Rate zonal sedimentation of proteins in one hour or less.

Rate zonal sedimentation gives information about the shape and size of proteins, and is useful for investigating protein-protein interactions. However, rate zonal sedimentation experiments typically last approximately 1 day. In contrast, this report describes a rate zonal sedimentation method requiring 1 h or less. This was accomplished by centrifuging small density gradients (200 microliters) prepared with sucrose or OptiPrep in a fixed-angle rotor at high relative centrifugal force. By using small gradient volumes, the sample dilution that occurs with larger gradients and with many chromatographic techniques was also avoided. For a variety of proteins, plots of S20,w versus distance sedimented during centrifugation in a TLA 120.2 rotor were linear. As a practical application, sedimentation of the heterotrimeric stimulatory G protein and its dissociated alpha-subunit were determined. The results were similar to those obtained with 17- to 22-h centrifugations in an SW 50.1 rotor and agreed with previously published values. Long periods of centrifugation might preclude the study of some unstable proteins or the investigation of protein-protein interactions whose affinities are to low to survive the lengthy centrifugations required to carry out traditional rate zonal sedimentation experiments. A rate zonal sedimentation technique that rivals many chromatographic methods in celerity will help to circumvent these problems.

Does subunit dissociation necessarily accompany the activation of all heterotrimeric G proteins?

Heterotrimeric (alpha beta gamma) G proteins mediate a variety of signal transduction events in virtually every cell of every eukaryotic organism. The predominant hypothesis is that dissociation of the alpha-subunit from the G beta gamma-subunit complex necessarily accompanies the activation of these proteins, and that the alpha-subunit is primarily responsible for regulating the response of effector molecules. However, there is increasing evidence that both the alpha-subunit and the beta gamma-subunit complex function in regulating effector activity. Furthermore, data for some G proteins suggest that they function as activated heterotrimers rather than as dissociated subunits.

Chloride effects on Gs subunit dissociation. Fluoroaluminate binding to Gs does not cause subunit dissociation in the absence of chloride ion.

The stimulatory guanine nucleotide binding protein (Gs) is heterotrimeric ( alpha beta gamma), and mediates activation of adenylyl cyclase by a ligand-receptor complex. The alpha subunit of Gs (Gsalpha) has a guanine nucleotide binding site, and activation occurs when tightly bound GDP is displaced by GTP. Together, GDP and fluoroaluminate (AlF4-) form a transition state analog of GTP that activates Gs. The work of other investigators suggests that AlF4- causes subunit dissociation when it activates Gs. We have observed that in solution AlF4- did not cause Gs subunits to dissociate unless NaCl was also present. The effect of NaCl was concentration dependent (10-200 mM). Omitting F-, Al3+, or Mg2+ prevented the NaCl-induced dissociation of Gs subunits. Na2SO4 could not substitute for NaCl in causing subunit dissociation, but KCl could, suggesting that the anion was responsible for the effect. Gs subunit reassociation occurred when the concentration of Cl- was reduced even though the concentrations of AlF4- and Mg2+ were maintained. The absence of Cl- did not prevent AlF4- binding to Gsalpha. We have concluded that AlF4-, a ligand which is capable of activating G proteins, can bind to Gs in solution without causing subunit dissociation.

Comparison of the anti-inflammatory effect and gastrointestinal tolerability of aceclofenac and diclofenac.

Aceclofenac (CAS 89796-99-6) and diclofenac (CAS 15307-79-6) are orally effective non-steroidal anti-inflammatory drugs. Their anti-inflammatory and potential gastrointestinal damaging effects were compared following single and repeated administration (5 days). Both drugs exerted an anti-inflammatory activity and showed a similar gastrointestinal tolerability in the rat.

GTP binding to Gs does not promote subunit dissociation.

The stimulatory G protein (Gs) mediates activation of adenylyl cyclase. Gs is a heterotrimeric protein (alpha beta gamma) that is activated when guanosine triphosphate (GTP) or a non-hydrolyzable GTP analogue displaces tightly bound guanosine diphosphate (GDP) from the guanine nucleotide-binding site of the alpha-subunit (Gs alpha). Divalent cations such as magnesium are also required for Gs activation. Subunit dissociation can accompany Gs activation and is thought to be critical for this process. We investigated the effects of MgCl2 and various purine nucleotides on Gs-subunit dissociation and activation. Subunit dissociation was assayed by measuring the amount of G protein beta-subunit that was co-precipitated by Gs alpha-specific antiserum. Gs activation was determined by its ability to reconstitute adenylyl cyclase activity in S49 cyc-membranes that lack Gs alpha. High concentrations of MgCl2 caused bound GDP to dissociate from Gs and inactivated the protein unless high concentrations of GDP or GTP were present in solution. MgCl2 caused a concentration-dependent dissociation of Gs subunits. GTP gamma S (a non-hydrolyzable GTP analogue) shifted the MgCl2 concentration-response curve for subunit dissociation to lower concentrations of MgCl2, suggesting that GTP gamma S promoted subunit dissociation. On the other hand, GDP and GTP were equally effective in shifting the curve to higher concentration of MgCl2. These results suggest that GTP, the compound that activates Gs in vivo, was no more effective at promoting Gs subunit dissociation than was GDP.

Cell-free synthesis of functional type IV adenylyl cyclase.

Type IV adenylyl cyclase was synthesized in a cell-free coupled transcription and translation system. Radiolabeled type IV adenylyl cyclase was specifically immune precipitated with anti-ACIV antibodies. The molecular weight of in vitro translated type IV adenylyl cyclase was 110,000, similar to that for type IV adenylyl cyclase produced in the baculovirus system [B. Gao and A. G. Gilman, (1991) Proc. Natl. Acad. Sci. USA 88, 10178-10182]. Dimyristoyl phosphatidylcholine was required for efficient stimulation of activity by both forskolin and GS, with a maximum specific activity of 700 +/- 100 nmol cAMP.min-1.mg-1 attained with both effectors combined. Both bovine brain GS and in vitro translated GS alpha activated in vitro translated type IV adenylyl cyclase; however, G beta gamma only enhanced stimulation in the presence of in vitro translated GS alpha. Forskolin maximally activated at concentrations from 200 to 400 microM in the absence or presence of GS. The in vitro translated product was very stable as production of cAMP by forskolin/GS activated type IV adenylyl cyclase was linear for up to 90 min at 30 degrees C.

Pharmacokinetics of ketoprofen enantiomers in monkeys following single and multiple oral administration.

Pharmacokinetic studies are reported after single oral administration of 3 mg/kg of stereochemically pure (S)-ketoprofen [(S)-KP] and (R)-ketoprofen [(R)-KP] to three male Cynomolgus monkeys and after repeated administration for 6 months of 3, 15 and 75 mg/kg/day of (S)-KP to both male and female monkeys. A high-performance liquid chromatographic (HPLC) analysis was performed without derivatization of the samples, using a chiral column. The pharmacokinetic parameters for (S)-KP after administration of (S)-KP and for (R)-KP after administration of (R)-KP were, respectively, elimination half-life 2.32 +/- 0.36 and 1.64 +/- 0.40 h; oral clearance 3.50 +/- 0.66 and 7.50 +/- 3.20 ml/min/kg; apparent volume of distribution 0.74 +/- 0.24 and 1.16 +/- 0.76 liter/kg; mean residence time 1.79 +/- 0.77 and 1.41 +/- 0.65 h; area under the concentration/time curve 14.16 +/- 2.93 and 7.31 +/- 2.98 micrograms.h/ml. Forty-nine percent unidirectional bioinversion of (R)-KP to (S)-KP was observed in this species and the pharmacokinetic parameters for the (S)-KP resulting from this inversion were also calculated. In the study of 6-month repeated administration of (S)-KP, linear pharmacokinetic behavior and no evidence of drug accumulation were observed at the three dose levels.

Regulation of glycogen synthase activity in isolated rat adipocytes by levamisole.

The effect of levamisole on glycogen synthase activity in isolated adipocytes was studied. The addition of levamisole to these cells resulted in an acute concentration-dependent increase in glycogen synthase activity. In contrast, epinephrine, dibutyryl cyclic AMP (DcAMP) and cysteamine decreased glycogen synthase activity. The stimulatory effect of levamisole on the activity of the enzyme was not affected by the presence of epinephrine but was diminished when either DcAMP or cysteamine was present. The results of this study suggest that levamisole increases adipocyte glycogen synthase activity by a mechanism that can be reversed by the elevation of cAMP.

Regulation of pyruvate dehydrogenase activity in rat fat pads and isolated hepatocytes by levamisole.

The effect of levamisole on the catalytic activity of the pyruvate dehydrogenase (PDH) complex in rat adipose tissue segments and isolated hepatocytes was studied. Levamisole, an anthelmintic and immunotherapeutic agent with concentration-dependent oxidant/antioxidant properties, increased the catalytic activity of the pyruvate dehydrogenase complex in these systems. The activity of this enzyme complex may be regulated in part by protein-thiol modification. The treatment of rat adipose tissue segments and isolated hepatocytes with and without levamisole (0.1-3.0 mM) for 30 min resulted in a reversible increase in the catalytic activity of this enzyme complex. Levamisole at 1.5 mM and 2.0 mM were as potent as insulin (1.0 mU/ml) in adipose tissue and as dichloroacetate (2.0 mM) in isolated hepatocytes, respectively, in increasing the catalytic activity of the PDH complex. The stimulatory effects seen with levamisole were not accompanied by decreases in adenosine triphosphate (ATP) levels. The results of the present investigation suggest that the pyruvate dehydrogenase complex in adipose tissue and liver may be useful models for studying the mechanism of action of levamisole.

Regulation of glucose transport in isolated adipocytes by levamisole.

When isolated rat adipocytes were incubated with increasing concentrations of levamisole (0.5-5 mM), basal glucose oxidation decreased by almost 50% and insulin-stimulated glucose oxidation decreased by 90%. The decrease in glucose oxidation correlated with an inhibition of glucose transport, since levamisole at 5.0 mM decreased basal 3-O-methylglucose transport by 60% and insulin-stimulated transport by 80%. Diamide-stimulated glucose transport was also inhibited approximately 80% by 5.0 mM levamisole. Levamisole at concentrations up to 5.0 mM had no effect on phosphofructokinase activity. The present results suggest that levamisole inhibits glucose utilization by inhibiting glucose transport in a concentration-dependent manner.

In vitro and in vivo studies with flutrimazole, a new imidazole derivative with antifungal activity.

1-[(2-Fluorophenyl)(4-fluorophenyl)phenylmethyl]-1H-imidazole (flutrimazole, UR-4056, CAS 119006-77-8) is a new topical imidazole antifungal agent which displays potent broad-spectrum in vitro activity against dermatophytes, filamentous fungi and yeasts, saprophytic and pathogenic to animals and humans (MIC = 0.025-5.0 micrograms/ml). In most of these studies the activity of flutrimazole was comparable to that of clotrimazole and markedly higher than that of bifonazole (MIC differences of greater than or equal to 1 order of magnitude). Tested against Scopulariopsis brevicaulis (8 strains), both flutrimazole and clotrimazole exhibited fungistatic and fungicidal activity, and clotrimazole appeared something less active (MIC = 0.3-2.5 micrograms/ml) than flutrimazole (MIC = 0.15-0.6 micrograms/ml). In animal experiments, topical application of 1% and 2% flutrimazole, as a cream or solution, was highly effective in models of rat vaginal candidiasis and guinea-pig trichophytosis, giving a rate of cured or cured plus markedly improved animals higher than 80%. Flutrimazole shares the mode of action of other imidazole or triazole-containing antifungals, i.e. inhibition of fungal lanosterol 14 alpha-demethylase, as it strongly inhibits ergosterol biosynthesis in a cell-free homogenate of Candida albicans, with an IC50 value of 0.071 mumol/l.

Percutaneous absorption and skin distribution of [14C]flutrimazole in mini-pigs.

The percutaneous absorption and skin distribution of a skin cream containing 1% 14C-labelled 1-[(fluorophenyl) (4-fluorophenyl) phenylmethyl]-1H-imidazole (flutrimazole, UR-4056, CAS 119006-77-8) was studied in minipigs. The same dose of flutrimazole was administered i.v. and topically (as a cream) on scarified skin according to a crossover protocol. Samples of urine and faeces were taken at various intervals after administration, and radioactivity was measured. The percentage of radioactivity accumulated in urine after topical and intravenous administration were 1.46% and 41.7%, respectively. In faeces, the percentage of radioactivity observed was 6.0% after intravenous administration, and none was detected after topical application. In order to study the distribution and penetration of [14C]flutrimazole, the cream was applied to intact and scarified skin. At various intervals after administration, skin samples were taken. The samples for the autoradiographic studies were cut transversely, and for the measurement of the levels of radioactivity at different skin depths, slices were cut parallel to the cutaneous layers. The results obtained indicate that [14C]flutrimazole penetrates quickly into the different epidermic layers and is retained mainly in the strata spinosum, granulosum and basale. The stratum basale possibly acts as a selective barrier preventing the penetration of the compound into the dermis. The percentage of radioactivity in the stratum corneum is lower than that detected in all the other epidermic layers taken together. The stratum corneum offers low resistance to penetration by the flutrimazole, which very probably crosses the epidermic strata by a transcellular route.

Pharmacokinetic study of [14C]flutrimazole after oral and intravenous administration in dogs. Comparison with clotrimazole.

This trial involved a comparative study using 6 Beagle dogs on the pharmacokinetics of 14C-labelled 1-[(2-fluorophenyl)(4-fluorophenyl)phenylmethyl]-1H-imidazole (flutrimazole, CAS 119006-77-8) and [14C]clotrimazole labelled in the imidazole ring. On the basis of a cross-over trial, each animal received a dose of 5 mg/kg (approx. 100 microCi) [14C]flutrimazole and [14C]clotrimazole, both intravenously and orally. The levels in plasma, urine and faeces of the total radioactivity, unchanged drug and the [14C]imidazole formed by metabolization of the unchanged drug were determined. Flutrimazole presented a biological half-life (t1/2) of 14.4 +/- 3.8 h and a clearance (Cl) of 6.7 +/- 0.8 l/h, while the values for clotrimazole were very different: t1/2 4.6 +/- 0.8 h and Cl: 13.6 +/- 1.0 l/h. After oral administration a fraction of absorbed dose (f) of 78 +/- 21% and bioavailability of 8.9 +/- 6.1% were calculated for flutrimazole. For clotrimazole, these were: 52 +/- 10% and 4.9 +/- 1.9%, respectively. Both drugs showed a significant first-pass effect, with 90% of the absorbed dose being metabolized before reaching the systemic circulation. The total recovery of radioactivity in faeces and urine 5 days after i.v. and oral administration was 58% and 68%, respectively, for [14C]flutrimazole, and 81% and 79% for [14C]clotrimazole. In both cases, most of the radioactivity was recovered in the faeces. The high radioactivity obtained in faeces after i.v. administration of both drugs confirms biliary elimination. For both flutrimazole and clotrimazole, less than 1% of the total recovered in the urine after i.v. administration was recovered as unchanged drug.(ABSTRACT TRUNCATED AT 250 WORDS)

A pharmacokinetic study of E-4441, a new quinolone, in the rat, mouse and cynomolgus monkey.

The purpose of this present work has been to study the pharmacokinetical profile of E-4441 in the rat, mouse and cynomolgus monkey. Analytical determination of the levels in plasma, urine and organs was affected by two different techniques: a microbiological agar diffusion assay with Bacillus subtilis, and HPLC with preliminary extraction in chloroform (pH 8.5). The kinetic behaviour of the unchanged substance was similar in the three animal species studied. Absorption and excretion took place rapidly (T1/2a = 2-20 min, T1/2el = 25-225 min). In mouse there is a linear relationship between administered dose and the area under the curve (AUC) of plasma levels. The absolute bioavailability of unchanged E-4441 is of 20% and the urinary excretion represents 1 to 2% of the administered oral dose. The organs in which the highest concentrations of substance were found were bile, liver and kidney, while the substance could not be detected in brain, testis, ovary or adipose tissue.

Anti-ulcer activity and other pharmacological properties of lozilurea.

As a result of trials on a large series of compounds, one of these, N' -3-chlorobenzyl-N'-ethylurea (lozilurea, ITA 312) has shown marked anti-ulcer activity. It has shown itself to be active against chemically and neurogenically induced gastric and duodenal lesions in various experimental animal models. It has no major anti-secretory action. The experimental data obtained suggest that the mechanism of action of lozilurea consists in increasing the protective function of the mucus barrier. In the screening trials carried out in order to detect the side effects of lozilurea, it has shown sedative, antipyretic and vasodilatory actions.

Influence of plafibride, an antiplatelet and hypolipemic agent, on prostacyclin and thromboxane synthesis, 3',5'-cyclic AMP phosphodiesterase activity and serum clearance of a lipid emulsion.

The activity of N-2-(p-chlorophenoxy)-isobutyryl-N'-morpholinomethylurea (plafibride, ITA 104) on arachidonic acid metabolism, the 3',5'-cyclic AMP-phosphodiesterase and the serum clearance of a lipid emulsion is reported in order to clarify its mechanism of action on platelet aggregation and lipid metabolism. Plafibride did not act on the arachidonic acid metabolism as far as platelet aggregation was concerned, since it did not modify the generation of prostaglandin endoperoxides nor prostacyclin. Neither did it act on the generation of thromboxane A2. Plafibride inhibited the activity of 3',5'-cyclic AMP-phosphodiesterase, which is one of the principal mechanisms of inhibition of platelet aggregation. The serum c clearance of a commercial lipid emfy the generation of prostaglandin endoperoxides nor prostacyclin. Neither did it act on the generation of thromboxane A2. Plafibride inhibited the activity of 3',5'-cyclic AMP-phosphodiesterase, which is one of the principal mechanisms of inhibition of platelet aggregation. The serum clearance of a commercial lipid emulsion was increased by plafibride which also possessed a strong hypotriglyceride activity. The correlation between platelet antiaggregant and hypolipemic activity of plafibride is discussed in this paper.

Pharmacological activities and side effects of plafibride.

This paper reports on pharmacological properties of N-2-(p-chlorophenoxy)-isobutyryl-N'-morpholinomethyl-urea (plafibride, ITA 104) and its possible side effects. This work was carried out on CNS, ANS and PNS, cardiovascular system, respiratory and gastroenteric apparatus as well as anti-inflammatory activity and gastric tolerance. The most evident secondary effects were: a certain sedation, as a light tranquillizing agent, a hypothermic effect when it was administered at high doses, a certain beta-blocking and antiarrhythmic activity probably due to its local anaesthetic action. All the side effects appeared at high doses, much higher than the therapeutic ones.

Toxicological studies of plafibride. Part 4: Interaction of plafibride with other drugs.

The possible pharmacological interaction was studied between N-2-(p-chlorophenoxy)-isobutyryl-N'-morpholinomethylurea (plafibride, ITA 104) and a series of products with analgesic, anticoagulant, antidepressant, antidiabetic, Beta-blocking, diuretic, hypotensive, nootropic, tranquillizing and vasodilator activities. In the experiment the LD50 of each product was compared with that found for the joint administration of the product and plafibride. Plafibride increased the acute toxicity of reserpine and potentiated its hypothermic effect in the mouse; it did not alter its effect on arterial pressure in dog and rat nor its effect on palpebral ptosis in the mouse. Although plafibride did not potentiate the acute toxicity of warfarin, its anticoagulant activity was potentiated, possibly due to the movement of warfarin from its binding sites to the plasma proteins.