Double-blind, vehicle-controlled randomized twelve-month neurodevelopmental toxicity study of common aluminum salts in the rat.

This good laboratory practice (GLP) study of aluminum salts in Sprague-Dawley rats was conducted according to double-blind, vehicle-controlled randomized design by exposing offspring to aluminum citrate in-utero, through lactation, and then in drinking water post-weaning. Three dose levels were used: 30, 100, 300 mg Al/kg bw/day, in addition to control groups that received either water or a sodium citrate solution (27.2 g/L). Endpoints were assessed in both female and male pups: behavioral (motor activity, T-maze, auditory startle, the Functional Observational Battery (FOB) with domains targeting autonomic function, activity, neuromuscular function, sensimotor function, and physiological function), cognitive function (Morris swim maze), brain weight, clinical chemistry, hematology, tissue/blood levels of aluminum and neuropathology. The most notable treatment-related effect observed in the offspring was renal pathology, most prominently in the male pups. Higher mortality and significant morbidity were observed in the male pups in the high Al-citrate dose group; leading to euthanization of this group at day 89. There was evidence for dose-response relationships between neuromuscular measurements-hind-limb and fore-limb grip strength-and Al-treatment in both males and females, although some of the effects may be secondary to body weight changes. No consistent treatment-related effects were observed in ambulatory counts (motor activity) in the different cohorts. No significant effects were observed for the auditory startle response, T-maze tests (pre-weaning day 23 cohort) or the Morris water maze test (day 120 cohort). None of the lesions seen on histopathological examination of brain tissues of the day 364 group was reported as treatment-related and, as these were also seen in the control group, were likely due to aging. In conclusion, these results indicate that concentrations of aluminum in the drinking water that are required to produce minimally detectable neurobiological effects in the rat are about 10,000 times higher than what is typically found in potable drinking water.

Pharmacokinetics and tissue residues of Telazol in free-ranging polar bears.

A pharmacokinetic and tissue residue study was conducted to assess the risks associated with human consumption of polar bears in arctic Canada that have been exposed to the immobilizing drug Telazol, a mixture of tiletamine hydrochloride and zolazepam hydrochloride. Twenty-two bears were remotely injected with about 10 mg/kg of Telazol. Following immobilization, serum samples were collected serially at regular intervals until the bears awakened. Sixteen of the bears were relocated and killed under permit by local hunters at various times from 0.5 to 11 days after dosing. Serum, kidney, muscle and adipose tissue samples were collected immediately after death. All samples were stored at -70 C until analysis by HPLC. The concentration-time data of tiletamine and zolazepam in serum during the immobilization period were fitted to curves by computer and the pharmacokinetic parameters assessed. In addition, the serum and tissue samples collected at the time of death were analyzed for both parent drugs, for one metabolite of tiletamine (CI-398), and for three metabolites of zolazepam (metabolites 1, 2 and 4). A one-compartment model with first-order absorption and elimination best fit the time-series data for the drugs in serum during the immobilization period. This model gave half-lives (mean +/- SE) for tiletamine and zolazepam of 1.8+/-0.2 h and 1.2+/-0.08 h, respectively, clearance values of 2.1+/-0.3 l x h(-1) x kg(-1) and 1.1+/-0.1 l x h(-1) x kg(-1), and volumes of distribution of 5.2+/-0.6 l/kg and 1.8+/-0.2 l/kg. The concentrations of both drugs and their metabolites declined rapidly to trace levels by 24 h post-dosing, although extremely low concentrations of some metabolites were encountered sporadically over the entire sampling period. In particular, zolazepam metabolite 2, remained detectable in fat and muscle tissue at the end of the study, 11 days after dosing. It was concluded that during immobilization, both tiletamine and zolazepam levels decline rapidly in a monoexponential fashion, and their pharmacokinetic parameters in polar bears are similar to those observed in other species. Tissue levels of the drugs and their metabolites declined sufficiently rapidly that individuals eating meat from exposed bears would be unlikely to experience pharmacological effects from the drugs. Nevertheless, slight exposure to the drugs and/or their metabolites might be possible for an indeterminate time after dosing.

Isolation and identification of the major urinary metabolite of 4-(4-fluorophenoxy)benzaldehyde semicarbazone after oral dosing to rats.

4-(4-Fluorophenoxy)benzaldehyde semicarbazone (1) is a novel anticonvulsant affording excellent protection in the rat oral maximal electroshock (MES) screen as well as having an apparent protection index of over 300. The metabolism of this compound was studied by examining the urine or rats dosed orally with 50 mg/kg of 1 which revealed that most of the drug was converted into one metabolite 2. The structure of 2 was shown by mass spectrometry to be 1-[4-(4-fluoro-phenoxy)benzoyl]semicarbazide which was confirmed by an independent synthesis. Compound 2 was bereft of activity in the rat oral MES screen when nine times the ED50 dose of 1 was administered. This datum provided strong evidence that the anticonvulsant activity of 1 and related compounds is due to the intact molecules and is not produced by breakdown products in vivo.

Postnatal Deficiencies of Zinc and Taurine Alter Electroretinograms, Oscillatory Potentials and Morphology of the Rat Retina.

The study objective was to evaluate the retinal response to deficiencies of zinc and taurine present throughout the period of postnatal retinal development. At parturition, Sprague-Dawley dams were assigned to one of four treatments in a 2 × 2 factorial design with two levels of zinc (4.5 and 50 µg/g) and two levels of taurine (0 and 2 µmol/g). Guanidinoethyl sulfonate, a taurine transport inhibitor, was added to the drinking water of the rats receiving 0 µmol/g taurine. Male pups (n = 10) were weaned on to their respective diets at postnatal day 22. Dark adapted electroretinograms and oscillatory potentials (OP) were recorded in the pups at 48-57 days of age. At maximal light intensity, the amplitudes of the a- and b-waves were depressed by deficiency of either nutrient, but the influence of combining these treatments was less than additive; the same pattern was evident for Vmax, the maximum amplitude obtained when the b-wave was plotted as a function of light intensity. This type of interaction was also evident for the amplitudes of OP1, OP3 and OP4. Zinc deficiency independently decreased the amplitude and increased the latency of OP5, and increased the latencies of OP3 and OP4. Light and transmitting electron microscopic examination revealed the most pronounced retinal degeneration in the rats deficient in both zinc and taurine. Tibia zinc and liver taurine concentrations provide evidence that these nutrients also interact in other tissues. The findings of this study demonstrate retinal damage with deficiencies of zinc and taurine during postnatal life. These nutrients interact in at least some of their functions in the retina through an as yet unidentified mechanism.

Long-term surgical preparation of portal vein-drained viscera for determination of their nutritional requirements in preruminant calves.

OBJECTIVE: To develop a surgical preparation to study the nutrient concentration difference across the portal vein-drained viscera of preruminant calves over a 2-week period. ANIMALS: 9 healthy preruminant male Holstein calves. PROCEDURE: A bilateral subcostal approach was used to reach the portal area to provide access for proper placement of an ultrasonic transit time flow probe around the portal vein. The umbilical vein was used as an entry point for the portal vein catheter. The femoral artery was also catheterized. Calves were observed daily, and food intake was recorded. Body weight was recorded weekly. The calves were euthanatized, and necropsy was performed 2 weeks after surgery. RESULTS: Of the 9 calves, 7 recovered without surgical complications. Within 24 hours of surgery, 1 calf developed an intestinal hernia at the flank incision that was surgically repaired without further complications. One calf was euthanatized a week after surgery because it developed septicemia secondary to catheter-related infection. CONCLUSION: The bilateral subcostal approach provided access to the portal area, and the umbilical vein was useful as an entry point. Application of an ultrasonic flow probe provided consistent measurements of blood flow over a 2-week period. CLINICAL RELEVANCE: These results may have implications for development of treatment to promote gastrointestinal tract healing in calves with diarrhea.

Identification and determination of phase I metabolites of propafenone in rat liver perfusate.

Propafenone (PF) is a class 1C antiarrhythmic agent. To study the mechanisms of PF interactions with dietary nutrients in isolated, perfused rat livers, metabolites of PF in liver perfusate were identified and an analytical method was developed for these metabolites plus parent drug. Identification of phase I metabolites was performed using HPLC/MS equipped with a Lichrospher RP-18 column and tandem mass spectrometry (MS/MS) with electrospray and atmospheric pressure chemical ionizations. Three major metabolite peaks, whose protonated molecular ions were m/z 358, 358 and 300, were identified as a propafenone derivative hydroxylated in the omega-phenyl ring (omega-OH-PF), 5-hydroxypropafenone (5-OH-PF), and N-despropylpropafenone (N-des-PF). The levels of omega-OH-PF, 5-OH-PF, N-des-PF and PF were determined simultaneously by HPLC with UV detection at 210 nm and a mobile phase of 0.03% triethylamine and 0.05% phosphoric acid in water-acetonitrile-methanol (45:20:35, v/v/v) after extraction with 5 ml diethyl ether at pH 10.0 and evaporation of solvent under nitrogen. The results revealed that omega-OH-PF, which was not found in humans, was the major metabolite of PF in rat liver perfusate, not 5-OH-PF which is the major metabolite in human plasma.

Multifocal retinopathy of Great Pyrenees dogs.

Forty-four related Great Pyrenees dogs were examined ophthalmoscopically. Focal retinal elevations, multiple gray-tan-pink subretinal patches, and discrete areas of tapetal hyper-reflectivity were seen in 19 dogs, ranging from 13 weeks to 10 years of age. These lesions varied in size from focal spots that were barely visible with the indirect ophthalmoscope to areas that were larger than the optic disc. Complete blood cell counts, serum biochemical profiles, urinalyses, and blood pressure measurements were completed on four affected dogs and all were within normal reference ranges. Photopic and scotopic electroretinography was completed and the a-wave and b-wave amplitudes and latencies were similar for affected and age-matched nonaffected Great Pyrenees and other normal dogs. Electroretinograms that were examined twice during a 3-year period on three affected adult dogs did not reveal significant progressive deterioration of the a or b-wave parameters. Fluorescein angiography was completed on four affected dogs of ages 1 (n = 2), 5, and 6 years. These angiograms were repeated in three of these dogs 1 year later. The blood ocular barrier was intact in these dogs but there was blocked choroidal fluorescence. Postmortem examination, light microscopy, scanning and transmission electron microscopy were performed on three affected puppies and two affected adult dogs. These examinations revealed that the lesions in the puppies were limited to bilateral multiple areas of retinal pigment epithelial vacuolation, hypertrophy, and apparent separation from Bruch's membrane, and multiple serous retinal detachments. The affected adult dogs had focal retinal degeneration and retinal pigment epithelial hypertrophy, hyperplasia and pigmentation. Pedigree analysis and test mating confirm that this condition is inherited, probably as an autosomal recessive trait. This condition develops at approximately 13 weeks of age and the focal areas of retinal detachment and retinal pigment epithelial vacuolation progress to permanent and stable focal areas of retinal degeneration, and retinal pigment epithelial hypertrophy and pigmentation.

Inhibition of metoprolol metabolism by amino acids in perfused rat livers. Insights into the food effect?

A mixture of amino acids inhibits propranolol metabolism in perfused rat livers. To obtain mechanistic information about the interaction, a related but less tissue-bound drug, metoprolol, was used to determine Vmax and K(M) for parent drug and two metabolites in the presence and absence of amino acids. Six groups of 4 livers from 24 male Sprague-Dawley rats were perfused in the single-pass mode at 3 ml/min/g liver for 130 min with oxygenated buffer containing 3.74, 4.49, 5.61, 7.48, 18.7, or 44.9 microM metoprolol. From 50 to 90 min, a balanced amino acid mixture was included in the buffer. Samples of liver effluent taken every 5 min were analyzed by HPLC for metoprolol and two metabolites, alpha-hydroxymetoprolol and O-demethylmetoprolol. Steady-state concentrations of drug determined before, during, and after amino acids were used to determine Vmax and apparent K(M) values by nonlinear curve-fitting under each condition. Amino acids reversibly reduced the Vmax values of metoprolol and both metabolites by approximately 50% without significantly affecting apparent K(M) values. As a result, large increases in availability occurred, especially at low metoprolol inlet concentrations (> 90%). Amino acids also increased oxygen consumption until the effluent buffer was almost depleted. Possible mechanisms influencing Vmax include direct inhibition of metabolic enzymes by amino acids or cosubstrate (NADPH or oxygen) limitation. Amino acid-mediated pericentral oxygen depletion in the hepatic sinusoids could result in inhibition of drug-metabolizing enzymes, and is consistent with a reduction of Vmax and oxygen depletion in the effluent buffer during amino acid coinfusion. We postulate that one or more of these mechanisms could contribute to the interaction between food and high first-pass drugs observed in humans.

Interaction between propranolol and amino acids in the single-pass isolated, perfused rat liver.

Propranolol (PL) bioavailability has been shown to increase substantially when it is administered with a protein-rich meal. A change in metabolic capacity or tissue uptake, induced by amino acids (AAs) released as a result of digestion of dietary protein, is a possible contributing mechanism to the food effect. This hypothesis was tested in isolated, perfused rat livers in the single-pass mode. Rac-PL (20 micrograms/ml) was infused to steady-state at 3 ml/min/g liver for 150 min. A balanced mixture of I-AA was coinfused from 70 to 110 min. The AA reversibly increased the steady-state concentration of PL by 18% and reduced steady-state concentrations of 4-hydroxypropranolol, N-deisopropylpranolol, PL glycol, naphthoxylactic acid, and naphthoxyacetic acid by an average of 41% and propanolol conjugates by almost 100%, indicating metabolic inhibition. In a second experiment, PL was coinfused with AAs from the beginning of the experiment, and tissue binding was compared with control livers. There was no significant effect of AAs on PL tissue binding. In a third study, the effect of four different concentrations of AAs coinfused from 70 to 110 min was assessed. The percentage change in PL and phase I metabolite levels was linearly correlated to the influent AA concentration. The large magnitude, reversibility, lack of pathway specificity, and concentration dependence of the AA interaction in the perfused liver are also features of food interaction in humans. These similarities constitute evidence that metabolic inhibition by AAs originating from dietary protein could contribute to the PL-food interaction.

Propranolol disposition in the single-pass isolated, perfused rat liver in the presence and absence of insulin and glucagon.

The large increase in propranolol (PL) bioavailability when administered with food cannot be entirely explained by a transient increase in hepatic blood flow. A change in metabolic capacity or tissue uptake induced by changes in insulin and/or glucagon associated with food ingestion may contribute to the food effect. This hypothesis was tested in three groups of four isolated, perfused rat livers in the single-pass mode. PL (20 micrograms/ml) was infused to steady state at 30 ml/min for 120 min, then washed out for 30 min. Insulin or glucagon at 2 x 10(-9) M, or saline (control), was introduced at 70 min. Although neither insulin nor saline perturbed effluent PL or metabolite steady-state concentrations, glucagon caused a transient (15-min) reduction in PL, N-deisopropylpropranolol, propranolol glycol, and naphthoxylactic acid, indicating increased PL uptake, but not a change in metabolic activity. PL uptake was 668 +/- 108 micrograms/g liver tissue overall, and additional uptake after initiation of glucagon infusion was significant at 29 +/- 11.6 micrograms/g liver tissue (4% of initial uptake). Although the increase in PL uptake was small under the described conditions, this interaction with glucagon may contribute to the food effect. In this model system, hormonal effects on PL metabolism were not observed.

Simplified high-performance liquid chromatographic method for propranolol and five metabolites in liver perfusate, rat serum and dog plasma.

A new method has been developed for the analysis of propranolol plus five major metabolites in perfusate from isolated liver preparations, and also in rat serum and dog plasma. The regional isomers of hydroxypropranolol are clearly separated within a total run time of less than 15 min. The basic and neutral metabolites are extracted and analysed together, while the acidic metabolites are extracted in a second step. The new assay is more simple and time efficient than previously published methods.

Anticonvulsant properties of some Mannich bases of conjugated arylidene ketones.

Thirty 1-aryl-5-dimethylamino-1-penten-3-one hydrohalides and related compounds were prepared as candidate anticonvulsants and evaluated in maximal electroshock seizure (MES), subcutaneous pentylenetetrazole threshold, and neurotoxicity screens. Following administration by the intraperitoneal route, many of the compounds were active in the MES screen, whereas only 10% of the Mannich bases afforded protection in the subcutaneous pentylenetetrazole test. Quantitation of half of the compounds prepared revealed that many had activity comparable with that of clinically useful drugs in the MES screen. The anticonvulsant properties of eight of the compounds following oral administration were reduced considerably or abolished compared with those following intraperitoneal administration. Various synthetic strategies for future development of potential anticonvulsants are outlined.

3,5-Bis-arylidene-1-methyl-4-piperidone methohalides and related compounds with activity against L 1210 cells and DNA binding properties.

A series of 3,5-bis-arylidene-1-methyl-4-piperidone methohalides 2 and two related analogues in general demonstrated activity against L 1210 leukemia cells in vitro and bound to a synthetic DNA, poly[d(AT)]. Plots of various physicochemical constants of the aromatic substituents in series 2 versus the IC50 figures revealed correlations between the aryl MR and pi values but not the sigma constants. The delta Tm values of six members of series 2 were correlated with the MR figures of the aryl substituents but not the sigma nor pi values of the aromatic atoms and groups. Some suggestions for future molecular modification with a view to increasing cytotoxicity are presented.

Treatment of retinopathy of prematurity with argon laser photocoagulation.

Fifteen eyes of nine infants were treated for retinopathy of prematurity by confluent photocoagulation of the avascular retina using an argon laser indirect ophthalmoscope. All treated eyes presented at or beyond threshold of stage 3 retinopathy of prematurity with "plus" disease. Three treated eyes had retinopathy of prematurity in zone I. Early complete regression of extraretinal proliferation was seen in 13 eyes. At 6 month's [corrected] minimum follow-up, 11 (73%) of the treated eyes demonstrated a favorable outcome, while four eyes (27%) progressed to an unfavorable outcome. No intraocular hemorrhages occurred during any laser treatment.

Interactions between hydralazine and oral nutrients in humans.

To help clarify whether food or enteral nutrients decrease hydralazine relative bioavailability, eight subjects were given oral hydralazine under four nutritional conditions: fasted (F), with a standard breakfast (SB), with a bolus of enteral nutrients (EB), and with a slow infusion of enteral nutrients administered by nasogastric tube (EI). The area under the curve and maximum concentration values were much higher under the fasted and enteral infusion conditions than under the standard breakfast or enteral bolus conditions, indicating that the absorption and/or disposition kinetics of hydralazine may be altered by food. The median (range) values for these parameters were 2,641 (385-4,747) and 87 (4.5-224) for F; 1,189 (202-1,737) and 15 (3.5-33.9) for SB; 999 (227-3,576) and 11 (2.5-50) for EB; and 3,068 (313-4,917) ng/ml/min and 113 (3.6-235) ng/ml for EI. Furthermore, the rate of nutrient administration, but not necessarily the physical form, of the nutrients appears to be a significant factor in determining the magnitude of the food effect. The nutrient interaction should be accounted for in patients receiving hydralazine and enteral nutrition concomitantly.

A chronic conscious dog model for direct transhepatic studies in normal and pancreatic islet cell transplanted dogs.

A chronic conscious, large mammal model for repeated transhepatic studies over a period of 6-8 weeks has been developed for application to the study of the hepatic effects of pancreatic hormone secretion and glucose metabolism, studies of the hepatic mechanisms associated with high first-pass drug metabolism and food interactions, and studies of insulin balance in dogs that have undergone previous pancreatectomy and islet cell auto-transplantation. The preparation of specialized blood sampling catheters and blood flow probes, surgical preparations, pre- and postoperative care, catheter maintenance, and possible complications are described. Data from an oral glucose meal (OMT) are presented. Frequent blood samples from portal, hepatic and jugular veins, and carotid artery were collected and analyzed for plasma insulin concentrations (IRI) and glucose. IRI fluxes were determined for each time period by interpolation of flux X time curves. Total IRI flux was determined as the sum of the areas under the curves for each sampling period. Hepatic insulin extraction was calculated for each sampling interval. By this method of analysis, it was possible to determine hepatic IRI extraction during non-steady-state conditions. The techniques described in the development of this complex animal model, which allows for repeated transhepatic studies in the same conscious subject, may also have application in other chronic studies involving organs other than the liver.