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.

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.

The use of human hepatocytes to select compounds based on their expected hepatic extraction ratios in humans.

PURPOSE: The present investigation retrospectively evaluates the use of human hepatocytes to classify compounds into low, intermediate or high hepatic extraction ratio in man. METHODS: A simple approach was used to correlate the in vivo hepatic extraction ratio of a number of compounds in man (literature and in-house data) with the corresponding in vitro clearance which was determined in human hepatocytes. The present approach assumes that, for compounds eliminated mainly through liver metabolism, intrinsic clearance is the major determinant for their in vivo hepatic extraction ratio and subsequently their bioavailability in man. The test compounds were selected to represent a broad range of extraction ratios and a variety of metabolic pathways. RESULTS: The present data show that in vitro clearances in human hepatocytes are predictive for the hepatic extraction ratios in vivo in man. Most of the test compounds (n = 19) were successfully classified based upon human hepatocyte data into low, intermediate or high hepatic extraction compounds, i.e. compounds with potential for high, intermediate or low bioavailabilities in humans. CONCLUSIONS: The present approach, validated so far with 19 test compounds, appears to be a valuable tool to screen for compounds with respect to liver first-pass metabolism at an early phase of drug discovery.

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.

Interspecies scaling of tolcapone, a new inhibitor of catechol-O-methyltransferase (COMT). Use of in vitro data from hepatocytes to predict metabolic clearance in animals and humans.

1. In the present study, in vivo pharmacokinetic data in animals were combined with in vitro metabolic data from animal and human hepatocytes to predict the human systemic plasma clearance and the kinetic profile of tolcapone, a compound metabolized by phase II reactions. 2. The integration of in vitro metabolic data from hepatocytes into allometric scaling gave satisfactory predictions of metabolic clearance in humans for tolcapone (74.2 ml/min predicted versus 118 ml/min observed). 3. Using combined time transformations and in vitro metabolic rates, the range of values predicted from the various animal species (90.4 to 242 ml/min, 0.60 to 2.2 h and 7.3 to 121 for clearance, half-life and volume of distribution, respectively) were in good agreement with the observed values in humans (118 ml/min, 1.3 h and 8.6 h, respectively). 4. Compared to the conventional correction factors (e.g. maximum life span, brain weight), in vitro metabolic data provide a more rational basis for extrapolating the metabolic clearance in humans.

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.

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.

Distribution of remikiren, a potent orally active inhibitor of human renin, in laboratory animals.

1. Whole-body autoradiography was used to compare the distribution of remikiren in the squirrel monkey, in which the compound is a potent inhibitor of renin, with the rat and the guinea-pig in which it is less active. 2. Following intravenous administration, drug-related material was rapidly and extensively taken up by the tissues of all three species. Consistent with rapid biliary elimination, high levels of radioactivity were found in the bile duct/gall bladder/intestinal contents. Of the remaining organs, the kidney consistently showed the highest concentrations of drug-related material. 3. Radio-hplc analysis of the kidney samples demonstrated that the majority of the retained material was present as intact remikiren, even at 24 h after administration. A similar degree of retention by the kidney was also found after oral dosing. 4. Uptake of remikiren by the kidney may act as a reservoir for the drug, resulting in the prolonged duration of pharmacological activity, which, despite the high plasma clearance of the drug, has previously been observed in primates.

Pharmacokinetics of remikiren, a potent orally active inhibitor of human renin, in rat, dog and primates.

1. An hplc method with fluorescence derivatization was developed for the quantification of remikiren in plasma (limit of quantification 2 ng/ml). This was used to determine the pharmacokinetics in various species of primate, in which the compound is a potent inhibitor of renin, as well as in the rat and dog in which it is less active. 2. After intravenous administration the mean residence time was < or = 1.5 h in all species, and the plasma clearance approached the corresponding hepatic blood flows. 3. Studies in the bile-duct cannulated rat and dog demonstrated that the high clearance was due to a combination of rapid metabolism, plus biliary and renal excretion of intact drug. 4. Consistent with the high hepatic clearance, oral bioavailability was low ( < or = 6%) in each species. However, all of the species tested absorbed a small proportion of an oral dose extremely rapidly, to give peak concentrations generally within 5 min of administration. 5. 'Simultaneous' collection of blood samples from the hepatic portal vein and aorta of rat confirmed that shortly after oral dosing the intact drug did cross the liver; however, the later collections contained predominantly more polar metabolites. 6. The rapid absorption of intact remikiren is consistent with the transient blockade of plasma renin activity, previously observed in primates after oral administration. However, the high clearance appears inconsistent with the subsequent prolonged decrease in blood pressure, suggesting that the latter effect is mediated through a 'tissue' compartment.

Interspecies scaling of bosentan, a new endothelin receptor antagonist and integration of in vitro data into allometric scaling.

PURPOSE: The goal of this study was to find a rational and reliable method of using animal data to predict the clearance of metabolised drugs in humans. METHODS: One such approach is to use in vitro liver models (e.g. hepatocytes and microsomes) to determine the relative capacities of the various animal species and humans to metabolise the test compound. These data can then be combined with the in vivo clearances in animals, to calculate the in vivo clearance in humans using allometric scaling techniques. In this study, this approach was evaluated with a new endothelin receptor antagonist, bosentan, which is eliminated mainly through metabolism and is characterized by very large interspecies differences in clearance. Therefore, this compound provided a stringent test of our new extrapolation method for allometric scaling. RESULTS: The results obtained with bosentan showed that adjusting the in vivo clearance in the different animal species for the relative rates of metabolism in vitro gave a far better prediction of human clearance than an empirical correcting factor (brain weight). CONCLUSIONS: This approach provided a more rational basis for predicting the clearance of metabolised compounds in humans.

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.

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.

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.

Animal pharmacokinetics and metabolism of the new antimalarial Ro 42-1611 (arteflene).

Ro 42-1611 (arteflene) is a new synthetic structural analogue of yingzhaosu, a Chinese traditional herbal drug, now under development for treatment of malaria. The in vivo activity of arteflene in a mouse animal model was 4-5 fold higher after parenteral than after oral administration. Pharmacokinetics of the drug were investigated in mice, rats, dogs, marmosets and cynomolgus monkeys. Plasma concentrations of arteflene were determined using a specific HPLC-UV method; the limit of quantification was 45 ng/ml using 0.5 ml plasma. The oral bioavailability was very low and variable (0.6% in mice, 4-5% in rats, 2.5 +/- 1% in dogs, < or = 0.5% in marmosets and < 0.5% in cynomolgus) as expected from the high metabolic clearance and the relative short apparent half-life (1.4-4.7 h). However, a metabolite (MA) was observed in plasma of all species indicating that drug was absorbed but underwent extensive first-pass metabolism. MA was also detected in samples of human plasma, collected during an oral tolerability study in healthy volunteers. After incubation of 14C-arteflene with liver microsomes of mice, rats, dogs and humans, the same major metabolite was detected and both samples were identical to Ro 47-6936 which was chemically synthesized as a reference compound. The in vitro activity of Ro 47-6936 was tested against Plasmodium falciparum and found to be about 1/4 that of the parent drug. Therefore, this metabolite makes a significant contribution to the biological activity in vivo, partially explaining the high activity of arteflene after oral administration in spite of its low bioavailability. Moreover, comparison of the metabolic patterns from human, rat and dog microsomes indicated that the dog is an appropriate species for toxicological evaluations.

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.

A potentially new metabolic pathway: ethyl esterification of acitretin.

1. The acidic retinoid, acitretin, was esterified to etretinate (ethyl ester) by rat and human liver 12,000 g supernatant. The amount of etretinate formed was increased by adding ethanol to the rat preparation. 2. This esterification almost certainly involves enzymic catalysis, and the amounts of etretinate formed were increased by the use of fresh rat liver. 3. Co-administration of acitretin and ethanol to rats resulted in a maximum plasma concentration of etretinate at approximately 1 h after dosing. Secondary maxima were induced by administering ethanol alone at 5 and 8 h after dosing with acitretin. 4. Comparison of acitretin and etretinate concentrations in rat portal and jugular vein plasma after ethanol administration indicated that the ester was formed mainly systematically, rather than during absorption. 5. The results of our study in the rat could indicate that the presence of etretinate in plasma of some patients being treated with acitretin may result from the intake of alcohol.