Changes in noradrenergic sensitivity to tumor necrosis factor-alpha in brains of rats administered clonidine.

Tumor necrosis factor-alpha (TNF alpha) and the imidazoline clonidine modulate norepinephrine (NE) release from noradrenergic nerve terminals in the central nervous system. The present study demonstrates an intrinsic association between presynaptic alpha 2-adrenergic receptor sensitivity and TNF alpha responsiveness in governing this NE release. Superfusion and electrical field stimulation were applied to a series of rat hippocampal brain slices in order to study the regulation of [3H]-NE release. The alpha 2-adrenergic agonist clonidine and the cytokine TNF alpha concentration-dependently inhibit [3H]-NE release; whereas, the alpha 2-adrenergic antagonist idazoxan potentiates [3H]-NE release. The fractional release of [3H]-NE during field stimulation of control hippocampal slices was decreased by the addition of TNF alpha in a concentration-dependent manner, an effect which was potentiated by the alpha 2-adrenergic antagonist idazoxan; whereas, TNF alpha attenuated the concentration-dependent potentiating effect of idazoxan. Furthermore, constitutive TNF alpha, demonstrated to be present in several brain areas, was significantly decreased following administration of the alpha 2-adrenergic agonist clonidine (0.6 mg/kg, i.p., twice daily) to rats for either 1 or 14 days, without a change in TNF alpha mRNA accumulation. We next investigated whether the presynaptic sensitivity to TNF alpha was changed after clonidine administration to rats. TNF alpha enhanced, rather than inhibited, [3H]-NE release after 1 day of clonidine administration, while a suppressed sensitivity to TNF alpha was observed in the hippocampus after 14 days of clonidine administration. In addition, in the presence of idazoxan, TNF alpha potentiation of [3H]-NE release after 1 day clonidine administration was reversed to a decreased inhibition as compared to control slices exposed to idazoxan. Therefore, the temporary reversal in the presynaptic TNF alpha response after 1 day of clonidine administration illustrates a mechanism of action for its persistent antihypertensive effect, its transient sedative and antihyperpathic effects, and its acute ability to promote antidepressants. These results demonstrate a novel role for an immune mediator in the central nervous system, and demonstrates that presynaptic TNF alpha responsiveness is intimately associated with adrenergic receptor sensitivity.

Beta-adrenergic receptor regulation of macrophage-derived tumor necrosis factor-alpha production from rats with experimental arthritis.

Prostaglandin E2 (PGE2) and beta-adrenergic agonists can suppress lipopolysaccharide-induced tumor necrosis factor-alpha (TNF) production from elicited macrophages. We assessed the responsiveness of rat peritoneal macrophages to PGE2 and the beta-adrenergic agonist isoproterenol during immunologically-mediated arthritis. We assessed macrophage sensitivity to these mediators from resident macrophages and macrophages elicited with either streptococcal cell wall or complete Freund's adjuvant. Peritoneal macrophages were obtained from female Lewis rats that were (1) injected with complete Freund's adjuvant and non-arthritic (CFA); (2) injected with streptococcal cell wall and arthritic (ART); (3) injected with streptococcal cell wall and non-reactive (NON) and (4) non-elicited resident macrophages (RES). When challenged with graded concentrations of lipopolysaccharide (0.1 to 10,000 ng/ml), macrophages obtained from each group of rats released TNF in a concentration-dependent manner, with macrophages from arthritic rats (ART) producing the greatest amount of TNF (p < 0.001). While PGE2 suppressed lipopolysaccharide (100 ng/ml) stimulated TNF production in a concentration-dependent manner in all groups, the greatest sensitivity to PGE2 was observed with macrophages obtained from rats which received streptococcal cell wall when compared to both complete Freund's adjuvant-elicited and resident macrophages (p < 0.05). The beta-adrenergic agonist isoproterenol also inhibited lipopolysaccharide-stimulated TNF production from macrophages in all groups. In addition, the specific beta 2-adrenergic antagonist, ICI 118.551, shifted isoproterenol concentration-effect curves to the right (p < 0.01). Minimal responsiveness to isoproterenol was observed with resident peritoneal macrophages. Maximum isoproterenol-induced inhibition of TNF production was observed with complete Freund's adjuvant-elicited macrophages, and significantly less in macrophages of streptococcal cell wall-injected rats. Of particular interest, macrophages obtained from streptococcal cell wall-injected rats, which became arthritic, were significantly less sensitive to isoproterenol than those which did not develop arthritis (p < 0.02). In addition, these changes in sensitivity were not reflected by changes in the sensitivity of both CFA and ART groups to dibutyryl cAMP. The present study demonstrates a shift in the balance between inhibitory mediator responses in rats inoculated with one of two different adjuvants. These investigations support the role of PGE2 and a neurotransmitter as immunomodulating compounds which may effectively maintain an inflammatory lesion such as arthritis.

Adrenergic regulation of macrophage-derived tumor necrosis factor-alpha generation during a chronic polyarthritis pain model.

Increases in the levels of proinflammatory cytokines, such as TNF alpha, have been intricately linked with arthritis and the pathogenesis of several models of neuropathic pain. In addition, arthritis (as well as other types of persistent pain) is associated with increased sympathetic activity and alterations of other responses in autonomic nervous activity. Adrenergic regulation of LPS-stimulated TNF production by M phi isolated from rats with streptococcal-cell-wall (SCW)-induced arthritis has been examined. Serum TNF levels and the cellular composition of peritoneal exudates have also been assessed. M phi were obtained from: (1) normal control rats, (2) animals injected with complete Freund's adjuvant (CFA), 3 rats injected with SCW and arthritic, and (4) those injected with SCW, which failed to develop arthritis. Serum levels of TNF alpha in rats that develop arthritis are significantly greater (2.4 fold) than levels from the other groups. The proportion of OX19-positive T cell subpopulations are the same in peritoneal exudates from all groups. Immunocytochemical staining also reveals differences between M phi subgroups in the degree of activation. Peritoneal exudates from rats that develop arthritis contain a greater proportion of the high TNF producing subclass of M phi, as identified by positive ED3 staining (p < 0.001). In contrast, Ia antigen presenting M phi (OX6-positive) in the peritoneal exudate cells are only elevated in rats administered CFA. The selective blockade of adrenergic receptors by idazoxan or propranolol demonstrates that the constitutive involvement of either alpha 2 or beta-adrenergic regulation of M phi-derived TNF production is pronounced in rats with arthritis (p < 0.001). These investigations demonstrate a distinctive pattern of peripheral M phi populations in rats that develop chronic polyarthritic pain. We believe that identification of interactions between the adrenergic responses and proinflammatory cytokines will lead to the development of improved strategies to treat patients with chronic pain.

A highly sensitive tool for the assay of cytochrome P450 enzyme activity in rat, dog and man. Direct fluorescence monitoring of the deethylation of 7-ethoxy-4-trifluoromethylcoumarin.

The O-deethylation of 7-ethoxy-4-trifluoromethylcoumarin (EFC) by liver microsomes has been assessed as a method for monitoring the activity of cytochrome P450. The principle advantage of this substrate is the formation of a fluorescent product 7-hydroxy-4-trifluoromethylcoumarin (HFC) which can be assayed directly in the reaction medium. For rat microsomes the deethylated product was confirmed as the main metabolite, the reaction rate was linear with respect to both time and microsomal protein concentration and was independent of small changes in the added co-factors. A linear formation rate for the deethylated metabolite was also confirmed with dog and human microsomes. The intra-assay precision for rat, dog and human microsomes was 3, 5 and 4%, respectively. Hanes transformations of the dog and human data showed two phases, in contrast to a linear decline seen for the rat. Hybrid parameters for Vmax and Km, calculated from the apparently linear portions of these curves, gave interday SD for the Vmax of rat, dog and man of 2, 14 and 4%, respectively, and approximately 15% for the Km in all species. The Vmax in rat, dog and human microsomes was 1.4 +/- 0.2, 4.3 +/- 1.5 and 0.9 +/- 0.5 nmol HFC/min/nmol P450, respectively. The Km was 11.0 +/- 3.1, 67 +/- 19 and 6.8 +/- 2.5 microM, respectively. Direct evidence that at least two isoenzymes (cytochrome P450 1A2 and 2E1) metabolize EFC was obtained by experiments with competitive, suicide and immuno-inhibitors. Compared with ethoxycoumarin, the involvement of P450 2E1 in O-deethylation seemed similar in the rat. In conclusion, EFC provides a straightforward and reproducible assay for microsomal enzyme activity, requiring at most 25 pmol/mL of cytochrome P450.

Estimation of oral drug absorption in man based on intestine permeability in rats.

Predicting the fraction of an oral dose absorbed in humans is of considerable interest at an early stage of a research program in the pharmaceutical industry. Models described in the literature to predict the oral absorption in man include: the permeability in Caco-2 cells, absorption from a perfused segment of rat intestinal lumen and uptake into everted rings. The present study used an isolated and vascularly perfused rat small intestine to determine the permeability values of eleven compounds across the intestinal epithelium. A good correlation was obtained between the permeability values determined in this model and the proportion of an oral dose absorbed in humans. Compared to the other models, the present one could allow the appearance in the artificial bloodstream and the intestinal metabolism of a compound to be studied simultaneously.

A newly discovered xenobiotic metabolic pathway: ethyl ester formation.

Formation of etretinate, ethyl ester of acitretin, can be confirmed in vitro and in vivo using acitretin as the substrate. Etretinate was identified by LC/MS. The in vitro incubation was performed using rat and human liver 12,000 g supernatant, and the in vivo experiment was conducted in rats after oral dosing of acitretin. The ethyl ester formation was greatly enhanced by addition of or dosing with ethanol.

Mechanistic studies on the ethyl-esterification of acitretin by human liver preparations in vitro.

Studies have been performed with human liver microsome preparations in vitro, to investigate the reaction mechanisms involved in the conversion of acitretin to the corresponding ethyl ester, etretinate. The results indicate that: Three fresh samples of human liver, which had been stored in liquid nitrogen for up to 8 months, all produced traces of etretinate (5.8 +/- 0.8 ng/ml) in the presence of ethanol but not when the acitretin was added in acetone, or when the sample was denatured by preheating. Studies with pooled human liver microsomes, to identify the cellular location of the enzymes and the co-factors involved in this esterification, indicate a primary requirement for both ethanol and CoA + ATP with a secondary potentiation in the presence of an NADPH regenerating system. A possible explanation for these finding is that the microsomal ligase enzymes form an intermediate ester between CoA and acitretin, which is then trans-esterified by the ethanol. The low formation with CoA + ATP may indicate that second stage of this process occurs spontaneously, with the NADPH potentiation suggesting that it could also be mediated enzymically.

A new extrapolation method from animals to man: application to a metabolized compound, mofarotene.

Allometric scaling (a technique which uses data obtained in laboratory animals to predict human pharmacokinetics) works well for drugs that are cleared intact, but is less successful with extensively metabolised compounds. This paper describes a new method to improve the accuracy of such projections, by integrating metabolic data obtained in vitro (e.g. with liver microsomes or hepatocytes) into these calculations. The approach was used prospectively, to predict the clearance of mofarotene (Ro 40-8757) in humans from in vivo kinetic data obtained in mouse, rat and dog. This compound was selected to illustrate this approach because it is exclusively eliminated through metabolism. Without the metabolic correction or using empirical correcting factors, the values predicted for man were 2.7 and 0.6 ml/min/kg. This fell outside the range subsequently obtained in healthy volunteers dosed orally with 300 mg of mofarotene (7.5 +/- 4.0 ml/min/kg, n = 12). However, inclusion of the microsomal or hepatocyte data gave values of 5.1 and 4.2 ml/min/kg, respectively, illustrating that the integration of in vitro metabolic data improves the accuracy of kinetic extrapolations. In contrast to the existing empirical techniques, this approach offers a rational basis to predict clearance of metabolized compounds in human.

Comparative in vivo and in vitro studies of phenytoin protein binding and in vitro lipolysis in plasma of pregnant and nonpregnant rats.

This investigation was designed to determine the cause of the changes in drug protein binding that occur in rat plasma, particularly in plasma from pregnant animals, during in vitro drug-protein binding measurements. In vivo estimates of phenytoin binding in plasma were obtained from steady-state CSF-plasma concentration ratios in pregnant and nonpregnant rats. Immediate ultrafiltration of heparin- or EDTA-anticoagulated plasma yielded phenytoin free fraction values that were in good agreement with in vivo estimates for nonpregnant rats but that were about one-third higher than in vivo estimates for pregnant animals. In vitro free fraction values tended to increase during incubation of plasma and/or during equilibrium dialysis. The concentrations of the four major endogenous free fatty acids were similar in plasma of pregnant and nonpregnant rats if determined immediately after blood collection. Six hours of incubation at 37 degrees C caused fatty acid concentrations to increase about fivefold and twofold in heparin-anticoagulated plasma from pregnant and nonpregnant animals, respectively. The corresponding increases in EDTA-anticoagulated plasma were only about twofold and 1.14-fold, respectively. These changes were associated with decreased plasma protein binding of phenytoin. The in vivo differences between pregnant and nonpregnant rats with respect to phenytoin binding in plasma are not due to differences in fatty acid concentrations, but the in vitro differences are due primarily to corresponding differences in free fatty acid concentrations if extensive in vitro lipolysis occurs.

Effect of folic acid on the pharmacokinetics of acutely administered phenytoin in pregnant and nonpregnant rats.

The concentrations of both total and free phenytoin in the plasma of epileptic women tend to decrease during pregnancy, suggestive of a pregnancy-associated increase in the metabolic clearance of the drug. On the other hand, the metabolic clearance of free (unbound) phenytoin decreases during pregnancy in rats. One possible reason for this species difference is the routine dietary supplementation of folic acid in human pregnancy and the apparent ability of folic acid to lower phenytoin plasma concentrations even in nonpregnant humans. The purpose of this investigation was to determine the effect of treatment with folic acid on the pharmacokinetics of phenytoin in pregnant and female nonpregnant rats. In one experiment, the treated animals received folic acid in the drinking water, approximately 100-150 micrograms/kg/d, for 19 d. There was no apparent difference between the treated and untreated rats in the pharmacokinetics of a 10-mg/kg iv dose of phenytoin (which was administered to the pregnant rats on the 20th day of gestation), regardless of pregnancy status, In another experiment, pregnant and female nonpregnant rats received either folic acid, 400 micrograms/kg/d, or an equal volume of the solvent only, by gastric intubation for 19 d. The next day (which was the 20th day of gestation for the pregnant rats), the animals received an intravenous injection of phenytoin, 30 mg/kg. Again, pretreatment with folic acid had no apparent effect on the pharmacokinetics of phenytoin in both pregnant and nonpregnant rats. However, the results of this investigation confirm previous observations of dose-dependent phenytoin pharmacokinetics in rats and of decreased clearance of free phenytoin in late pregnancy.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics of drug action in disease states III: Effect of pregnancy on the relationship between phenytoin concentration and antiseizure activity in rats.

The purposes of this investigation were to determine the effect of pregnancy on the susceptibility of female rats to experimentally induced seizures and on the relationship between serum phenytoin concentration and antiseizure activity. Pregnant rats (on the 18th day of gestation) were more susceptible than nonpregnant female rats to seizures produced by maximal electroshock or by a body-weight-based dose of pentylenetetrazol. There was no apparent difference between pregnant (20th day of gestation) and nonpregnant rats in the relationship between seizure protection (percent of animals protected) and the serum concentration of total (free plus protein-bound) phenytoin. The relationship between concentration and effect was essentially the same 20 min after an injection of phenytoin and 2 h after the start of a constant-rate infusion preceded by a loading dose of the drug. Since the protein binding of phenytoin is appreciably decreased in late pregnancy, the serum concentration of free phenytoin required for seizure protection tended to be higher in pregnant than in nonpregnant rats. This may be due to the increased susceptibility of pregnant rats to seizure stimuli.

Interspecies scaling of interferon disposition and comparison of allometric scaling with concentration-time transformations.

Interspecies scaling is used to extrapolate pharmacokinetic parameters from animals to humans, through the application of physiologically based models, by empirical allometric procedures, or using concentration-time transformations. The aim of this study was to compare the accuracies of the last two methods for predicting the pharmacokinetic parameters and concentration-time curves in humans. In the first part of this study, interspecies scaling techniques were applied to a hypothetical drug (extracellular distribution and elimination through glomerular filtration), to examine the influence of various laboratory animals (mouse, rat, cynomolgus monkey and dog) on the parameters predicted for man. The same techniques were also applied to interferon-alpha A, using the literature data for various animal species. The kinetic parameters predicted in man were then compared to the values published for man. Our theoretical example showed that, for allometric scaling, each species has a very different influence on the prediction in human. With the approach using concentration-time transformations, however, each animal species potentially makes a similar contribution to the prediction for man. Based on the pharmacokinetic data published for interferon-alpha A in laboratory animals, allometric equations underestimated the observed values of CL and Vdss in man by 2-3-fold, and the prediction of t1/2 was likely to be unreliable, due to a poor correlation. The use of equivalent time, kallynochron, and apolysichron transformations improved the pharmacokinetic predictions for all three parameters in man. In conclusion, concentration-time transformations make more adequate use of the data available in the different species of laboratory animals, to give better predictions of the pharmacokinetic parameters in man.

Integration of in vitro data into allometric scaling to predict hepatic metabolic clearance in man: application to 10 extensively metabolized drugs.

In this study, we investigated rational and reliable methods of using animal data to predict in humans the clearance of drugs which are mainly eliminated through hepatic metabolism. For 10 extensively metabolized compounds, adjusting the in vivo clearance in the different animal species for the relative rates of metabolism in vitro dramatically improved the predictions of human clearance compared to the approach in which clearance is directly extrapolated using body weight. Using hepatocyte data to normalize the in vivo clearances led to lower median deviations between the observed and predicted clearances in man compared to the approach normalizing data with brain weight (30-40% vs 60-80%, respectively). In addition, the approach integrating in vitro data appeared to be superior with respect to the range of deviations: approximately 2-fold underestimation, in the worst case, was observed by using in vitro data, whereas normalizing data by brain weight led to up to 10-fold underestimation of clearance in man. In addition, the integration of in vitro data provides a more rational basis to predict the metabolic clearance in man and may be applicable to compounds undergoing phase I and phase II metabolism as well.

Animal pharmacokinetics and interspecies scaling from animals to man of lamifiban, a new platelet aggregation inhibitor.

Relating pharmacokinetic information obtained in animal species to man (interspecies scaling) can play an important role in enabling understanding of the differences and similarities between species, and helping to predict the kinetic profile of a new compound in man. Interspecies scaling techniques have been applied to lamifiban (Ro 44-9883), a new selective and potent nonpeptidic inhibitor of human glycoprotein IIb-IIIa intended for use in clinical treatment of, for example, acute coronary syndrome. The pharmacokinetic profile of lamifiban in man was predicted from animal data (in rats, dogs and cynomolgus monkeys) by using allometric scaling and concentration-time transformations. These extrapolations for lamifiban were performed prospectively, to help design the first pharmacokinetic studies in man. The approach based on equivalent time was preferred for our prospective predictions, in view of the high values found for the allometric exponents. Using allometric scaling, clearance (CL), half-life (t1/2) and volume of distribution (Vd) were overestimated by approximately two- to fourfold. Compared with allometric scaling, the transformation based on equivalent time improved the prediction for all human pharmacokinetic parameters. For t1/2 and CL, the observed values for man were within the range predicted from the various animal species. Of the individual animal species, the cynomolgus monkey gave the most reliable predictions of these two parameters, as well as accurately predicting the Vd value.

Effect of heparin or salicylate infusion on serum protein binding and on concentrations of phenytoin in serum, brain and cerebrospinal fluid of rats.

The purpose of this investigation was to determine if administration of heparin causes displacement of an acidic drug from serum protein binding sites in vivo as has been suggested by several groups of investigators. Rats were injected and infused with phenytoin to produce steady-state serum concentrations of about 25 micrograms/ml. After 2 hr, some of the animals also received injections and infusions of either heparin or salicylic acid. Salicylic acid (about 300 micrograms/ml), a classical inhibitor of phenytoin protein binding, reduced the steady-state serum concentration of total (free plus bound) phenytoin but had no significant effect on the steady-state serum concentration of free phenytoin or on the concentrations of phenytoin in cerebrospinal fluid and brain. The concentrations of phenytoin in cerebrospinal fluid were almost identical to the free phenytoin concentrations in serum. Similar effects were observed with respect to 5-(p-hydroxyphenyl)-5-phenylhydantoin and were found to be due to displacement of this major metabolite of phenytoin from serum protein binding sites by salicylate. Heparin administration caused an apparent increase of the steady-state plasma concentration of free phenytoin when determined as the product of the concentration of total phenytoin and the free fraction measured by in vitro equilibrium dialysis. Since heparin treatment had no significant effect on the total concentrations of phenytoin in plasma, cerebrospinal fluid and brain, it is concluded that the apparent displacement of phenytoin from plasma proteins occurs in vitro, after collection of blood samples from heparinized animals.

Effect of pregnancy on the pharmacokinetics of phenytoin in rats.

Phenytoin, in single doses of 10 or 30 mg/kg, was administered by i.v. injection to nonpregnant (congruent to 200-300 g) and 20 days pregnant inbred Lewis rats. The plasma protein binding of phenytoin was determined under conditions which minimized in vitro lipolysis and consequent artifactual results. The absolute (milliliters per minute) plasma clearance of total (free plus bound) phenytoin by the pregnant rats was increased (10-mg/kg dose) or not significantly different (30-mg/kg dose) compared to concurrent nonpregnant controls. The relative (milliliters per minute per kilogram) plasma clearance of total phenytoin was not significantly changed (10-mg/kg dose) or was decreased (30-mg/kg dose) in pregnancy. The relative apparent volume of distribution of free drug (but not of total drug) was essentially the same in the pregnant and nonpregnant animals and was independent of dose. The plasma clearance of free (unbound) phenytoin decreased with dose and was decreased during pregnancy, at both doses studied (more so at the larger dose). The elimination kinetics of p- hydroxyphenytoin , the major metabolite of phenytoin which inhibits phenytoin metabolism in rats, were similar in pregnant and nonpregnant rats and so were the plasma concentrations of this metabolite after phenytoin administration. The relatively more pronounced effect of pregnancy on the elimination of the larger dose of phenytoin may reflect a greater inhibitory effect of p- hydroxyphenytoin during pregnancy. This does not occur in humans (due to much lower plasma concentrations of the metabolite) and that may account for possible differences in the effect of pregnancy on phenytoin pharmacokinetics in rats and humans.

A comparison of the size of families of roman catholics and non-catholics in Great Britain.

Abstract In a recent study of family size ideals in the D.S.A. it was found that in the 1960'S the mean ideal family size of Catholics was about half a child higher than the mean ideal size of non-Catholics. This note describes an analysis of similar data for married women in Great Britain, derived from an investigation undertaken in 1966 for the Population Investigation Committee. A difference in ideal family size, which was of the same order as the American difference, was found; and, in addition, the actual fertility of Catholics was compared with that of others.