Anti-allergy and anti-asthma drugs. Disposition in infancy and childhood.

Six classes of drug may be prescribed in the treatment of airway hyperreactivity and allergy. Use of the methylxanthine theophylline requires that plasma drug concentrations be monitored because of its pharmacokinetic properties and narrow therapeutic range. beta 2-Selective adrenergic agonists, glucocorticoids, sodium cromoglycate and the quaternary antimuscarinic ipratopium achieve specificity of drug action on the bronchi with minimal side effects by local delivery as aerosols or 'microfine' powders. Glucocorticoids, sodium cromoglycate and ipratropium bromide may also be applied locally to the nasal mucosa in allergic rhinitis, and sodium cromoglycate (cromolyn sodium) may be applied to the eye. The 'antihistamines'--H1-receptor antagonists--are not used to treat bronchial hyperreactivity but are frequently used systemically for treating allergic conditions. Two new agents, terfenadine and astemizole, appear to be specific for H1-receptors and represent a new 'generation' of antihistamines that produce sedation only infrequently. Terfenadine exhibits a bimodal elimination phase (slow component +/- 22 hours) and astemizole has an active metabolite with a half-life of 12 days. The half-lives of most other antihistamines lie in the 4- to 8-hour range (except chlorpheniramine, which has a longer half-life). However, data reflecting disposition of these drugs in children are scanty. There is a need for more powerfully predictive pharmacokinetic approaches, which is discouraged by the widely used modelling approach combining patient and drug characteristics in single variables. Separation of these could improve extrapolation from drugs for which data are available to those for which they are not.

Delayed, ferrous iron-dependent peroxidation of rat liver microsomes.

Measurement of both chemiluminescence (CL) and the formation of 2-thiobarbituric acid-reacting substances (TBAR) has been used to study the delayed, nonenzymatic lipid peroxidation (LP) initiated in rat liver microsomes by ferrous chloride. Following Fe2+ addition, the CL technique revealed a burst of light emission (peak, Phase II) which was preceded by a period of little or no detectable photon production (delay, Phase I) and succeeded by an increased emission (Phase III). Analysis of TBAR indicated a low rate of LP during the delay which increased more than fivefold during a 1-min period and which corresponded to the CL peak. The delay length depended on both the Fe2+ concentration and the microsome concentration; increased Fe2+ yielded longer delays while increased microsome concentration decreased the delay. As reported by others [J. R. Bucher, M. Tien, and S. D. Aust (1983) Biochem. Biophys. Res. Commun. 111, 777-784; J. M. Braughler, L. A. Duncan, and R. L. Chase (1986) J. Biol. Chem. 261, 10282-10289], Fe3+ also decreased the delay. The ferric-nitrilotriacetate (Fe3+-NTA) complex was found to be more efficient than "free" Fe3+ [Fe(NO3)3]; a 100 microM concentration of the 1:1 Fe3+-NTA complex eliminated the delay due to 100 microM Fe2+, whereas 400 microM Fe(NO3)3 reduced the delay from 17.5 to 2.5 min. Incubation under reduced O2 tension demonstrated a requirement for O2 during the delay. The use of antioxidants [butylated hydroxytoluene, (+)-catechin, promethazine, and uric acid] and inhibitors of the Haber-Weiss reaction (mannitol, Tris buffer, dimethyl sulfoxide, catalase, and superoxide dismutase) indicated that the initiating species has characteristics of a weak oxidizing radical capable of either hydrogen or electron abstraction from suitable target molecules. We hypothesize that the delay that is sensitive to the Fe2+:microsome ratio is due to reductive elimination of the initiating species by "free" Fe2+. The nature of the initiating species has yet to be determined; however, the argument is presented that the perferryl ion (Fe3+-O2-.) may possess the characteristics required for the initiator.

The pharmacokinetics of isoxicam in elderly patients with rheumatoid arthritis.

Nineteen patients, aged 60 years and over, with rheumatoid arthritis participated in a clinical trial to investigate the pharmacokinetics of isoxicam (a new non-steroidal anti-inflammatory drug) in this age group. The purpose of the study was to determine if the pharmacokinetics are different compared to a younger healthy population. The half-lives were independent of dosage, indicating linearity of pharmacokinetics. Furthermore, the half-lives after repeated dosing were not different from those found after single doses of 400 mg. This shows that there is neither undue accumulation of the drug nor induction of its own metabolism. These results are similar to the results obtained in other centres when isoxicam was administered to healthy subjects between 18 and 32 years.

Lowering of plasma isoxicam concentrations with acetylsalicylic acid.

The pharmacokinetics of isoxicam 200 mg administered orally in 10 healthy male volunteers was studied before and during administration of acetylsalicylic acid 3.9 g daily by mouth starting 5 days after isoxicam. There was a statistically significant decrease in plasma isoxicam concentrations, but no significant change in time to reach maximum plasma concentration or disappearance time. The mechanisms of this interaction is probably competitive displacement of isoxicam from albumin by acetylsalicylic acid or salicylate. These results are consistent with the known effect of ASA in producing competitive displacement of other protein bound antiinflammatory drugs.

Lipid peroxidation stimulated by iron nitrilotriacetate in rat liver.

The complex of ferric iron with nitrilotriacetate (iron-NTA) given i.p. is an unusually potent stimulus for lipid peroxidation (LP) in vivo, as monitored by exhaled alkanes. Localization of 59Fe-labeled NTA radioactivity in mouse liver and accumulation of thiobarbituric acid (TBA)-reacting material in liver after i.p. injection suggested that the effect of i.p. iron-NTA could be primarily hepatic. It was found that 100 microM iron-NTA added to a hepatocyte suspension gassed with air stimulated ethane production (3 +/- 1 pmoles/10(6) cells/min) versus an undetectable control, and at a sensitivity of 0.083 pmole/10(6) cells/min. Under similar conditions, hepatocytes stimulated by iron-NTA generated low level chemiluminescence (CL) in parallel with formation of TBA-reactants; the generation of CL was concentration related. Liver was homogenized and fractionated by ultracentrifugation: iron-NTA stimulated CL in whole liver homogenate as in intact cells. The greater part of this activity localized to the microsomal and mitochondrial fractions where NADH or NADPH was required. Using rat liver microsomes, it was shown that iron-NTA in the presence of NADPH stimulated two phases of CL with an initial phase maximum in 1-2 min (phase 1) which decreased abruptly to be followed by a prolonged rise (phase 2); NADH could replace NADPH. Ferrous iron (as chloride) caused a burst of CL, whereas ferric iron was inactive. However, complex differences exist between CL stimulated by Fe(II) and by iron-NTA in the presence of reducing equivalents. Under conditions resulting in the production of CL, a microsomal system with iron-NTA and reducing equivalent accumulated TBA-reactants in parallel with the stimulated CL and rapid increase in oxygen consumption. Both desferrioxamine and butylated hydroxyanisole were able to strongly inhibit the CL stimulated by iron-NTA. When iron-NTA and iron-ADP were compared in the microsomal system, similar responses were obtained but major differences characterized the effects of these iron chelates on whole cells with the ADP complex being relatively inactive. We conclude that iron-NTA stimulated free radical reactions in liver by undergoing cyclic oxidation and reduction and that these reactions utilized oxygen, generated CL, and formed TBA-reactants and ethane. At a subcellular level, the reactions of iron-NTA resembled those reported for iron-ADP.

Porphyria cutanea tarda, or the uroporphyrinogen decarboxylase deficiency diseases.

The term "porphyria cutanea tarda" originally described the dermatological manifestations of various chronic porphyrias. Its usage now is usually restricted to disorders associated with a deficiency of uroporphyrinogen decarboxylase (UROD), for which the term "UROD-deficiency" may be more appropriate. Four etiologic agents have been implicated in this condition: alcohol, oral estrogens, halogenated aromatic hydrocarbons, and iron. An inherited deficiency of UROD is also recognized, with increased susceptibility to these agents. Certain halogenated aromatic hydrocarbons can cause UROD-deficiency in animals and synergism with iron is demonstrable in this model. Neither ethanol nor estrogen has been shown to cause UROD-deficiency in animals. Treatment by venesection to reduce total body iron is safe and effective. The 4-aminoquinoline antimalarial drugs also provide effective treatment, possibly by lysis of affected liver cells. Unlike venesection, they may not reverse the biochemical lesion which causes porphyrins to accumulate. The mechanism of acquired UROD-deficiency is not clear but animal studies suggest a role for the hepatic mixed function oxygenases which initiate iron-dependent inactivation of UROD. Diagnosis is simple, often requiring only appropriate clinical data and testing of a random urine sample. Although not common, the disorder is the most frequently diagnosed disturbance of porphyrin metabolism in many countries, and further insight into its unusual pathogenesis may clarify the hepatotoxic effects of the 4 etiologic agents.

The role of contaminants in hexachlorobenzene toxicity.

Hexachlorobenzene (HCB) is hepatotoxic, causing a porphyria with an acquired deficiency of uroporphyrinogen decarboxylase. Conflicts have arisen regarding the toxicological significance of impurities in samples of HCB. In female rats, unpurified practical-grade HCB from Eastman Kodak Company was more porphyrogenic than a sample from Aldrich Chemical Company that had been recrystallized from benzene. To determine whether specific inducers of hepatic microsomal cytochrome P1-450, such as 2,3,7,8-tetrachlorodibenzo-para-dioxin (TCDD), contributed to the difference in toxicity of these samples, induction of porphyria and activities of the cytochrome P1-450-dependent mono-oxygenase, ethoxyresorufin-O-deethylase (ERR), were compared in rats receiving HCB (0.2% w/w) or TCDD (10 micrograms/kg initial dose, then 2.5 micrograms/kg weekly). Porphyria occurred in the former but ERR activity was induced about four times more in the latter. The relative potency of crude and purified HCB samples in inducing ERR activity was further evaluated in C57Bl/6 and DBA/2 mice; ERR activity was three times higher in C57Bl/6 than in DBA/2 mice treated with 0.01% w/w HCB. ERR activity was also higher in C57Bl/6 mice treated with impure than in those treated with purified HCB. These results show that undefined impurities can be important determinants of the biological effects of HCB samples and suggest that these effects are partly mediated via the Ah receptor. However, porphyria induced by HCB appears to be independent of HCB impurities and not solely dependent on induction of the cytochrome-P1-450 mono-oxygenases.

Interaction of human lactoferrin with the rat liver.

Binding of human lactoferrin (hLf) by purified rat liver plasma membranes was studied to clarify whether the liver possesses specific hLf receptors. The binding was rapid between 4 degrees and 37 degrees C, with a pH optimum close to 5.0. At 22 degrees C and in glycine-NaOH (5 mM, pH 7.4) containing 150 mM NaCl and 0.5% albumin, 1 microgram of membrane bound a maximum of 11.8 ng hLf. The dissociation constant of the interaction was 1.6 X 10(-7) M. Other proteins of high isoelectric points (lactoperoxidase, lysozyme, and particularly salmine sulfate) and a piperazine derivative inhibited hLf binding in a concentration-dependent manner. In contrast, monosaccharides (galactose, N-acetylgalactosamine, mannose, and fucose) were ineffective. By omitting NaCl from the incubation buffer, binding was increased 3.6-fold. Erythrocyte ghosts bound hLf less firmly and alveolar macrophages more firmly than hepatic plasma membranes. Liver cell fractionations performed after the intravenous injection of labeled hLf showed that approximately 88% of the hepatic radioligand was associated with parenchymal cells. When binding was expressed per unit of cell volume, however, more hLf was present in nonparenchymal than in parenchymal cells, implying that the above value was determined by the relative cell masses rather than affinities alone. It is concluded that the binding of hLf by hepatic plasma membranes is electrostatic, i.e., is mediated by the cationic nature of the ligand, and that it is explicable in terms of a "specific nonreceptor interaction" of the generalized type proposed by Cuatrecasas and Hollenberg (Adv. Protein Chem. 30: 251-451, 1976).

A simple bioassay for monocyte-derived hepatocyte stimulating factor: increased synthesis of alpha 2-macroglobulin and reduced synthesis of albumin by cultured rat hepatocytes.

Cytokines released from monocytes upon stimulation by lipopolysaccharide cause a number of cells to undergo proliferative and synthetic changes. At least one of these cytokines affects hepatocytes in vivo causing increased synthesis of a series of acute-phase proteins. We have established an in vitro micro-assay for hepatocyte stimulating factor (HSF) using primary cultures of normal rat hepatocytes. Measurement of increased synthesis of alpha 2-macroglobulin and decreased synthesis of albumin caused by exogenously added factor constitute a sensitive parameter for quantifying HSF. For comparing various cytokines preparations, we have defined a unit of HSF activity in terms of a stimulation index. We have used this assay to follow some preliminary attempts to isolate the factors responsible for stimulation of synthesis of acute-phase reactant by the liver.

The acute-phase response of cultured rat hepatocytes. System characterization and the effect of human cytokines.

Hepatocytes were isolated from adult livers and cultured for periods of up to 5 days as monolayers at an initial density of 10(6) cells/10cm2 in Williams E medium containing insulin, dexamethasone and 5% foetal-calf serum. The daily production of 11 plasma proteins was measured by electroimmunoassay and compared with the concentrations of the same proteins in the plasma of normal rats and of those with experimental inflammation. Hepatocytes from normal rats synthesized proteins in relative amounts which were similar to the relative proportions of the same proteins in the plasma of turpentine-injected animals. The pattern changed only slowly during 5 days in culture, but it did so profoundly either when the medium was devoid of dexamethasone or when human cytokines (from endotoxin-stimulated monocytes or unstimulated human squamous-carcinoma cell line COLO-16) were added. The cytokines consistently increased the synthesis of alpha 2-macroglobulin and fibrinogen and depressed that of albumin; variable increases in the synthesis of alpha 1-acute-phase globulin, alpha 1-acid glycoprotein, haptoglobin and alpha 1-proteinase inhibitor, and variable decreases in transferrin synthesis, were seen, whereas the synthesis of antithrombin III, alpha 1-macroglobulin and prothrombin remained virtually unaffected. The cytokine effects on protein synthesis required the presence of dexamethasone. The hepatocyte-stimulating activity derived from monocytes chromatographed on Sephadex G-100 corresponding to 30 000 Da, as opposed to the lymphocyte-activating factor, which was eluted as a molecule of approx. 15 000 Da. This suggests that both activities probably reside with distinct molecular species in the preparations of human cytokines.

Release of ferrous iron from ferritin by liver microsomes: a possible role in the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Nonheme iron is synergistic with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in producing hepatotoxicity in mice. Fe2+ rather than Fe3+ is the probable toxin and we speculated that TCDD, an inducer of microsomal electron transport, might favour reduction of iron. We have defined a system which will release Fe2+ from ferritin (Fe3+) under anaerobic conditions and in the presence of added flavin mononucleotide (FMN). The rate of reduction iron was proportional (a) to microsomal protein from 0.5 to greater than 3 mg/mL, (b) to the activity of NADPH-cytochrome c reductase over 0.1 U/mL, (c) to ferritin at concentrations exceeding iron concentrations greater than 200 mumol/L, and (d) to the concentration of FMN when it was less than 125 mumol/L. The system was approximately twice as active with NADPH as with NADH as electron donor. The linear phase of iron release did not commence immediately, but followed a delay (+/- 0.5 min) after adding FMN to an anaerobic mixture containing microsomes, ferritin, an NADPH-generating system, and an oxygen-scavenging system. When microsomes from untreated, phenobarbital-treated (3 days), or TCDD-treated (1 or 3 weeks) rats were compared, iron release correlated most closely with the cytochrome P-450 concentration. However, when the microsomal proteins were solubilized and the NADPH-cytochrome c reductase and cytochrome P-450 activities were separated, reduction of ferritin iron was shown to be a function only of the reductase fraction, except that the delay in initiating release of Fe2+ was increased with purified reductase and decreased when a monooxygenase system was reconstituted with cytochrome (phenobarbital or TCDD induced) and lipid.(ABSTRACT TRUNCATED AT 250 WORDS)

Sensitivity to suppression of cytotoxic T cell generation by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is dependent on the Ah genotype of the murine host.

Susceptibility of mice to a variety of toxic effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) is genetically determined by the Ah locus. To determine if immunotoxicity following TCDD exposure was also regulated by the Ah locus, we tested the ability of low dose TCDD (4 ng/kg) to suppress the generation of allospecific cytotoxic T cells (CTL) by lymphocytes from "susceptible" C57B1/6, and "resistant" DBA/2, and from C57B1/6XDBA/2J F1 hybrid mice in vitro. To determine if TCDD acted directly on the "susceptible" lymphoid cells, the immune response of C57B1/6 leads to DBA/2 and DBA leads to C57B1/6 bone marrow chimeras was also measured. C57B1/6 and F1 mice proved susceptible to suppression consistent with the dominant effect of Ah. Susceptibility to suppression in chimeric mice, however, was determined by the Ah genotype of the host and not by the genotype of the grafted lymphomyeloid cells. Mixing experiments demonstrated that suppression of CTL generation by TCDD was due to suppressor T cells. The frequency of CTL precursors was not affected by TCDD. These results are consistent with the idea that TCDD acts by an Ah locus-dependent mechanism to indirectly promote development of suppressor T cells that block the generation of CTL from their precursors.

The role of proteolytic enzymes derived from crude bacterial collagenase in the liberation of hepatocytes from rat liver. Identification of two cell-liberating mechanisms.

Crude bacterial collagenase was chromatographed on DEAE-cellulose to yield three peaks with proteolytic activity: an arginine esterase (DEAE-1), gelatinase (DEAE-2) and a caseinolytic activity (DEAE-3). The arginine esterase and gelatinase activity fractions were slightly contaminated with each other but neither possessed caseinolytic activity; the caseinolytic fraction was devoid of arginine esterase and gelatinase activities. In addition, crude collagenase was fractionated by ZnII-affinity chromatography to produce a gelatinase peak (ZnII peak 1), which was free from arginine esterase and caseinolytic activities. The four fractions were compared to crude collagenase in their ability to liberate rat hepatocytes by using either liver slices or a standard perfusion technique. Compared to crude collagenase (0.05-0.1% w/v), which produced 70-80% liver digestion with approximately 80% cell viability, digestion with equivalent quantities of the isolated enzymic activities was relatively poor. Gelatinase activity (ZnII peak 1) was wholly ineffective and DEAE-1 and DEAE-2 each possessed only slight digestive properties. Hepatocyte liberation by the caseinolytic activity, DEAE-3, was partially successful (30-40% digestion, 25-30% viability) but only a portion of liver tissue was digested regardless of the quantity of DEAE-3 used. However, by mixing certain fractions before perfusion two gelatinase-dependent, cell-releasing mechanisms were identified: (a) DEAE-3 with ZnII peak 1 and (b) DEAE-1 mixed with either DEAE-2 or ZnII peak 1. Each system compared creditably with the digestive properties of an equivalent activity of crude collagenase. At present we are attempting to determine any differences between hepatocytes produced by the two enzymic mechanisms.

Variability in the human drug response.

Variations in response to drugs may be pharmacodynamic, implying inter-individual differences in the response of receptors in equal concentrations of drug, or pharmacokinetic, implying that individuals receiving the same dose of drug will have different concentrations of drug in their body fluids. Either type of variation can be inherited or acquired. Variations in receptor sensitivity do occur but few instances, inherited or acquired, have well documented clinical relevance. If the dose response relationship for the drug in question is not steep, or if the therapeutic index is low, drug concentration in the region of the receptor will not be critical and causes of kinetic variation are unlikely to be clinically significant. However, it is the many causes of kinetic variation which are best described. These include effects due to drug formulation and changes in the absorption, distribution, metabolism and excretion of drugs. If a consideration of dynamics suggests that drug concentration will determine therapeutic efficacy, analysis and prediction of variability due to these factors is desirable. Prediction requires an accurate description of the system but commonly used pharmacokinetic models may fail when prediction is a goal. The variables, volume of distribution (Vd) and rate constant of elimination (Ke) are hybrid in that they arise from the interaction of patient and drug characteristics. Important events including macromolecular binding and altered blood flow may not be represented. More data is required to determine the clinical significance of pharmacodynamic variation but better analytical tools are required to deal with kinetic variation when this is important. Specifically, pharmacokinetic models should represent physiological variables and levels of unbound drug in body fluids should receive greater emphasis.