Variation in MR signal intensity across normal human knee cartilage.

Signal intensity (SI) of individual pixels on sagittal magnetic resonance (MR) images of normal human knee cartilage was quantified to investigate whether it was related to cartilage proteoglycan content. In five subjects, images were acquired with spin-echo sequences with a TR msec/TE msec of 1,000 or 700/20 and a three-dimensional gradient-echo (GRE) sequence (60/15). In a sixth subject, the GRE sequence alone was used with 15 degrees, 30 degrees, and 50 degrees flip angles. In all subjects, SI was maximal in pixel layers of the medial zone and minimal at both cartilage edges, resulting in the presence of a bell-shaped curve of interpixel (zonal) SI variation across the cartilage thickness. The magnitude of SI was dependent on the pulse sequence and flip angle, but the bell shape of the SI variation curve was independent of them. For example, in the medial tibial cartilage, the peak SI was highest with the 1,000/20 spin-echo sequence, intermediate with the 700/20 sequence, and lowest with the GRE sequence. The differences were statistically significant. The bell-shaped SI variation curve resembled the curve for zonal variation in cartilage proteoglycan content but not the curves for collagen or free water content. The physiologic basis for this resemblance and the potential usefulness of the findings for early diagnosis of diseases such as osteoarthritis are discussed.

Determination of in vivo hepatic extraction ratio from in vitro metabolism by rat hepatocytes.

Using isolated rat hepatocytes, the in vitro intrinsic hepatic clearance of three drugs, namely, antipyrine (AP), propranolol (P), and CGS 13429 was determined. The hepatic extraction ratios of AP, P, and CGS 13429 were then calculated to be 0.10, 0.90, and 0.88, respectively. To determine the in vivo hepatic extraction ratio in the rat, AP, P, or CGS 13429 was infused separately at a rate of 40, 20, and 20 micrograms/min.kg, via jugular and hepatic portal veins. The steady state plasma levels of AP, P, and CGS 13429 were 5.30 +/- 0.33 micrograms/ml, 608 +/- 76 and 380 +/- 46 ng/ml after jugular vein (iv) infusions, and 4.96 +/- 0.46 micrograms/ml, 162 +/- 26, and 148 +/- 23 ng/ml after hepatic portal vein (hpv) infusions, respectively. The blood to plasma ratios of AP, P, and CGS 13429 were 1.0, 0.78, and 1.0, respectively. The in vivo hepatic extraction ratio, calculated as (Css,iv--Css,hpv)/Css,iv from steady state blood levels following iv and hpv infusions for AP, P, and CGS 13429, were 0.065, 0.73, and 0.61, respectively. These results suggest that in vitro metabolism studies by hepatocytes may have potential application, during drug discovery, to distinguish drugs of low and high metabolic hepatic extraction ratio in vivo.

A pilot study of the bioavailability and pharmacokinetics of 2',3'-dideoxycytidine in patients with AIDS or AIDS-related complex.

Eight patients with AIDS or ARC received four single doses of 2',3'-dideoxycytidine (ddC). The treatments included 0.5 and 5 mg oral tablets, a 0.5 mg oral solution, and a 0.5 mg intravenous infusion. Blood samples were collected for 4 to 6 h after each dose. Plasma concentrations of ddC were determined by a specific gas chromatographic-mass spectrometric (GC-MS) assay. A combination of the low dose and the assay sensitivity of 2 ng/ml limited data treatment and comparison. Mean Cmax of 8.5, 7.6, and 79.0 ng/ml occurred at mean tmax of 1.1, 1.3, and 0.9 h for the 0.5 mg oral solution, the 0.5 mg tablet, and the 5 mg tablet, respectively. A mean clearance of 5.57 ml/min/kg and volume of distribution of 0.64 L/kg were determined from the 0.5 mg intravenous infusion. Half-life values ranged between 0.95 and 2.0 h and appeared to be independent of the dose and route of administration. The bioavailability values calculated for the oral tablets were variable, ranging from 54 to 127%. Single doses of ddC were well tolerated in this population. The results of this pilot study indicate that ddC is rapidly and extensively absorbed when administered as an oral tablet or solution to fasting AIDS or ARC patients. It is also rapidly eliminated with a half-life of 1-2 h. There are no apparent differences in the absorption or elimination of ddC between 0.5 and 5 mg oral doses.

Uptake and digestion of 125I-labelled bovine serum albumin by the rat visceral yolk sac cultured in vitro as a closed vesicle.

A system for culturing the rat visceral yolk sac in vitro as a closed vesicle--the 'giant' yolk sac--has been employed to investigate the vectorial nature of the uptake and digestion of exogenous protein substrates. Uptake of 125I-labelled formaldehyde-denatured bovine serum albumin by such yolk sacs was found to be similar to that observed in yolk sacs removed directly from the mother at 17.5 days' gestation, provided that homologous serum was used as a culture medium. However, unlike the control yolk sacs, giant yolk sacs tended to accumulate substrate within the tissue with increasing culture time. The concentration of digestion products released to the inside of the closed vesicle was found to be greater than that released to the surrounding culture medium at time intervals up to five hours. Giant yolk sacs preloaded with 125I-labelled bovine serum albumin were found to release material to the culture medium or the inside of the vesicle almost entirely in the acid-soluble (digested) form. This system is a useful model for studying the polar nature of epithelia, particularly those involved in the uptake and transport of nutritional and/or informational macromolecules.

Flumazenil: US clinical pharmacology studies.

Flumazenil, a benzodiazepine antagonist, blocks the central effects of benzodiazepines by competitive interaction at the receptor site. Two double-blind, placebo-controlled, randomized studies in healthy volunteers (110/study) were performed to determine the minimal effective dose of flumazenil required to reverse the sedative, psychomotor and amnesic effects of benzodiazepines used to produce conscious sedation. Conscious sedation was produced by i.v. diazepam (12-30 mg) in one study and i.v. lorazepam (0.045 mg kg-1) in the other. Intravenous flumazenil (0.001, 0.003, 0.007 or 0.014 mg kg-1) or placebo was administered after diazepam or lorazepam. Assessment of sedation, psychomotor performance and recall/recognition were made both before and after the benzodiazepine as well as serially after flumazenil or placebo. Doses as low as 0.007 and 0.014 mg kg-1 flumazenil consistently reversed diazepam- and lorazepam-induced effects, respectively. The duration of reversal produced by varying doses of flumazenil (0.2, 0.6, 1.0 or 3 mg) was evaluated in 50 volunteers in a double-blind, placebo-controlled, parallel group study. A constant level of conscious sedation was produced by a continuous infusion of midazolam. Assessments of sedation and psychomotor performance were assessed both before and at varying times after the administration of flumazenil or placebo. Preliminary results indicate that the duration of reversal produced by 3.0 mg flumazenil was longer than that produced by any of the lower doses. While the mean duration of reversal produced by the lower doses was comparable, the 0.2 mg dose resulted in the greatest between subject variability and only partial rather than complete reversal. Two further double-blind, placebo-controlled studies were done in healthy volunteers (45/study) to evaluate the safety of flumazenil 1.0 mg or placebo given i.v. to reverse midazolam-induced sedation in subjects who had been treated for up to 14 days with either oral diazepam or triazolam. No clinically significant changes were noted in laboratory test values, electrocardiograms or vital signs monitored for up to 36 h after flumazenil or placebo in any pre-treatment group.

Pharmacokinetics of single and multiple ascending doses of the antiallergic agent tiacrilast in man.

Five groups of six healthy subjects received single oral doses of 150, 300, 450, 600 or 750 mg tiacrilast 150 mg capsules, followed 24 h later by the same dose given every 6 h for 7 days, in a study designed to assess the pharmacokinetics of single and multiple doses of tiacrilast. Plasma samples were obtained at specified times after the initial dose, after 4 days of multiple dosing and after the last dose of tiacrilast. Samples were assayed for unchanged drug by a specific HPLC method. Wide variability was seen in the plasma concentration-time data. Plasma concentrations and pharmacokinetic parameters were nearly proportional to dose over the 150 to 750 mg dose range studied. Moreover, there was no evidence of unexpected accumulation of the drug in the plasma during multiple dosing and food did not appear to alter the bioavailability of tiacrilast to any clinically significant extent. The apparent elimination half-life was similar after single and multiple doses and ranged from 1 to 3 h.

The pharmacological profile and initial clinical evaluation of tiacrilast (Ro 22-3747): a new antiallergic agent.

Tiacrilast (Ro 22-3747) is an allergic mediator release inhibitor which has demonstrated potent oral activity in two IgE-mediated animal models of immediate hypersensitivity: the rat passive cutaneous anaphylaxis test (ID50 of 0.65 mg/kg) and a model in which anaphylactic bronchospasm is induced in passively sensitized rats (ID50 of 0.022 mg/kg). In addition to oral efficacy, in the latter model Ro 22-3747 was 23-fold more potent than cromoglycate by the aerosol (nebulization) route of administration. In vitro studies have confirmed that the mechanism of action of Ro 22-3747 in the in vivo models is through allergic mediator release inhibition since Ro 22-3747 was a potent inhibitor of antigen-induced (IgE-mediated) histamine release from passively sensitized rat peritoneal cells in vitro (IC50 values of 0.25 and 1.5 microM for Ro 22-3747 and cromoglycate, respectively), and Ro 22-3747 did not display end organ antagonism to histamine, serotonin, or the leukotrienes. Clinical evaluations of Ro 22-3747 at a 350 mg oral dose have been conducted in patients with allergic asthma and allergic rhinitis. In a limited study in allergic asthmatics, Ro 22-3747 demonstrated significant inhibitory activity relative to placebo in reducing acute airway responses to inhaled pollen extracts in patients with ragweed sensitivity (measured by changes in log PD20 FEV1 and log PD35 SGaw). The activity seen, however, was less than that observed with cromoglycate (20 mg) administered by inhalation.(ABSTRACT TRUNCATED AT 250 WORDS)

The giant yolk sac: a model for studying early placental transport.

Nine and a half day rat embryos can be cultured for 48 hours in whole heat-inactivated rat serum using the roller culture method described by New, Coppola & Terry (1973). We have prolonged the culture period, usually by seven days. Although the embryo dies almost immediately during this extended culture period, the yolk sac continues to grow and reaches a diameter of approximately 2 cm; we have called this the giant yolk sac. The morphology of the giant yolk sac is very similar to that of control yolk sacs (17 1/2 or 18 1/2 days in vivo), the main difference being the greatly enlarged vacuolar volume in the endodermal cells of the giant yolk sac, which have been studied morphometrically. The pinocytic nature of the giant yolk sac has been demonstrated by its ability to take up colloidal gold. Its rate of uptake of 125I-polyvinylpyrrolidone in whole serum gassed with 95% O2; 5% CO2 has been shown to be similar to the rate of uptake found in control yolk sacs under the same incubation conditions. Acid phosphatase activity was found to be similar in the giant yolk sac and control yolk sacs using both histochemical and biochemical methods. Giant yolk sacs without a contained dead embryo can be produced by removing the embryonic pole of the egg cylinder prior to incubation. They exhibit all the features detailed above. Finally it is shown that the fluid from within the extra-embryonic coelom of the giant yolk sac has some capacity to support the growth and development of 9 1/2 day rat embryos when a source of bulk protein is also provided. This model, therefore, seems to be very useful for the study of transport in a placental system. Its full potential requires further study.

Evidence for the different responses of delta9-, delta6- and delta5-fatty acyl-CoA desaturases to cytoplasmic proteins.

Microsomes prepared from the livers of 4-week-old rats were, after extraction with 0.1 M potassium phosphate buffer, pH 7.4, unable to catalyse either the delta6 desaturation of alpha-linolenic acid (9c.12c.15c., 18 : 3) into 6c.9c.12c.15c., 18 : 4 or the delta5 desaturation of eicosatrienoic acid (8c.11c.14c., 20 : 3) into arachidonic acid (5c.8c.11c.14c., 20 : 4). Both these enzymes only showed full activity after incubation of the microsomes with either the 100 000 X g supernatant fraction or with purified bovine catalase. Bovine serum albumin, while capable of restoring 50% of the delta5 desaturase activity has no effect on the delta6 desaturase. In contrast the delta9 desaturase activity of microsomes was never completely lost after extraction with buffer but could be stimulated by optimum concentrations of both bovine serum albumin and catalase. The significance of the different responses of the three desaturases to the cytoplasmic components is discussed.