The acute toxicity of various monomer and dimer contrast media was assessed after intracerebroventricular (ICV) injection to mice and intracisternal (ICI) injection to rats. RESULTS: In mice, median lethal dose (LD50) values for monomer contrast media apart from iohexol were higher than those for dimer contrast media. In rats, iopentol and iopromide were more neurotoxic than all other contrast media. The signs of toxicity for all contrast media included convulsions, dyspnea, hypoactivity, and sedation. Hypertonic D-mannitol solution was tolerated as well as artificial cerebrospinal fluid. Neither the hydrophilicity of the molecules nor the physicochemical properties of their solutions explain the toxicities satisfactorily. CONCLUSIONS: Neurotoxicity of monomer or dimer contrast media depends more on chemical structure characteristics other than hydrophilicity than on the physicochemical characteristics of their solutions.

Clinical safety of SonoVue, a new contrast agent for ultrasound imaging, in healthy volunteers and in patients with chronic obstructive pulmonary disease.

RATIONALE AND OBJECTIVES: To evaluate the safety profile of SonoVue, a new echo-contrast agent based on stabilized sulfur hexafluoride (SF6) microbubbles, in healthy volunteers and in patients with chronic obstructive pulmonary disease (COPD). METHODS: Safety and tolerability of SonoVue were evaluated in 66 healthy volunteers during two placebo-controlled phase I studies (a single intravenous ascending-dose study in 36 volunteers given SonoVue doses of 0.003 to 0.12 mL/kg and a multiple-dose study in 30 subjects given cumulative doses of 0.15 to 0.6 mL/kg) and in 12 patients with COPD of various degrees of clinical severity, who were given SonoVue at a dosage of 4 mL (corresponding to 0.057 mL/kg in a 70-kg patient). Adverse events were monitored up to 48 to 72 hours after administration. All volunteers underwent extensive safety assessments (monitoring of vital signs, electrocardiogram, blood oxygen saturation, laboratory assessments, and Mini-Mental test) up to 24 to 72 hours after administration. In addition, patients with COPD underwent specific lung function tests, such as forced expiratory volume, forced vital capacity, and forced midexpiratory flow. RESULTS: No serious adverse events occurred throughout the study. All nonserious adverse events were minor, mild, and rapidly self-resolving. No difference in the incidence of adverse events was observed among the various dosages of SonoVue and between SonoVue and placebo. There were no clinically significant changes in any of the safety assessments. No statistically significant differences between SonoVue and placebo were observed in mean forced expiratory volume, forced vital capacity, or forced midexpiratory flow levels. No substantial changes from baseline in blood oxygen saturation were observed for either study agent at any postinjection time point. CONCLUSIONS: SonoVue showed a good safety profile both in healthy subjects and in patients with COPD.

Safety and pharmacokinetics of a new liposomal liver-specific contrast agent for CT: results of clinical testing in nonpatient volunteers.

RATIONALE AND OBJECTIVES: To evaluate the safety and pharmacokinetics of BR21, a liposome-encapsulated iomeprol formulation, in nonpatient volunteers. METHODS: This was a single-blind, placebo-controlled, ascending dose study in 30 adult, male nonpatient volunteers, randomized to receive a single intravenous bolus (2 mL/s) of BR21 (0.5, 1.0, 1.5, 2.0, and 2.5 mL/kg, four volunteers per dose level) or matched volumes of placebo (0.9% saline, 10 volunteers). The safety controls performed consisted of preand postdose complete physical examinations, measurement of vital signs, electrocardiographic controls, clinical laboratory investigations (hematology, serum chemistry, and urinalysis), and monitoring of adverse events. The safety controls and monitoring of subjects for adverse events continued up to 7 days after the dose. For pharmacokinetic analysis, the determination of total iomeprol content was performed by a high-performance liquid chromatography assay procedure in blood, urine, and fecal samples collected before the dose and serially after the dose, up to 120 hours. RESULTS: No serious adverse events occurred throughout the study. All nonserious adverse events were minor and mild in intensity and rapidly resolved without treatment. No difference in the incidence of adverse events was observed among the various doses of BR21 and between BR21 and placebo. There were no clinically significant changes in vital signs, electrocardiographic parameters, or clinical laboratory findings. Iomeprol blood level decay can be described by a three-exponential function, consistent with a distribution phase (range, t1/2 0.12-0.21 hours), a fast elimination phase (range, t1/2 1.2-1.5 hours), and a slow elimination phase from a deep compartment (range, t1/2 3.3-4.5 hours). There was an apparent linearity in the relation between the area under the curve and the dose. Urinary elimination of unchanged iomeprol accounted for 89% to 90% of injected dose within 24 hours. CONCLUSIONS: BR21 appeared to be safe and well tolerated in nonpatient subjects. Its pharmacokinetic profile was compatible with nonspecific distribution into the extracellular fluid space and specific distribution into a deep compartment.

Differential binding in vitro to glucocorticoid receptors of deflazacort and prednisolone.

Deflazacort, a new steroidal anti-inflammatory agent, has remarkable glucocorticoid activity in both animals and humans. Its biologically active form, 21-desacetyl deflazacort, displaces [3H]dexamethasone from its cytosol receptor sites in rat kidney, thymus and liver in vitro. Although less active than prednisolone in its binding to glucocorticoid receptors, deflazacort 'stabilizes' the resulting steroid-receptor complex more effectively than the former in the kidney and thymus but not in the liver. This property might explain the greater activity of deflazacort than of prednisolone in the rat.

Ex vivo binding to glucocorticoid receptors in the thymus of the adrenalectomized rat.

The interaction of glucocorticoids with thymic cytosol receptors in the adrenalectomized rat was studied by a method based on their capacity to deplete cytosol receptors when administered in vivo. Three h after a single oral administration of reference steroids at various dose levels, thymus cytosol aliquots were incubated with a saturating concentration of [3H]dexamethasone (30 nM) for 24 h at 0-2 degrees C with and without 2000 nM unlabeled dexamethasone. Bound radioactivity was determined by dextran-coated charcoal adsorption. The depletion of cytosol receptors was dose-dependent for each glucocorticoid, with the following ED50 (mg/kg): fluocinolone acetonide 0.032, dexamethasone 0.09, triamcinolone acetonide 0.12, betamethasone 0.24, deflazacort 0.55, triamcinolone 1.6, prednisolone 4.0, hydrocortisone 17.0. A striking correlation (r 0.991) between ex vivo receptor binding and thymolytic effect was observed. When data from in vitro competition studies were compared with those obtained in ex vivo experiments, the latter were correlated more tightly with the biological response.

In vivo stereospecific [3H]spiperone binding in rat brain: characteristics, regional distribution, kinetics and pharmacological properties.

The time course of [3H]spiperone distribution in the three major pools (specifically and non-specifically membrane-bound and soluble) of different brain areas, was studied in rats given a tracer amount of the drug. In addition, the stereospecificity, dissociation kinetics and pharmacological nature of the in vivo bound [3H]spiperone were investigated. The data show that [3H]spiperone binding sites in the striatum, olfactory tubercles and hypophysis differ clearly from those of the cortical regions. In the prevalently dopaminergic areas the amount of ligand bound to membranes is, up to 24 h post-treatment, proportional to the total 3H present. However a more correct analysis of the data was obtained in all the experiments when membrane-bound was measured instead of total radioactivity. Thus assay of the in vivo specifically bound [3H]spiperone appears essential for a correct evaluation of the density, affinity, regional distribution, pharmacological nature and kinetics of the drug-receptor interaction.

Evidence for a role of progesterone in the control of uterine ornithine decarboxylase in the pregnant hamster.

Changes in uterine ornithine decarboxylase (ODC) activity throughout pregnancy and the importance of progesterone in the regulation of this enzyme during the early post-implantation period have been studied in the hamster. Soon after implantation, from day 5 to day 6 of pregnancy, ODC activity rapidly increased. It reached a plateau on day 7, then abruptly fell on day 8 and remained low until the end of pregnancy. DL-alpha-difluoromethyl-ornithine (DFMO), an irreversible inhibitor of ODC, induced pregnancy termination as a consequence of the reduction of uterine ODC activity. When pregnancy arrest was induced by removing endogenous progesterone by administration of prostaglandin F2 alpha (PGF2 alpha) or by ovariectomy, the ODC rise was completely abolished, and exogenous progesterone was able to entirely counteract this effect on the enzyme activity and the termination of pregnancy. These results suggest that progesterone play a significant role in the rise of uterine ODC activity, which appears to be essential for the early post-implantation events needed for pregnancy maintenance.

In vitro interaction of premazepam with benzodiazepine receptors in rat brain regions.

Premazepam (PRZ) in vitro competitively displaced 3H-diazepam (DIA), 3H-flunitrazepam (FLU) and 3H-RO 15-1788 from their binding sites on rat brain synaptosomes, with a potency intermediate to other benzodiazepines (BDZs), and Hill coefficients near 1 in different brain regions. Incubation at 37 degrees C reduced premazepam's affinity for BDZ receptors to a lower extent than other benzodiazepines and had no effect on the Hill coefficient. The IC50 of PRZ on 3H-RO 15-1788 and 3H-FLU binding was markedly reduced by GABA in rat cortex, like those of reference classical BDZs, but was GABA-independent in the cerebellum. The IC50 of the BDZ antagonist, RO 15-1788 was unaffected by GABA in both brain areas. The possibility that PRZ behaves as a partial agonist in the cortex and as an antagonist in the cerebellum is discussed.

On the mechanism of action of a new pregnancy-terminating agent. Part I: Effects of prostaglandin F2 alpha metabolism in the rat and the hamster.

Studies have been carried out to evaluate the effects of 3-(2-ethyl-phenyl)-5-(3-methoxyphenyl)-1H-1,2,4 triazole (DL 111-IT) a new non-hormonal post-implantation anti-fertility agent, on metabolism of prostaglandin F2 alpha (PGF2 alpha MET) by the utero-placental unit (UPU) and the lung. In rats treated with a single (100 mg/kg) subcutaneous minimal 100%-effective dose, DL 111-IT showed a slight and transient inhibitory effect on UPU PGF2 alpha MET, whereas after multiple (2.5 mg/kg/day for 3-5 days), equally effective doses, no effect was observed. In addition, PGF2 alpha levels in both plasma and the UPU were not changed. In rat lung, PGF2 alpha MET was markedly inhibited either after single or multiple injections and the inhibition was prolonged for at least 5-6 days after administration. In hamster, on the contrary, doses up to twenty times greater than 100%-effective ones caused only a slight inhibition of PGF2 alpha MET. The effects of DL 111-IT seem therefore to be dose and species dependent, but not to be related to its pregnancy-terminating activity, thus excluding the involvement of this effect as a part of its mechanism of action.

Contragestational effects of DL-alpha-difluoro-methylornithine, an irreversible inhibitor of ornithine decarboxylase, in the hamster.

In the hamster, there are remarkable changes in ODC activity in various uterine reproductive tissues during gestation. When DL-alpha-difluoromethylornithine (DFMO), a highly selective irreversible inhibitor of ODC, is given during the early post-implantation period, when ODC activity in the conceptus is rapidly rising and reaches its highest values, it causes pregnancy arrest. Low doses of DFMO are contragestational only during a short period of gestation (day 7 and 8), with primary action on the embryo. Higher doses given soon after implantation (day 5) induce early pregnancy termination by a primary effect on decidual tissue. Biochemical investigations in animals treated on day 5 or day 7 showed that DFMO induces a rapid and dramatic fall in ODC activity in the products of conception. These findings strengthen and extend previous evidence that ODC and polyamines play an essential role in the maintenance of early pregnancy in rodents.

On the mode of action of a new contragestational agent (DL 111-IT).

Non-hormonal compounds belonging to 3,5-diphenyl-1H,1,2,4 triazoles and chemically related classes (triazoles in short) are known to be endowed with high contragestational activity in rodents, dogs and primates. The data herein reported for one of the leaders of this family of compounds (DL 111-IT) along with those previously reported for some analogues, demonstrate that triazoles cause pregnancy arrest by a direct action on conception product. This action is expressed through a progressive slowing down of the conceptus development with a consequent onset of a state of anoxia, pregnancy arrest, degeneration of placentae and adnexae and their absorption or expulsion. The selective time of gestation during which they elicit the antifertility effect (early post-implantation period) and the histological examinations revealed that the target of their action are the ectoplacental and decidual cells. Biochemical studies on conceptus product demonstrate that, although they do not bind to progesterone receptors or inhibit ornithine decarboxylase activity, triazoles induce an early and marked increase in the number of cytosol progesterone receptors accompanied by a steep decrease in the ornithine decarboxylase activity in the product of conception. These findings are discussed in relation to the possibility that triazoles may trigger pregnancy arrest by interfering with the chain of events by which progesterone regulates the mitotic activity of decidual and trophoblastic cells.

A new non-hormonal pregnancy-terminating agent.

DL 111-IT, 3-(2-ethylphenyl)-5-(3-methoxyphenyl)-1H-1,2,4 triazole, a compound belonging to a new class of non-hormonal antifertility agents, when given subcutaneously, intramuscularly, intravaginally or orally terminates pregnancy in the rat, the mouse, the hamster and the dog. Time-course and dose-activity studies indicate that its effectiveness is dependent on dose, vehicle, route and time of pregnancy. DL 111-IT has no pre-implantation activity. The most effective time for treatment is the early post-implantation period. The compound has an antifertility effect through a slow and continuing action that results in the degeneration and subsequent resorption or expulsion of conceptuses. As a result, there must be sustained availability of active principle to arrest the pregnancy. Administered parenterally in a proper vehicle (oily) and with a suitable schedule of treatment (x 2-5 days), it demonstrates a very high pregnancy terminating activity (ED50: 0.04-0.7 mg/kg/day). Multiple intravaginal and oral administrations are also effective but the daily doses required are 10-20 and 40-100 times higher than the parenteral ones. Studies of the mechanism of action indicate that the site of action is the utero-placental complex. In fact, in pregnant rats, mice, hamsters and dogs, both plasma progesterone levels and the ineffectiveness of progesterone therapy rule out luteolysis as a basis for the activity. Moreover in hypophysectomized, ovariectomized animals whose pregnancies were maintained with proper hormonal treatments, DL 111-IT terminates pregnancy and adrenalectomy does not prevent its effect, which suggests that pituitary, ovaries and adrenals are not required for the antifertility action.

Hepatic transport of the magnetic resonance imaging contrast agent gadobenate dimeglumine in the rat.

RATIONALE AND OBJECTIVES: Gadobenate dimeglumine is a new octadentate gadolinium (III) complex salified with meglumine. The compound is currently under evaluation as an intravenously administered paramagnetic contrast agent for magnetic resonance (MR) imaging. We investigated the mechanisms involved in the biliary excretion of gadobenate ion, the contrast-effective ion in gadobenate dimeglumine. METHODS: Biliary and urinary excretion of gadobenate ion injected intravenously to rats at 0.25 mmol/kg was studied following pretreatment with bromosulfophthalein (BSP) disodium salt, sodium taurocholate (TC), or oxyphenonium bromide (OP) and at various times after common bile duct ligation. Gadobenate ion was assayed by high-pressure liquid chromatography in bile and urine. Plasma bilirubin levels after duct ligation were measured by colorimetric assay. RESULTS: The 90-min excretion of gadobenate ion into bile accounted for 35.5 +/- 3.7% and excretion into urine for 45.7 +/- 3.5% of the injected dose (mean +/- SD). Pretreatment with BSP reduced recovery of the compound in bile to less than 1% and increased urinary excretion to 65.6 +/- 4.7%. Gadobenate dimeglumine had a substantial choleretic effect that was completely abolished by pretreatment with BSP. Pretreatment with TC and OP did not change the biliary or urinary excretion of gadobenate ion. Surgical cholestasis led to a massive increase in plasma bilirubin levels from 3.9 +/- 2.2 (day of surgery) to 129 +/- 37 mumol/L (4 days after common bile duct ligature) and decreased 6-hr cumulative biliary excretion of gadobenate ion from 45 +/- 16% to 5.3 +/- 4.2% of the injected dose. Urinary excretion increased correspondingly from 49 +/- 15% to 83 +/- 12%. CONCLUSION: The transport of gadobenate ion from plasma to bile occurs in the rat mainly through the BSP/bilirubin transport systems.

Toxicological safety assessment of iomeprol, a new X-ray contrast agent.

The toxicology of pharmaceutical formulations of iomeprol, a new nonionic iodinated radiographic contrast agent, was studied in rodents (mice, rats and guinea-pigs) and non-rodents (rabbits and dogs). When injected intravascularly the acute toxicity of iomeprol, both in terms of median lethal dose and symptoms, was comparable to that of analogous triiodinated nonionic contrast media (CM). Intravenous daily dosing for 4 weeks showed that iomeprol was well tolerated at doses as high as the maximum dose anticipated for clinical use. Moreover, the compound did not possess reproductive, developmental, or genetic toxicity. Tissue tolerability was completely superimposable on those of reference CM such as iopamidol and iohexol. Finally, no antigenic potential was detected either in mice or guinea-pigs. These favourable toxicological characteristics bode well for iomeprol as an intravascular radiographic contrast agent.

Pharmacokinetics and tissue distribution of iomeprol in animals.

The pharmacokinetics of iomeprol, a new triiodinated nonionic radiographic contrast agent, has been studied in rats, rabbits and dogs. Following intravenous administration, iomeprol did not bind measurably to plasma proteins and was rapidly excreted in unchanged form, mostly through the kidneys. The compound was rapidly distributed to the plasma and thence to the extracellular spaces. Iomeprol did not accumulate in specific organs or tissues except for those involved in its elimination, i.e. the kidneys and the liver. However, 24 h after administration, less than 1% of the injected dose remained in the tissues. The overall profile was very similar to the published profiles of other radiographic contrast agents used in uroangiography.