Thromboxane B2 uptake and metabolism in isolated perfused lung. Identification and comparison with prostaglandin F2 alpha.

Thromboxane B2 was metabolised in isolated perfused guinea-pig lungs to a product identified by negative ion chemical ionisation mass spectrometry as 13,14-dihydro-15-ketothromboxane B2. Conversion was measured by radio TLC and was greater in guinea-pig than rat lungs (29.1 vs. 13.8% at 10 ng/ml), but similar in lungs from normal and sensitized guinea-pigs. Thromboxane B2 metabolism was less than that of prostaglandin F2 alpha but, like it, was prevented at 5 degrees C and reduced by cycloheximide pretreatment. Tissue to medium ratio in perfused guinea-pig lungs was 3.4 for thromboxane B2, but 0.2 for insulin (showing that thromboxane B2 is accumulated within the lung) and was altered after experimental manipulations. Neither lung slices, crude homogenates, cytosolic and microsomal fractions nor purified prostaglandin 15-hydroxydehydrogenase metabolised thromboxane B2 in vitro, although prostaglandin F2 alpha was extensively inactivated. Quantitative partition coefficient and albumin-binding data confirm that thromboxane B2 lacks prominent lipophilicity, implying that cellular uptake in lung must be carrier-mediated. We conclude that thromboxane B2 is a substrate for pulmonary degradation which may form a route for the biological inactivation of thromboxane A2. Its resistance to prostaglandin 15-hydroxydehydrogenase as conventionally tested remains paradoxical and is discussed.

Evaluation of 99mTc-RP128 as a potential inflammation imaging agent: human dosimetry and first clinical results.

UNLABELLED: 99mTc-RP128 is a bifunctional peptide chelate designed to target the tuftsin receptor, expressed by neutrophils, monocytes, and macrophages. Studies in animal models of both infectious and noninfectious inflammation have shown a positive correlation between accumulation of 99mTc-RP128 and quantitative measures of inflammation. A phase 1 trial was conducted with the objective of determining the safety, biodistribution, and human dosimetry of 99mTc-RP128 in eight healthy volunteers. For evaluation of the potential of 99mTc-RP128 for imaging sites of inflammation, 10 patients with active rheumatoid arthritis were studied. METHODS: Normal biodistribution was determined using the conjugate view method up to 24 h after intravenous injection of 280 MBq 99mTc-RP128. Dosimetry calculations were based on standard MIRD methodology, using the International Commission on Radiological Protection model 30 of the gastrointestinal tract and a voiding bladder model with an interval of 4.8 h. For rheumatoid arthritis patients, whole-body scans and spot views of the hands, knees, and feet were obtained at 1 and 3 h after injection of 475 MBq 99mTc-RP128. RESULTS: 99mTc-RP128 was cleared rapidly from the blood by renal excretion, and no major organs showed significant accumulation. The synovia of the major joints were visualized for all subjects. The effective dose equivalent and the effective dose were calculated to be 0.011 and 0.0094 mSv/MBq, respectively. The highest dose was to the bladder wall, which received 0.076 mGy/MBq. In all rheumatoid arthritis patients, we observed a markedly increased uptake in several affected joints. Painful and swollen joints were detected with a sensitivity of 76% and 69%, respectively. Seventy-three percent of the joints with radiographic signs of erosion were scintigraphically positive. In some patients, lines of increased activity were observed and were considered to correspond to uptake in the synovium lining tendon sheaths in the wrists and hands. CONCLUSION: This study shows that 99mTc-RP128 is safe and can successfully be used to visualize clinically affected joints in patients with long-standing rheumatoid arthritis. A proposed radioactive dose of 450-500 MBq will produce an effective dose well within the range of effective doses for commonly used radiopharmaceuticals.

Biodistribution and dosimetry of (99m)Tc-RP527, a gastrin-releasing peptide (GRP) agonist for the visualization of GRP receptor-expressing malignancies.

UNLABELLED: The aim of this study was to determine the human biodistribution and radiation dosimetry of (99m)Tc-RP527, a promising radioligand for the visualization of gastrin-releasing peptide (GRP) receptor-expressing human malignancies. METHODS: Whole-body scans were obtained up to 48 h after intravenous injection of 555 MBq (99m)Tc-RP527 in each of 6 subjects. Blood samples were taken at various times up to 48 h after injection. Urine was collected up to 48 h after injection for calculation of renal clearance and whole-body clearance. Time-activity curves were generated for the thyroid, heart, breasts in women, testes in men, and liver by fitting the organ-specific geometric mean counts, obtained from regions of interest, on the respective images as a function of the time after injection. The MIRD formulation was applied to calculate the absorbed radiation dose for various organs. RESULTS: The serial whole-body images showed rapid hepatobiliary excretion, resulting in low background and potentially high-contrast imaging of the thoracic region. Imaging of abdominal tumors may prove problematic, however, because of the extensive bowel activity. (99m)Tc-RP527 was predominantly cleared by the kidneys and to a lesser extent by the gastrointestinal tract. The mean excretion in the urine (+/-SD) at 48 h after injection was 58.3 +/- 5.4 percentage of the injected activity corrected for decay to the time of injection. The highest absorbed doses were received by the excretory organs (i.e., the urinary bladder and gallbladder wall). The average effective dose of (99m)Tc-RP527 was estimated to be 0.0095 mSv/MBq. CONCLUSION: The biodistribution of (99m)Tc-RP527 revealed low lung, myocardial, and liver uptake, which allowed early imaging of the supradiaphragmatic region with a favorable dosimetry (including effective dose) for administered activities required for SPECT imaging.

Steroid inhibition of oedema formation in the rat skin.

1. A model has been developed to compare the inhibitory effects of the topical steroid, betamethasone-17-valerate, to those of systemically administered betamethasone upon oedema responses induced by 5-hydroxytryptamine (5-HT), platelet activating factor (PAF) and zymosan-activated serum (ZAS) +/- prostaglandin E1 (PGE1), measured in the rat skin by use of 125I-labelled human serum albumin. 2. Systemic betamethasone had a selective, time- and dose-dependent inhibitory effect upon oedema treatment, with 1 mg kg-1 and a 3 h pretreatment having the greatest effect of the doses and times employed. 3. Topical betamethasone inhibited the oedema responses to all of the stimuli showing no apparent selectivity. 4. Topical betamethasone inhibits inflammatory stimuli in a different manner from systemic betamethasone. The broad spectrum of inhibition suggests that topical betamethasone acts by affecting a fundamental feature of the inflammatory response common to all of the stimuli.

Serum corticosterone, interleukin-1 and tumour necrosis factor in rat experimental endotoxaemia: comparison between Lewis and Wistar strains.

1. Circulating corticosterone, interleukin-1 (IL-1) and tumour necrosis factor-alpha (TNF alpha) activities in serum of Lewis and Wistar rats were measured following injection of lipopolysaccharide (LPS). IL-1 was measured as 'lymphocyte activation factor' (LAF) activity following precipitation of inhibitory activity with polyethylene glycol. TNF alpha activity was measured as cytotoxic activity. 2. Compared to the Wistar, the Lewis rat had higher circulating LAF and TNF activities following LPS, and release of both cytokines was prolonged in this strain. 3. Corticosterone increases in response to LPS were less in the Lewis than in the Wistar rat following the initial peak at 1 h; basal corticosterone was lower in the Lewis rat. 4. Adrenalectomized Lewis rats had even greater amounts of circulating LAF and TNF activities following LPS than did intact animals; the effect of adrenalectomy was not however mimicked by acute treatment with the steroid receptor antagonist, RU486, suggesting that endogenous corticosteroids did not acutely control cytokine release. 5. Although in vivo administration of anti-murine IL-1 alpha antiserum significantly lowered LAF activity of serum, circulating corticosterone in response to LPS was not affected. Similarly, treatment with anti-murine TNF alpha monoclonal antibody (mAb) abrogated TNF activity without affecting corticosterone, suggesting that other mediators may be responsible for corticosterone release following LPS. 6. This 'overproduction' of inflammatory cytokines together with lower circulating corticosterone may contribute to the susceptibility of the Lewis rat to diseases such as adjuvant arthritis or experimental allergic encephalomyelitis.

Glucocorticoid-and non-glucocorticoid induction of lipocortins (annexins) 1 and 2 in rat peritoneal leucocytes in vivo.

1. We have studied the occurrence, distribution and disposition of lipocortins (annexins) 1, 2 and 5 in mixed peritoneal leucocytes obtained from rats in which glucocorticoid levels were altered by adrenalectomy, administration of the glucocorticoid antagonist, RU486, or by injection of dexamethasone or hydrocortisone, as well as from rats in which the peritoneal cells were elicited by inflammatory stimuli. 2. In cells obtained from untreated rats with an intact adrenal cortex, lipocortins 1, 2 and 5 were readily detectable: the majority of each of the proteins was apparently located intracellularly with much smaller amounts in the membrane. Lipocortin 1 and to a lesser extent lipocortin 5 were also seen in a Ca(2+)-dependent association with the external plasma membrane. Following administration of RU486 (2 x 20 mg kg-1) the amounts of lipocortin 1 and 2 in cells were greatly reduced. Conversely, injection of hydrocortisone (1 mg kg-1) or dexamethasone (0.08 mg kg-1) caused an increase in the amount of lipocortin 1 and 2 in peritoneal cells within 30 min. Lipocortin 5 was unchanged by any manipulation of glucocorticoid levels. 3. Lipocortins 1 and 2 were elevated in both intracellular and membrane-associated fractions of macrophages elicited by intraperitoneal injection in inflammogens. This phenomenon also occurred in adrenalectomized animals. 4. Our data indicate that glucocorticoids control the synthesis of some members of the lipocortin family in rat mixed peritoneal cells but also suggest the existence of a separate system for controlling the generation of this protein. The significance of these observations is considered in relation to the mechanism of glucocorticoid hormone action on eicosanoid production.

Antipyretic actions of human recombinant lipocortin-1.

The effect of human recombinant lipocortin-1 (hrLC-1) on the pyrogenic actions of the synthetic polyribonucleotide polyinosinic:polycytidylic acid (poly I:C) has been studied in conscious rabbits. Poly I:C (2.5 micrograms kg-1) given i.v. produced a biphasic fever with a first peak after 90-105 min and a second peak between 225-240 min. hrLC-1 (50 micrograms kg-1) given i.v. simultaneously with the poly I:C produced a significant reduction in the febrile response but without complete suppression. The thermal response index over 5 h (TRI5) was 4.69 +/- 0.51 for poly I:C given with saline and the TRI5 for poly I:C given with hrLC-1 was 2.66 +/- 0.45 (values are for n = 5 +/- s.e. mean, P less than 0.05). hrLC-1 administered alone had no effect on body temperature and its antipyretic activity was lost on heating. In a separate series of experiments 1 h pretreatment with dexamethasone (1 mg kg-1) given i.v. reduced the pyrogenic response (TRI5) to poly I:C (2.5 micrograms kg-1) from 4.87 +/- 0.54 without dexamethasone to 2.00 +/- 0.25 (n = 5, P less than 0.05) and dexamethasone given alone had no effect on body temperature. These data demonstrate that LC-1 possesses antipyretic actions and raises the possibility that the antipyretic actions of dexamethasone are mediated through the induction of LC-1.

The local anti-inflammatory action of dexamethasone in the rat carrageenin oedema model is reversed by an antiserum to lipocortin 1.

1. A local pre-injection of 1 micrograms dexamethasone sodium phosphate strongly inhibited (> 60% inhibition at 3 h; P < 0.001 at all time points) the development of carrageenin-induced paw oedema in the rat induced by a subplantar injection of 0.1 ml, 2% carrageenin. 2. Coinjection of a polyclonal rabbit antiserum raised against human 1-188 recombinant lipocortin 1, which also recognised the rat protein, reversed the inhibitory action of dexamethasone (P < 0.05 at 4 h and 5 h). At the highest volume used (40 microliters) control antisera were without any effect. 3. These data further support the concept that lipocortin 1 is involved in the anti-inflammatory mechanism of action of the glucocorticoids.

Sex steroids affect glucocorticoid response to chronic inflammation and to interleukin-1.

The influence of gender and sex hormones upon both the hypothalamic-pituitary-adrenal (HPA) axis and the immune and inflammatory responses is well recognized, but it is not clear to what extent the two effects are interdependent. We have investigated this interaction using a chronic inflammation model. Corticosterone levels were measured in mature BALB/c male and female mice, which were intact, sham-operated or gonadectomized. No significant differences were found between groups in baseline corticosterone, but systemic inflammation (cotton-induced granulomas) resulted in stimulation of the HPA axis in a reproducible pattern. Corticosterone levels were higher in sham-operated females than in males, but gonadectomy had opposing effects in the two genders, resulting in reduced levels in females but significantly increased levels in males. A similar pattern emerged after stimulation by ether exposure or injection of interleukin-1 beta. In the chronic inflammatory model, replacement of ovariectomized females with physiological levels of progesterone restored a response similar to that of intact females. Physiological levels of 5 alpha-dihydrotestosterone prevented the increase in corticosterone levels caused by castration in males and also resulted in reduced corticosterone levels in sham-operated females. Oestradiol treatment did not affect corticosterone levels. Release of interleukin-1 by peritoneal macrophages from intact and gonadectomized mice with chronic inflammation followed a similar pattern, females releasing more than males. These data suggest a complex inter-relationship between sex steroids, inflammatory stimuli and the HPA axis, such that females have a greater tendency than males to generate activating signals and in addition have a greater sensitivity to such factors.

A novel binding assay for phospholipase A2.

We have devised a rapid and simple assay for estimating the binding of pancreatic phospholipase A2 to a bilayer lipid membrane. The binding was observed to be extremely rapid at 37 degrees and was absolutely dependent upon Ca2+. Amongst several drugs known to inhibit the catalytic activity of phospholipase only mepacrine at high concentrations (500 microM) and chlorpromazine (100 microM) were active. Treatment of the enzyme with p-bromophenacylbromide did not inhibit binding. Several alcohols potentiated binding whereas detergents tended to inhibit. Amongst several purified proteins tested, only the steroid-induced anti-phospholipase protein lipocortin prevented binding. The use of this assay in screening for antiphospholipase agents is discussed.

Inhibition of prostaglandin 15-hydroxydehydrogenase by sulphasalazine and a novel series of potent analogues.

The ability of sulphasalazine, its colonic metabolites and various analogues to inhibit prostaglandin inactivation by two purified preparations of type INAD+-dependent prostaglandin 15-hydroxydehydrogenase or in various 100,000 g cytosolic supernatants was investigated using PGF2 alpha as substrate and radio-TLC. Bovine lung and human placental PGDH were inhibited in a dose-dependent and apparently non-competitive manner by sulphasalazine and most of the 26 salazine/sulphasalazine analogues tested, but the potencies of the analogues varied considerably. In a survey of structure-activity effects testing 30 drugs at a fixed dose (50 microM) in six test systems, it was established that only two aromatic rings are needed and that optimal PGDH inhibition requires -CH2COOH and -OH at positions 1 and 2 in the salicyl C ring system. Homosalazine was thus established as the type compound of a novel series of powerful PGDH inhibitors. Electronegative substituents meta or para in ring B produce compounds with greater than 150 X inhibitory potency of sulphasalazine, and a significant linear correlation (r = 0.82, P less than 0.002) was found between the inhibitory activity and the Hammett sigma substituent constant in this series of ten homosalazine analogues.

The detection and quantitation of inflammation in the central nervous system during experimental allergic encephalomyelitis using the radiopharmaceutical 99mTc-RP128.

RP128 is a novel agent which readily chelates 99mTc to form a radiopharmaceutical which binds in vivo to the tuftsin receptor located specifically on neutrophils and monocyte-macrophages, therefore removing the need for in vitro cell labelling prior to intravenous administration. We have assessed the ability of 99mTc-RP128 to detect central nervous system (CNS) inflammation in experimental allergic encephalomyelitis (EAE), an animal model of the human disease multiple sclerosis. The radiopharmaceutical was recorded at significantly increased levels in all EAE diseased CNS tissues, compared to normal and control samples, at 0.5, 1 and 3 h post-injection using a dual radioisotope technique to correct for non-extravasated tracer (P<0.05). Moreover, extravascular accumulation of the agent could be clearly demonstrated in inflammatory tissues with minimal loss of sensitivity when the secondary isotopic correction for blood volume was omitted. In addition, 99mTc-RP128 successfully monitored glucocorticoid suppression of inflammation (P<0.05), recording a typical dose-response to increasing steroid concentration. Clearly, 99mTc-RP128 can quantitatively detect CNS inflammation and assess responses to therapy indicating potential value as an imaging agent both clinically and as a research aid. Furthermore, the rapid in vivo labelling by 99mTc-RP128 of specific inflammatory cells combined with the ability to monitor the progress of anti-inflammatory therapeutics may recommend the agent for use in a variety of inflammatory conditions.

Dexamethasone inhibits platelet activating factor-induced inflammation in the paw but not the pleural cavity of rats.

The effect of drugs upon the inflammatory responses occurring after injection of 1 microgram platelet activating factor into the foot-pad and pleural cavity of the rat were investigated. Dexamethasone, but not indomethacin, BW755C, mepyramine or methysergide inhibited development of paw oedema; none of these drugs were effective in the pleurisy model. The triazolobenzodiazepine derivative WEB 2086 inhibited responses in both models. We conclude that the development of oedema in response to platelet activating factor in the rat is not dependent upon eicosanoid synthesis; and possible reasons for the difference in steroid sensitivity between the two sites are discussed.

Inhibition by ethanol and mepacrine of phospholipase-dependent prostaglandin release from the isolated perfused rat lung.

To investigate the possibility that millimolar concentrations of ethanol have a membrane-directed inhibitory effect on phospholipase A2 and prostanoid generation (suggested from previous platelet experiments), we studied the release of prostacyclin, thromboxane A2 and prostaglandin E2 from isolated perfused rat lung. Prostanoid release was evoked by arachidonic acid, bradykinin and ionophore A23187 and was measured after extraction by radioimmunoassay. In these experiments, prostanoid release is dependent upon biosynthesis from fatty acid precursors as there is no endogenous prostanoid storage pool. Arachidonic acid and bradykinin caused enhanced release of more prostacyclin than thromboxane A2 with much less prostaglandin E2 and no detectable prostaglandin F2 alpha, whereas A23187 released equal proportions of prostacyclin and thromboxane A2 with less prostaglandin E2. Ethanol at 50 mM resembled mepacrine (46 microM) in that prostanoid release in response to bradykinin and A23187 was highly significantly reduced with little effect on release induced by arachidonic acid. We suggest that ethanol, like mepacrine, interferes with prostaglandin generation by an action at the phospholipase step. This may be secondary to a physical effect on membrane configuration.

Site of anti-inflammatory action of dexamethasone in rabbit skin.

Systemic pretreatment of rabbits with dexamethasone results in a time-dependent inhibition of oedema responses caused by intradermal injection of both 'direct-acting' and 'neutrophil-dependent' stimuli. Local injection of actinomycin D, an inhibitor of RNA synthesis, inhibits this effect. Our studies suggest that a major action of dexamethasone in this model may be a local inhibition of increased permeability of the vascular endothelium.

A study of phospholipase A2-induced oedema in rat paw.

The inflammaogenic action of four extracellular phospholipases A2 was tested in the rat paw oedema model. Subplantar injection of microgram amounts of the venom phospholipases A2 from Vipera russeli, Naja mocambique mocambique and honey bee, or the porcine enzyme produced a rapid but transient oedematous response. The venom enzyme from Vipera was the most potent in this respect, the pancreatic enzyme the least. Pretreatment of the enzymes with para-bromophenacylbromide profoundly inhibited the ability of the enzymes to produce oedema. The inflammatory response produced by the phospholipases was insensitive to indomethacin, BW755C or the LTD4 antagonist L649,923 but was inhibited by the local administration of methysergide or, by pretreatment of the rats with dexamethasone. The PAF-antagonist BN 52021, but not WEB2086, was an effective inhibitor. Degranulation of mast cells seem the most likely explanation for the inflammatory action of these enzymes in the rat paw.

Is there a role for agonist gastrin-releasing peptide receptor radioligands in tumour imaging?

Gastrin-releasing peptide (GRP) has been shown to be a tumour growth stimulating agent for a number of normal and human cancer cell lines. The tumour growth effect is a direct result of GRP binding to membrane G-protein coupled GRP receptors (GRP-R) on the cell surface. Available data on the role of GRP and GRP-R in human lung, prostate, breast, colorectal and gastric carcinoma are reviewed and it is suggested that radiolabelled agonists are preferable to antagonists for imaging and therapy as they appear to be internalised, yielding a higher target/background ratio. The use of rhenium or indium radiolabels for therapy may provide a new approach to GRP/bombesin expressing tumours.

Development of specific antibody and in vivo response to antigen in different rat strains: effect of dexamethasone and importance of endogenous corticosteroids.

Endogenous glucocorticoids undoubtedly play a role in the control of immune responses: their contribution to inter-strain variation is unknown. The development of specific IgG and IgE was measured following inoculation with ovalbumin in Lewis, Fischer, Wistar and Brown Norway rats. The Lewis gives a smaller IgG and IgE response than the other strains and the response in vivo to antigen injected into the paw correlates with the titre of specific antibody. Treatment with the steroid receptor antagonist RU486 (mifepristone) following inoculation reveals that in the Lewis, and to a lesser extent in the Brown Norway, the development of a specific IgG response is limited by endogenous corticosteroids. The IgG response in different strains is differently sensitive to treatment with the synthetic glucocorticoid dexamethasone, the Lewis being particularly resistant. The importance of control by endogenous corticosteroids should not be overlooked in contributing to strain differences in immune response.

Effects of endothelin-1 on the rat isolated heart.

This study shows that bolus injections of endothelin-1 (ET-1) (1-30 pmol) produce transient vasodilator and prolonged coronary vasoconstrictor actions. The initial effect on cardiac contractility was a positive inotropic action, but with repeated doses a negative inotropic action developed. Verapamil (0.1 microM) antagonized the vasoconstrictor action of Bay K 8644 but did not affect ET-1-induced vasoconstriction. In contrast, removal of extracellular calcium did block the vasoconstrictor action of ET-1. This suggests that vasoconstriction is due to activation of receptor-rather than potential-operated calcium channels. The ET-1-induced vasoconstriction was not due to the release of platelet-activating factor (PAF) or thromboxane A2 since it was not inhibited by WEB 2086 (0.5 microM), fluribiprofen (2 microM), or BW755C (7 microM). In addition, thromboxane B2 could not be detected in the effluent from the heart. The vasoconstrictor action of ET-1 was potentiated by passage of air through the coronary vascular bed, suggesting that an intact endothelium normally opposes this vasoconstrictor effect.