Immunostimulation by a partially modified retro-inverso-tuftsin analogue containing Thr1 psi[NHCO](R,S)Lys2 modification.

The tuftsin retro-inverso analogue H-Thr psi[NHCO](R,S)Lys-Pro-Arg-OH was synthesized through a novel procedure for the high-yield incorporation of isolated retro-inverso bonds into peptide chains and the use of the new Meldrum's acid derivative (CH3)2C(OCO)2CH(CH2)4NHCOCF3 followed by its efficient coupling in solution to trimethylsilylated H-D-Thr(t-Bu)NH2. Closely related peptide impurities were eliminated both from the crude final peptide and the fully protected tetrapeptide amide precursor via ion-exchange and reversed-phase displacement chromatography, respectively. The tuftsin retro-inverso analogue proved to be completely resistant to enzymatic degradation in vitro, either against isolated aminopeptidases or human plasma proteolytic enzymes. When administered either orally or intravenously, it was significantly more active than normal tuftsin in increasing the number of specific antibody secreting cells in spleen of mice immunized with sheep erythrocytes. Furthermore, the analogue exerted an enhanced stimulatory effect on the cytotoxic activity of splenocytes against YAC-1 tumor cells. Finally, retro-inverso-tuftsin was about 10-fold more potent than the native peptide in reducing rat adjuvant arthritis. The resistance of the retro-inverso analogue to peptidases might explain the increased in vivo activities and allows its further immunopharmacological characterization.

The effect of adrenalectomy on interleukin-1 release in vitro and in vivo.

1 Peritoneal macrophages (M phi) collected from adrenalectomized (ADX) rats released more interleukin-1 (IL-1) activity and prostaglandin E2 (PGE2) than macrophages from sham-operated (SHO) rats. 2 The increase in IL-1 activity in the supernatants was confirmed by the increase of the cell-associated 33 kD IL-1 alpha precursor in ADX macrophages stimulated by lipopolysaccharide (LPS). 3 After the injection of Complete Freund's Adjuvant (CFA) to induce adjuvant arthritis, 60% of the ADX rats died, while no deaths occurred in the SHO group. 4 The in vivo administration of dexamethasone inhibited both IL-1 and PGE2 release by macrophages as well as protecting ADX animals from CFA-induced death. Indomethacin and BW 755C partially protected the animals from this lethal effect. 5 These results suggest that adrenalectomy induces an increased release of IL-1 both in vitro and in vivo, and are consistent with a feedback mechanism between IL-1 and glucocorticoid hormones.

Increase of extracellular brain calcium involved in interleukin-1 beta-induced pyresis in the rabbit: antagonism by dexamethasone.

1. This study investigates the role of extracellular brain calcium in the hyperthermia induced by interleukin-1 beta (IL-1 beta). 2. Intracerebroventricular (i.c.v.) injection of IL-1 beta (12.5 ng kg-1) in rabbits caused a prompt and sustained rise in cerebrospinal fluid (CSF) Ca2+ concentration ([Ca2+]) followed by enhanced prostaglandin E2 (PGE2) release and hyperthermia. 3. A linear and significant correlation was observed between the increase in [Ca2+] induced by IL-1 beta and the rise in body temperature. 4. Ventriculo-cisternal perfusion with artificial CSF containing the calcium chelator EGTA (1.3 mM) blocked the IL-1-induced PGE2 release and countered the febrile response. 5. I.c.v. administration of dexamethasone (Dex) (2.4 and 24 micrograms kg-1) 100 min prior to IL-1 beta, dose-dependently antagonized the cytokine-induced Ca2+ increase, the PGE2 release and the febrile response. 6. These results suggest that changes in extracellular brain calcium are involved in the regulation of body temperature. In this light, the antipyretic action of Dex may be related to its effect on Ca2+ uptake.

A novel anti-inflammatory peptide from human lipocortin 5.

1. A novel anti-inflammatory peptide (residues 204-212) of human recombinant lipocortin 5 (hrLC5) found on the high similarity region with uteroglobin is described. 2. Peptide 204-212 dose-dependently inhibited the contractions of rat isolated stomach strips elicited by porcine pancreatic phospholipase A2 (PLA2). Contractions caused by arachidonic acid (AA), prostaglandin E2 (PGE2) and 5-hydroxytryptamine were not affected. No direct enzyme inhibition was observed in a radiochemical assay. 3. PGE2 release by both human fibroblasts and rat macrophages was reduced by peptide 204-212 in a dose-dependent manner. 4. The development of carrageenin-induced oedema in rats was significantly inhibited by the local administration of peptide 204-212. 5. The pattern and potency of the biological effects of peptide 204-212 are similar to those of antiflammin 2, a lipocortin 1-derived peptide. 6. It is suggested that peptide 204-212 may represent the active site responsible for the anti-inflammatory properties of lipocortin 5.

Inhibition of interleukin-1 beta-induced pyresis in the rabbit by peptide 204-212 of lipocortin 5.

The intracerebroventricular administration of interleukin-1 beta (12.5 ng/kg) in rabbits caused a prompt rise of prostaglandin E2 concentration (+ 632.6 +/- 243.9%) in the cerebrospinal fluid followed by hyperthermia (+ 1.61 +/- 0.14 delta degrees C). The intracerebroventricular administration of an anti-inflammatory nonapeptide (amino acids 204-212, SHLRKVFDK) derived from lipocortin 5, thereafter referred to as lipocortin 5-(204-212)-peptide, inhibited in a significant manner both the increase in cerebrospinal fluid [prostaglandin E2] and the febrile response induced by the cytokine. This inhibitory effect is probably due to interference by the peptide with phospholipase A2 activity. A control peptide (FKRVHDLKS) formed by the same amino acids in a randomly shuffled sequence had no effect. These results show that, in addition to the anti-inflammatory effect previously reported, the peptide 204-212 of lipocortin 5 possesses, like glucocorticoids, anti-pyretic activity. The research on lipocortin-derived peptides may lead to the development of novel anti-inflammatory and anti-pyretic compounds.

Pharmacokinetics of flunitrazepam in rats studied by a radioreceptor assay.

In rat the kinetics of flunitrazepam (FNZ) was evaluated by a radioreceptor assay (RRA) after i.v. administration of 1 mg/kg and after oral administration of 1 and 3 mg/kg. The i.v. kinetics is biexponential and the g.i. absorption is very rapid (with a plasma peak at 0.25 hour) with a good bioavailability (69%); the apparent distribution volume is high, 4.8 L/kg; the half-life is equal to 3.5 hours; the elimination constant is equal to 0.8 h-1; the urinary excretion of FNZ-equivalent is negligible; the plasma total clearance is equal to 3.9 (L/kg)h-1. The concentrations of FNZ-equivalents after oral administration of 1 mg/kg show a peak at the 2-nd hour with a very high concentration in the following organs (in decreasing order): brain, kidneys, heart, liver; after 8 hours no FNZ-equivalents are present in these organs except in the brain, which shows detectable concentrations at the 32-nd hour. The peak concentrations of FNZ-equivalent in brain, kidneys and heart are higher than the corresponding peak concentration in plasma.

Inhibition of smooth muscle contraction and platelet aggregation by peptide 204-212 of lipocortin 5: an attempt to define some structure requirements.

Peptide 204-212 of lipocortin (LC) 5 inhibited porcine pancreatic phospholipase A(2) (PLA(2)) induced rat stomach strip contractions and ADP induced rabbit platelet aggregation in a concentration dependent manner (IC(30) of 10 muM and 400 muM, respectively). The first two amino acids are not necessary since the eptapeptide 206-212 was equipotent in both assays (IC(30) of 12.5 muM and 420 muM). Of the two pentapeptides 204-208 and 208-212 only the latter showed inhibitory activity in both models although the potency was much reduced (IC(30) of 170 muM and 630 muM) compared with that of the parent nonapeptide. Comparison of peptide 204-212 effects with those of its analogues on LC1 and LC2 indicate that lysine 208 and aspartic acid 211 are essential in order to maintain a fully active nonapeptide.

The release of interleukin-1-like activity by macrophages in two models of immunological inflammation in the rat.

Interleukin-1 (IL-1) is a putative mediator of inflammation released by activated macrophages in vitro. The IL-1 release by rat macrophages collected either from exudates in pertussis-induced air pouches or from the peritoneum during adjuvant arthritis has been investigated. In air pouch inflammation LPS-stimulated macrophages collected from sensitized animals release more IL-1 than cells from control rats at day 6 after challenge. This enhanced IL-1 release parallels the extent of mononuclear cell migration in the inflammatory lesion. In adjuvant arthritis LPS-stimulated macrophages collected from sensitized animals release more IL-1 than cells from control rats at days 16 and 23 after adjuvant injection. The secondary inflammation in arthritic rats was statistically significant at days 16 to 28. These results indicate that during immunological inflammation macrophages either from the inflamed area or from a non-inflamed region release more IL-1 than control cells. This release parallels the extent of the inflammation and may be important in its pathogenesis.

Persistent effects of interleukin-1 on smooth muscle preparations from adrenalectomized rats: implications for increased phospholipase-A2 activity via stimulation of 5-lipoxygenase.

Human recombinant interleukin-1 beta (hrIL-1 beta) potentiated CaCl2-induced contractions of isolated stomach strip preparations from adrenalectomized rats (ADXSS). Associated with this effect were increased prostaglandin E2 and peptidyl leukotriene (LT) synthesis. Unlike preparations from normal rat stomach strips or sham-operated animals, tissue responses and eicosanoid production of ADXSS failed to return to pre-hrIL-1 beta control levels after washout of the interleukin, these effects being abrogated by the lipoxygenase inhibitor nordihydroguaiaretic acid. Additionally, the LTD4 antagonist FPL55712 also prevented the increase in contraction and prostaglandin E2 synthesis caused by hrIL-1 beta without decreasing peptidyl LT synthesis. In separate experiments, hrIL-1 beta failed to increase the recovery of arachidonic acid-induced contractions after aspirin pretreatment in both ADXSS and rat stomach strips. The data indicate that the effects caused by hrIL-1 beta on ADXSS are possibly the result of an exacerbated phospholipase A2 activity particularly inasmuch as increases in cellular cyclooxygenase synthesis are unlikely in this experimental system. It appears likely that a peptidyl LT is involved in this process. Elevation of phospholipase A2 activity by interleukin-1 like drugs may be an important factor limiting the use of these agents as adjuvants particularly in patients with low blood glucocorticoid levels.

Evidence that interleukin-1 and lipoxygenase metabolites mediate the lethal effect of complete Freund's adjuvant in adrenalectomized rats.

Injection of complete Freund's adjuvant (CFA) into the right hind paw of adrenalectomized (ADX) rats caused a lethal effect, maximal after 3 days. Treatment of animals with polyclonal sera raised against either murine recombinant (mr) IL-1 alpha or mrIL-1 beta gave significant protection, suggesting the involvement of this cytokine in the observed lethality. Death was also caused by the intravenous injection of human recombinant (hr) IL-1 beta in ADX rats. No lethality was induced by either CFA or hrIL-1 beta in sham-operated (SHO) rats. The administration of dexamethasone or the dural cyclooxygenase/lipoxygenase inhibitor BW 755c significantly protected against the lethality induced by both CFA and hrIL-1 beta. The cyclooxygenase inhibitors, indomethacin and acetylsalicyclic acid, were ineffective. WEB 2086, a PAF-acether receptor antagonist, gave partial but not significant protection. These results suggest that activation of arachidonic acid metabolism is involved in the observed lethality, with lipoxygenase metabolites as possible final effectors. These experimental models of lethalities may be useful for the in vivo evaluation of drugs interfering with the synthesis and/or biological effects of IL-1.

Conceivable difference in the anti-inflammatory mechanisms of lipocortins 1 and 5.

Human recombinant lipocortins (LCT) 1 and 5 have been expressed in a yeast secretion vector and purified by ion exchange chromatography. The action of the proteins has been investigated in two models of experimental acute inflammation in the rat: carrageenin induced paw oedema and zymosan induced pleurisy. The effects of the proteins on PGE(2) release in vitro by rat macrophages stimulated with zymosan and on rat neutrophil chemotaxis induced by FMLP have also been assessed. LCT-1 significantly inhibited both paw swelling in carrageenin oedema and leukocyte migration in zymosan pleurisy. Moreover it showed a dose dependent, inhibitory effect on PGE(2) release. Neutrophil chemotaxis was only weakly affected by LCT-1. Conversely LCT-5 did not reduce carrageenin oedema and slightly inhibited PGE(2) release, but showed profound, dose dependent inhibitory activity on leukocyte migration in zymosan pleurisy and on neutrophil chemotaxis. These data suggest that LCT-1 acts mainly by interfering with arachidonic acid metabolism via the inhibition of phospholipase A(2). The anti-inflammatory activity of LCT-5, at variance with LCT-1, may be due to a direct effect on cell motility in addition to the interference with arachidonic acid metabolism.

4-substituted-5-acetyl-2-methyl-6-phenyl-3(2H)pyridazinones as PGE2 and IL-1 release inhibitors from mouse adherent macrophages.

A series of 4,5-functionalized 3(2H)-pyridazinones were evaluated as prostaglandin E2 (PGE2) and interleukin-1 (IL-1) release inhibitors from mouse adherent macrophages. Among the tested compounds only 2b was found to be devoid of activity in both the PGE2 and IL-1 tests, whereas the other compounds, showed a significant dose-dependent activity. Compounds 2a, 3 and 4 were able to inhibit PGE2 better than IL-1 release from stimulated macrophages. Compound 4, which showed an IC50 = 5.5 microM in the IL-1 test, appears to be a promising agent in this cell inflammation model. Structure-activity relationship (SAR) studies demonstrated the importance of the presence of a substituent characterized by a positive sigma constant at position 4 of the pyridazine system.

Prostaglandin E2 and bacterial lipopolysaccharide stimulate bioactive interleukin-1 release from rat hypothalamic explants.

While interleukin-1 (IL-1) is intimately involved in locally modulating the acute inflammatory response, it is also able to influence processes at remote sites, i.e., in an endocrine manner. While there is as yet little evidence that IL-1 can cross the blood-brain barrier, many effects such as fever, increased slow-wave sleep, anorexia and the modulation of neuroendocrine function suggest an action of circulating IL-1 at regulatory sites within the hypothalamus. However, there is accumulating evidence for IL-1 originating within the central nervous system (CNS), and it is currently unclear as to whether the neurally mediated manifestations of the acute inflammatory response are due to activation of central or peripheral (circulating) IL-1. In this study we have characterized the release of IL-1 from rat hypothalamic explants, and we have investigated the effects of putative modulators of IL-1 release, lipopolysaccharide (LPS) and the prostaglandins E2 (PGE2) and F2 alpha (PGF2 alpha). After 1 h of incubation, IL-1-like activity in hypothalamic supernatants ranged between 175 and 2,304 munits/mg of protein; this was substantially inhibited by the addition to the bioassay system of antibodies (1:200) against IL-1 alpha, but not against IL-1 beta. LPS and PGE2 significantly stimulated IL-1 release at 100 and 1 ng/ml respectively, whereas PGF2 alpha had no effect in the range of doses tested. It is therefore concluded that the control of hypothalamic IL-1 release may be investigated by means of acute rat hypothalamic explants.(ABSTRACT TRUNCATED AT 250 WORDS)

Anti-inflammatory effect of tuftsin and its retro-inverso analogue in rat adjuvant arthritis.

Tuftsin, an immunostimulating tetrapeptide derived from immunoglobulin heavy chain, and its retro-inverso analogue have been tested in rat adjuvant arthritis. The secondary lesion of rats injected with Freund's adjuvant was significantly inhibited by both the natural and retro-inverso tuftsin administered orally. The retro-inverso analogue was at least tenfold more potent than the parent molecule, suggesting an increased stability to degradation, because of its molecular modification. The anti-inflammatory effect of tuftsin may be due to its ability to modulate macrophage function.

Metabolic behavior and distribution of the synthetic nonapeptide fragment 163-171 of human IL-1 beta.

The pharmacokinetic parameters and distribution of the adjuvant synthetic nonapeptide VQGEESNDK, corresponding to the fragment in position 163-171 in human IL-1, were analyzed after administration to rabbit through different routes. The radiolabeled peptide did not bind to plasma proteins and, when inoculated i.v., it disappeared very rapidly from the circulation, with a t1/2 alpha of 1 min and a t 1/2 beta of 166 min. Upon administration through i.m., s.c. and oral route, the Cmax was reached between 30 and 90 min after inoculum and ranged between 7 and 4% of the administered dose. Organ distribution showed that most of the radioactivity was concentrated in kidneys and excreted in urine. From Sephadex G-10 chromatography, about 60% of the peptide recovered in the urine 4h after i.v. inoculum was intact, whereas it was more than 85% degraded when administered by other routes. The amount of intact peptide recovered in the urine correlated with the biological effectiveness through different routes, suggesting that the adjuvant effect in vivo is exerted by the intact peptide, rather than by its metabolites.