Effect of warming and flow rate conditions of blood warmers on red blood cell integrity.

BACKGROUND AND OBJECTIVES: Fluid warmers are routinely used to reduce the risk of hypothermia and cardiac complications associated with the infusion of cold blood products. However, warming blood products could generate haemolysis. This study was undertaken to compare the impact of temperature of blood warmers on the per cent haemolysis of packed red blood cells (RBCs) heated at different flow rates as well as non-flow conditions. MATERIALS AND METHODS: Infusion warmers used were calibrated at 41·5°C ± 0·5°C and 37·5°C ± 0·5°C. Cold RBC units stored at 4°C in AS-3 (n = 30), aged 30-39 days old, were divided into half units before being allocated under two different scenarios (i.e. infusion pump or syringe). RESULTS: Blood warmers were effective to warm cold RBCs to 37·5°C or 41·5°C when used in conjunction with an infusion pump at flow rate up to 600 ml/h. However, when the warmed blood was held in a syringe for various periods of time, such as may occur in neonatal transfusions, the final temperature was below the expected requirements with measurement as low as 33·1°C. Increasing the flow with an infusion pump increased haemolysis in RBCs from 0·2% to up to 2·1% at a flow rate of 600 ml/h regardless of the warming device used (P < 0·05). No relevant increase of haemolysis was observed using a syringe. CONCLUSIONS: The use of a blood warmer adjusted to 41·5°C is probably the best choice for reducing the risk of hypothermia for the patient without generating haemolysis. However, we should be cautious with the use of an infusion pump for RBC transfusion, particularly at high flow rates.

Antinociceptive pharmacology of N-[[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]methyl]-2-[2-[[(4-methoxy-2,6-dimethylphenyl) sulfonyl]methylamino]ethoxy]-N-methylacetamide, fumarate (LF22-0542), a novel nonpeptidic bradykinin B1 receptor antagonist.

The antinociceptive pharmacology of N-[[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]methyl]-2-[2-[[(4-methoxy-2,6-dimethylphenyl) sulfonyl]methylamino]ethoxy]-N-methylacetamide fumarate (LF22-0542), a novel nonpeptidic B1 antagonist, was characterized. LF22-0542 showed high affinity for human and mouse B1 receptors with virtually no affinity for the human B2 receptor; a selectivity index of at least 4000 times was obtained when LF22-0542 was profiled throughout binding or cell biology assays on 64 other G-protein-coupled receptor, 10 ion channels, and seven enzymes. LF22-0542 was a competitive B1 receptor antagonist and elicited significant antinociceptive actions in the mouse acetic acid-induced writhing assay, as well as in the second phases of formalin-induced nociception in mice and in both the first and second phases of the formalin response in rats. LF22-0542 was active after s.c. but not p.o. administration. In B1 receptor knockout (KO) mice, acetic acid and formalin responses were significantly reduced and LF22-0542 had no additional effects in these animals. LF22-0542 alleviated thermal hypersensitivity in both acute (carrageenan) and persistent inflammatory (complete Freund's adjuvant) pain models in rats. LF22-0542 produced a full reversal of experimental neuropathic thermal hypersensitivity but was inactive in reversing nerve injury-induced tactile hypersensitivity in rats. In agreement with this observation, B1 KO mice subjected to peripheral nerve injury did not show thermal hypersensitivity but developed nerve injury-induced tactile hypersensitivity normally. The data demonstrate the antihyperalgesic actions of a selective systemically administered B1 receptor antagonist and suggest the utility of this class of agents for the treatment of inflammatory pain states and for some aspects of neuropathic pain.

Activation of liver X receptors promotes lipid accumulation but does not alter insulin action in human skeletal muscle cells.

AIMS/HYPOTHESIS: The aim of this study was to investigate the effects of liver X receptor (LXR) activation on lipid metabolism and insulin action in human skeletal muscle cells prepared from control subjects and from patients with type 2 diabetes. SUBJECTS AND METHODS: Cultured myotubes were obtained from muscle biopsies of 11 lean, healthy control subjects and ten patients with type 2 diabetes. The mRNA levels of LXR isoforms and lipogenic genes were estimated by RT-quantitative PCR, and the effects of LXR agonists on insulin action were evaluated by assays of protein kinase B serine 473 phosphorylation and glycogen synthesis. RESULTS: Both LXRalpha and LXRbeta were expressed in human skeletal muscle and adipose tissue and there was no difference in their mRNA abundance in tissues from patients with type 2 diabetes compared with control subjects. In cultured muscle cells, LXR activation by T0901317 strongly increased expression of the genes encoding lipogenic enzymes, including sterol regulatory element binding protein 1c, fatty acid synthase and stearoyl-CoA desaturase 1, and also promoted triglyceride accumulation in the presence of a high glucose concentration. Importantly, these effects on lipid metabolism did not affect protein kinase B activation by insulin. Furthermore, LXR agonists did not modify insulin action in muscle cells from patients with type 2 diabetes. CONCLUSIONS/INTERPRETATION: These data suggest that LXR agonists may lead to increased utilisation of lipids and glucose in muscle cells without affecting the mechanism of action of insulin. However, the long-term consequences of triglyceride accumulation in muscle should be evaluated before the development of effective LXR-based therapeutic agents.

Neuroprotective effects of a postischemic treatment with a bradykinin B2 receptor antagonist in a rat model of temporary focal cerebral ischemia.

Bradykinin, an endogenous nonapeptide produced by activation of the kallikrein-kinin system, promotes neuronal tissue damage as well as disturbances in blood-brain barrier function through activation of B2 receptors. In a rat model of focal cerebral ischemia, blockade of B2 receptors before initiation of ischemia with the B2 receptor antagonist, LF 16-0687 Ms, afforded substantial neuroprotection. In order to assess the potential clinical value of this approach, we evaluated the effect of LF 16-0687 Ms given at reperfusion following focal cerebral ischemia on local cerebral blood flow (LCBF), neurological outcome, and infarct size. Sprague-Dawley rats were subjected to MCA occlusion for 90 min by an intraluminal filament. Animals were assigned to one of four treatment arms (n = 7 each): (1) vehicle, (2) LF 16-0687 Ms (1.0 mg/kg/day), (3) LF 16-0687 Ms (3.0 mg/kg/day), or (4) LF 16-0687 Ms (10.0 mg/kg/day) given at reperfusion and repetitively over 2 days. Neurological recovery was examined daily, and infarct volume was assessed histologically on day 7 after ischemia. Physiological parameters and local CBF were not influenced by the treatment. Significant improvement of neurological outcome was observed on postischemic day 3 in animals receiving 1.0 and 3.0 mg/kg/day of LF 16-0687 Ms (P < 0.05). Inhibition of B2 receptors significantly reduced infarct volume in all treated animals predominantly in the cortex. B2 receptor blockade with LF 16-0687 Ms showed neuroprotective effectiveness even when therapy was initiated upon reperfusion, i.e. 90 min after induction of ischemia. Therefore, blockade of B2 receptors seems to be a promising therapeutic approach after focal cerebral ischemia, which deserves further experimental and clinical evaluation.

Detrimental role of bradykinin B2 receptor in a murine model of diffuse brain injury.

Inhibition of the bradykinin B2 receptor type (B2R) has been shown to improve neurological outcome in models of focal traumatic brain injury. However, the involvement of B2R in trauma-induced diffuse injury has not yet been explored. This is an important point, since in humans a pattern of diffuse injury is commonly found in severely injured patients and has been associated with a poor neurological outcome and prognosis. Using the non-peptide B2R antagonist LF 16-0687 Ms and B2R null (B2R-/-) mice, we investigated the role of B2R in a model of closed head trauma (CHT). LF 16-0687 Ms given 30 min after injury reduced the neurological deficit by 26% and the cerebral edema by 22% when evaluated 4 h after CHT. Neurological function after CHT was improved in B2R-/- mice compared to B2R+/+ mice, although there was no difference in the development of brain edema. Treatment with LF 16-0687 Ms and B(2)R gene deletion decreased the accumulation of neutrophils at 24 h after CHT (50% and 36%, respectively). In addition, the inducible NO synthase (iNOS) mRNA level increased markedly, and this was reduced by LF 16-0687 Ms. Taken together, these data support a detrimental role of B2R in the development of the neurological deficit and of the inflammatory secondary damage resulting from diffuse traumatic brain injury. Therefore, blockade of bradykinin B2 receptors might represent an attractive therapeutic approach in the pharmacological treatment of traumatic brain injury.

Therapeutical efficacy of a novel non-peptide bradykinin B2 receptor antagonist on brain edema formation and ischemic tissue damage in focal cerebral ischemia.

OBJECTIVE: Bradykinin has been identified as a mediator of secondary brain damage in acute insults. We currently studied neuroprotective properties of a bradykinin B2 receptor antagonist (LF16-0687 Ms) in transitory focal cerebral ischemia to assess infarct formation and the development of brain edema. MATERIAL AND METHODS: 55 Rats were subjected to 90 min of MCA-occlusion. The receptor antagonist was administered at two dose levels, given from 30 min prior to ischemia over two days after ischemia. Ischemic tissue damage was quantified at day 7 after MCA-occlusion together with assessment of brain edema in separate experiments. Neurological recovery was studied daily. RESULTS: Animals receiving treatment (low dose) had a better functional recovery, particularly at days 3 and 4 (P < 0.05). Infarct formation was significantly attenuated in these animals in both total and cortical brain tissue by 50, or 80%, respectively. Postischemic brain swelling was significantly lowered, i.e. by 62%. CONCLUSIONS: Our findings provide further support for a mediator role of bradykinin in ischemic brain damage including edema formation, obviously by ligand binding to the bradykinin B2 receptor. The availability of a receptor antagonist may afford opportunity for translation of this experimental treatment into stroke patients.

Effects of LF 16-0687 Ms, a bradykinin B(2) receptor antagonist, on brain edema formation and tissue damage in a rat model of temporary focal cerebral ischemia.

Bradykinin, an endogenous nonapeptide produced by activation of the kallikrein-kinin system, promotes neuronal tissue damage as well as disturbances in blood-brain barrier function through activation of B(2) receptors. LF 16-0687 Ms, a non-peptide competitive bradykinin B(2) receptor antagonist, was recently found to decrease brain swelling in various models of traumatic brain injury. We have investigated the influence of LF 16-0687 Ms on the edema formation, neurological outcome, and infarct size in temporary focal cerebral ischemia in rats. Sprague-Dawley rats were subjected to MCA occlusion for 90 min by an intraluminal filament. Local CBF was bilaterally recorded by laser Doppler flowmetry. Study I: animals were assigned to one of three treatment arms (n=11 each): (a) vehicle, (b) LF 16-0687 Ms (12.0 mg/kg per day), or (c) LF 16-0687 Ms (36.0 mg/kg per day) given repetitively s.c. over 3 days. The neurological recovery was examined daily. The infarct volume was assessed histologically 7 days after ischemia. Study II: brain swelling and bilateral hemispheric water content were determined at 48 h post ischemia in eight rats, subjected to the low dose regimen as described above, and in eight vehicle-treated control animals. All treated animals showed tendency to exhibit improved neurological recovery throughout the observation period as compared to the vehicle-treated controls, while this improvement was only significant within the low dose group from postischemic days 3 to 4. Low dose LF 16-0687 Ms significantly attenuated the total and cortical infarct volume by 50 and 80%, respectively. Furthermore, postischemic swelling (-62%) and increase in water content of the infarcted brain hemisphere (-60.5%) was significantly inhibited. The present findings provide strong evidence for an involvement of bradykinin-mediated secondary brain damage following from focal cerebral ischemia. Accordingly, specific inhibition of bradykinin B(2) receptors by LF 16-0687 Ms attenuated postischemic brain swelling, improved the functional neurological recovery, and limited ischemic tissue damage, raising its potential for clinical evaluation in patients with acute stroke.

Pharmacological and functional characterization of the guinea-pig B2 bradykinin receptor stably expressed in CHO-K1 cell line.

In the present study, pharmacological properties of a bradykinin B(2) receptor amplified either from guinea-pig ileum or lung and homologous to the previously reported sequence except two amino-acid changes L(124)-->P and N(227)-->Y in the receptor protein were characterized. Tritiated bradykinin ([(3)H]-BK) specifically bound to the cloned guinea-pig B(2) bradykinin receptor stably expressed in Chinese hamster ovary cells (CHO-K1) with a K(D) value of 0.29+/-0.07 nM. In competition experiments, bradykinin (BK) affinity constant value was 0.21+/-0.05 nM while the two specific kinin B(1) ligands, des-Arg(9)-bradykinin (DBK) and des-Arg(9)-Leu(8)-bradykinin (DLBK) were unable to compete with [(3)H]-BK. As the specific peptide antagonist D-Arg-[Hyp(3),Thi(5),D-Tic(7),Oic(8)]-bradykinin (HOE140), (E)-3-(6-acetamido-3-pyridil)-N-[-N-[2,4-dichloro-3-[(2-methyl-8-quinolinyl)oxymethyl]phenyl]-N-methylaminocarbonylmethyl]acrylamide (FR173657) and 1-[[3-[2,4-dimethylquinolin-8-yl)oxymethyl] - 2,4 - dichloro - phenyl]sulfonyl] - 2(S) - [[4-[4-(aminoiminomethyl)-phenylcarbonyl]piperazin-1-yl]carbonyl]pyrrolidine (LF16-0335C) exhibited a high affinity for this receptor with K(i) values of 7.34+/-2.45 nM and 8.54+/-1.55 nM respectively. BK and kallidin (KD) increased inositol phosphates (IPs) levels with EC(50) values of 0.44+/-0.12 nM and 6.88+/-0.28 nM, respectively. Neither DLBK nor DBK (0.01 nM to 10 microM) stimulated or inhibited IPs turnover and as expected HOE140 did not raise IPs production. HOE140 (0.1 microM) and LF 16-0335c (1 microM) right shifted the BK response curve with pK(B) values of 9.2+/-0.4 and 8.4+/-0.3, respectively. The results indicate that this cloned guinea-pig receptor displayed typical pharmacological properties of a bradykinin B(2) receptor and support the existence of a single B(2) receptor in this species.

The importance of kinin antagonist treatment timing in closed head trauma.

BACKGROUND: Giving LF 16-0687 Ms (a bradykinin B2 receptor antagonist) 1 hour after closed head trauma (CHT) previously was reported to decrease brain edema at 24 hours and improve neurologic severity score (NSS) at 7 days. It is not certain whether a greater benefit could be achieved by treatment sooner after CHT. METHODS: To examine the latter possibility we studied a surrogate condition for the earliest possible administration of LF 16-0687 Ms after CHT, e.g., we examined brain edema and NSS when LF 16-0687 Ms was given 15 min before CHT in rats. RESULTS: LF 16-0687 Ms decreased brain water content (80.0 +/- 1.4%, mean +/- SD) at 24 hours and improved NSS (2 +/- 3, median +/- range) at 7 days after CHT in comparison to that with CHT + saline (82.9 +/- 1.3% and 8 +/- 4). CONCLUSION: Similarity of the present results to those previously reported indicates that the benefit of giving LF 16-0687 Ms 1 hour after CHT appears to represent the maximal benefit afforded by this drug.

Role of bradykinin B2 receptors in the formation of vasogenic brain edema in rats.

Bradykinin is a mediator of brain edema acting through B2 receptors. However, it is not known if bradykinin mediates the formation of cytotoxic or vasogenic brain swelling. To investigate this question we subjected rats to a cryogenic brain lesion over the left parietal cortex, a model well known to produce predominantly vasogenic brain edema. We inhibited bradykinin B2 receptors with the recently characterized nonpeptide B2 receptor antagonist, LF 16-0687. The animals were assigned to three groups (n = 10, each) receiving 10, or 100 microg/kg/min LF 16-0687 or vehicle (0.9% NaCl). Treatment started 15 min before trauma and was continued for 24 h. Another three groups of animals (n = 10, each) received 10 microg/kg/min LF 16-0687 starting 30 or 60 min after trauma or vehicle (0.9% NaCl) for 24 h. Animals were then sacrificed and swelling and water content of the brain were determined. In the vehicle treated group the traumatized hemisphere swelled by 9.3 +/- 1.1% as compared to the untraumatized contralateral side. Pretreatment with 10 microg/kg/min LF 16-0687 decreased brain swelling significantly to 6.4 +/- 1.3% (p < 0.05). Pre-treatment with 100 microg/kg/min was found to be less effective and did not result in a significant reduction of brain swelling (7.4 + 1.3%). Treatment with LF 16-0687 for 24 h (10 microg/kg/min) started 30 or 60 min after trauma did not reduce brain water content or hemispheric swelling. These results demonstrate that brain injury-mediated bradykinin production induces vasogenic brain edema by B2 receptor stimulation. Our findings further clarify the role of bradykinin in the pathophysiology of brain edema formation and confirm the therapeutic potency of bradykinin B2 receptor inhibition.

Control of conformational equilibria in the human B2 bradykinin receptor. Modeling of nonpeptidic ligand action and comparison to the rhodopsin structure.

A prototypic study of the molecular mechanisms of activation or inactivation of peptide hormone G protein-coupled receptors was carried out on the human B2 bradykinin receptor. A detailed pharmacological analysis of receptor mutants possessing either increased constitutive activity or impaired activation or ligand recognition allowed us to propose key residues participating in intramolecular interaction networks stabilizing receptor inactive or active conformations: Asn(113) and Tyr(115) (TM III), Trp(256) and Phe(259) (TM VI), Tyr(295) (TM VII) which are homologous of the rhodopsin residues Gly(120), Glu(122), Trp(265), Tyr(268), and Lys(296), respectively. An essential experimental finding was the spatial proximity between Asn(113), which is the cornerstone of inactive conformations, and Trp(256) which plays a subtle role in controlling the balance between active and inactive conformations. Molecular modeling and mutagenesis data showed that Trp(256) and Tyr(295) constitute, together with Gln(288), receptor contact points with original nonpeptidic ligands. It provided an explanation for the ligand inverse agonist behavior on the WT receptor, with underlying restricted motions of TMs III, VI, and VII, and its agonist behavior on the Ala(113) and Phe(256) constitutively activated mutants. These data on the B2 receptor emphasize that conformational equilibria are controlled in a coordinated fashion by key residues which are located at strategic positions for several G protein-coupled receptors. They are discussed in comparison with the recently determined rhodopsin crystallographic structure.

Synthesis and biological evaluation of bradykinin B(1)/B(2) and selective B(1) receptor antagonists.

We recently described a potent bradykinin B(2) receptor agonist (JMV1116) obtained by replacing the D-Tic-Oic dipeptide moiety of HOE140 by a (3S)-amino-5-(carbonylmethyl)-2,3-dihydro-1, 5-benzothiazepin-4(5H)-one (D-BT) moiety. This compound inhibited the specific binding of [(3)H]BK on membranes of CHO cells expressing the human cloned B(2) receptor with nanomolar affinity and contracted both isolated rat uterus and human umbilical vein. These data demonstrated that D-BT could be a good mimic of the Pro-Phe dipeptide. In the present study we characterized B(1) receptor antagonists containing the D-BT moiety. We prepared an analogue of compound JMV1116 deleting the C-terminal arginine residue. The resulting compound (1) had an affinity of 83 nM for the human cloned B(1) receptor. The most remarkable property of 1 is its ability to bind also the B(2) receptor with an affinity of 4.4 nM despite the absence of the C-terminal arginine residue. Modifications at the N-terminal part of 1 associated with the substitution of the thienylalanine residue by alpha-(2-indanyl)glycine resulted in analogues selectively binding to the B(1) receptor with an affinity in the picomolar range.

A rational approach to the design and synthesis of a new bradykinin B(1) receptor antagonist.

We have previously synthesized a potent and selective B(1) bradykinin receptor antagonist, JMV1645 (H-Lys-Arg-Pro-Hyp-Gly-Igl-Ser-D-BT-OH), containing a dipeptide mimetic ((3S)-amino-5-carbonylmethyl-2,3-dihydro-1, 5-benzothiazepin-4(5H)-one (D-BT) moiety) at the C-terminal. Analogues of this potent B(1) bradykinin receptor antagonist in which the central Pro(2)-Hyp(3)-Gly(4)-Igl(5) tetrapeptide has been replaced by constrained N-1-substituted-1,3,8-triazaspiro¿4. 5decan-4-one ring system were synthesized. Among these analogues, compound JMV1640 (1) was found to have an affinity of 24.10 +/- 9.48 nM for the human cloned B(1) receptor. It antagonized the ¿des-Arg(10)-kallidin-induced contraction of the human umbilical vein (pA(2) = 6.1 +/- 0.1). Compound 1 was devoid of agonist activity at the kinin B(1) receptor. Moreover, it did not bind to the human cloned B(2) receptor. Therefore, JMV1640 constitutes a lead compound for the rational search of nonpeptide B(1) receptor analogues based on the BK sequence.

Identification of a key region of kinin B(1) receptor for high affinity binding of peptide antagonists.

To investigate the molecular basis for the specificity of ligand recognition in human kinin B(1) (B(1)R) and B(2) (B(2)R) receptors, we constructed a series of chimeric receptors by progressively replacing, from the N to the C terminus, the human B(2)R domains by their B(1) counterparts. The chimeric construct possessing the C-terminal tail and the transmembrane domain VII (TM VII) of the B(2)R (construct 6) displayed 7- and 20- fold decreased affinities for the B(1) agonist [(3)H]desArg(10)-kallidin (desArg(10)-KD) and the B(1) antagonist [(3)H]desArg(10)-[Leu(9)]-KD respectively, as compared with the wild-type B(1)R. Moreover, the substitution of the B(1) TM VII by its B(2) homologue TM increased the affinity for the pseudopeptide antagonists, Hoe140 and NPC 567. High affinity for desArg(10)-KD binding was fully regained when the B(2) residue Thr(287) was replaced in construct 6 by the corresponding B(1) Leu(294) residue. When the B(2) residue Tyr(295) was exchanged with the corresponding B(1) Phe(302), high affinity binding for both agonist and antagonist was recovered. Moreover, the L294T and F302Y mutant B(1)R exhibited 69- and 6.5-fold increases, respectively, in their affinities for the B(2) receptor antagonist, Hoe140. Therefore we proposed that Leu(294) and Phe(302) residues, which may not be directly involved in the binding of B(1)R ligands and, hence, their Thr(287) and Tyr(295) B(2) counterparts, are localized in a receptor region, which plays a pivotal role in the binding selectivity of the peptide or pseudopeptide kinin ligands.

Inflammatory hyperalgesia induced by zymosan in the plantar tissue of the rat: effect of kinin receptor antagonists.

The Randall-Selitto paradigm (maximal tolerated pressure externally applied by a mechanical device) was used to develop a rat model of localized inflammatory hyperalgesia in order to compare the analgesic effects of bradykinin (BK) B1 and B2 receptor antagonists and of a non-steroidal anti-inflammatory drug (NSAID). Intra-plantar injection of zymosan (12.5 mg per paw) induced a considerable inflammation as evidenced from gross and histological evaluation and a mechanical hyperalgesia at 6 h. The contra-lateral paw of zymosan-treated animals or saline vehicle-injected paws did not exhibit a decreased pressure tolerance, relative to pre-injection measurements. Since the B1 receptor may be induced under inflammatory situations, we examined the amount of corresponding mRNA using quantitative RT-PCR. We found a significant increase of B1 receptor mRNA in the zymosan--but not the saline-injected paw at 6 h. Drugs were given subcutaneously 2 h before the 6 h readings to test their analgesic potential. The kinin B1 receptor antagonists [Leu8]des-Arg9-BK (3-30 nmol/kg) and R-715 (100 nmol/kg), the B2 receptor antagonists Hoe 140 (15 nmol/kg) and LF 16.0687 (3 and 10 mg/kg), as well as the NSAID diclofenac sodium (1 and 3 mg/kg) significantly reversed zymosan-induced hyperalgesia. We conclude that zymosan-induced hyperalgesia is a model suitable for the rapid evaluation of analgesic drugs with a peripheral site of action interfering either with kinin receptors or with prostanoid formation. In this regard, results of the present study confirm that blocking kinin B1 receptors is a novel approach for treatment of inflammatory pain.

LF16-0687 a novel non-peptide bradykinin B2 receptor antagonist reduces vasogenic brain edema from a focal lesion in rats.

Head injury world wide is still the most frequent cause of morbidity and mortality among the population under 45 years. Approximately 50% of patients dying from severe head injury have a therapy refractory intracranial pressure rise (Baethmann 1998). Traumatic brain edema, e.g. resulting from disruption of the blood-brain barrier is viewed as an important factor of the increased intracranial pressure. Bradykinin, an active peptide of the kallikrein-kinin system is considered to enhance brain edema formation which is attributed to its permeabilizing effect on the blood-brain barrier and on dilation of arterial blood vessels in the brain mediated by B2-receptors facilitating extravasation. Currently, LF16-0687, a novel non-peptide bradykinin B2 receptor antagonist was experimentally tested as to its therapeutical potential on vasogenic brain edema from a cortical focal lesion. Following trephination of the skull in anaesthesia, male Sprague-Dawley rats were subjected to a focal cold injury of the left parietal cortex. Animals of two experimental groups were receiving either LF16-0687 as high or low dose, whereas one group of untreated animals with trauma was treated with 0.9% NaCl as continuous infusion beginning 10 min before until 24 h after lesion. 24 h after trauma the brain was removed from the skull, and the cerebral hemispheres were separated in the median plane for gravimetric assessment of hemispheric swelling. No significant reduction of hemispheric brain swelling (+7.4 +/- 2.9%) was found in animals receiving high-dose LF16-0687 as compared to the untreated controls. Brain swelling, however was significantly attenuated by the low-dose treatment, i.e. to +6.4 +/- 1.3%; vs. +9.3 +/- 1.1% found in the controls, (p < 0.05). The current data confirm that blocking of bradykinin B2-receptors by LF16-0687 is significantly attenuating vasogenic brain edema from a focal cold lesion. The therapeutical properties of the antagonist on brain edema formation cannot be attributed to a lowering of the blood pressure. Rather, specific blocking effects of B2-receptors in the brain appear to be involved. In conclusion, the understanding of secondary brain damage including brain edema in head injury has been markedly enhanced by the discovery of pathophysiologically active mediator compounds playing a role in its various manifestations. The current data confirm a pathophysiological function of bradykinin in vasogenic brain edema mediated by activation of B2-receptors. Currently it is studied whether LF16-0687 also reduces brain swelling when given after an insult.

Effect of LF 16-0687MS, a new nonpeptide bradykinin B2 receptor antagonist, in a rat model of closed head trauma.

Bradykinin is an endogenous nonapeptide which potently dilates the cerebral vasculature and markedly increases vascular permeability. These effects are mediated by B2 receptors located on the vascular endothelium. Previous experimental studies have shown that blockade of the kallikreinkinin system, which mediates the formation of bradykinin, afforded a reduction of the brain edema that developed following a cryogenic cortical lesion. In the present study, we investigated the effect of LF 16-0687MS, a novel nonpeptide B2 receptor antagonist, on cerebral edema and neurological severity score (NSS) after closed head injury to rats. LF 16-0687MS or its vehicle (NaCl 0.9%) was continuously infused at 10, 30, and 100 microg/kg/min over 23 h starting 1 h after a focal trauma to the left hemisphere was induced using a weight-drop device. The extent of edema formation was evaluated 24 h after trauma from left and right hemispheres samples by measurement of specific gravity and water content. In a separate study, a neurological severity score based on scoring of behavioural and motor functions was evaluated 1 h and over 1 week after trauma. LF 16-0687MS at 100 microg/kg/min markedly reduced the development of brain edema as indicated by a 68% increase in specific gravity (p<0.05) and a 64% decrease of water content (p<0.05) in the left hemisphere. In addition the recovery of neurological function was significantly improved by 100 microg/kg/min LF 16-0687MS from day 3 to day 7 after CHT. In a separate experiment, we also showed that LF 16-0687MS at 100 microg/kg/min given either 1 h before or 30 min after CHT did not affect mean arterial blood pressure. These results show that blockade of bradykinin B2 receptors is an effective approach to reduce cerebral edema and to improve neurological outcome after a focal contusion to the cranium.

Specific nonpeptide photoprobes as tools for the structural study of the angiotensin II AT(1) receptor.

The aim of this work was to obtain photoactivatable nonpeptide antagonists of the angiotensin II AT(1) receptor. Based on structure-function relationships, two chemical structures as well as appropriate synthetic schemes were chosen as a frame for the design of radiolabeled azido probes. The feasibility of the strategy was first assessed by the synthesis of two tritiated ligands 21 and 22 possessing a high affinity for the AT(1) receptor and a low nonspecific binding to membrane or cell preparations. We then prepared two unlabeled azido derivatives 7 and 14 which retained a fairly high affinity for the AT(1) receptor. The latter compound proved to be suitable for receptor irreversible labeling and was prepared in its tritiated form 28. This tritiated azido nonpeptide probe displayed a K(d) value of 11.8 nM and a low nonspecific binding. It was suitable for specific and efficient covalent labeling of the recombinant AT(1A) receptor stably expressed in CHO cells. The electrophoretic pattern of the specifically labeled entity was strictly identical to that of purified receptor photolabeled with a biotinylated peptidic photoactivatable probe. This new tool should be useful for the mapping of the nonpeptide receptor binding site. These potential applications are discussed in light of the current knowledge of molecular mechanisms of G-protein coupled receptor activation and inactivation.

Pharmacological profile of LF 16-0687, a new potent non-peptide bradykinin B2 receptor antagonist.

LF 16-0687 (1-[[2,4-dichloro-3-[[(2,4-dimethylquinolin-8-yl)oxy] methyl]phenyl]sulfonyl]-N-[3-[[4-(aminoimethyl) phenyl] carbonylamino]propyl]-2(S)-pyrrolidinecarboxamide) has been selected from a large-scale medicinal chemistry program for further development. In competition binding studies using [3H]bradykinin (BK), LF 16-0687 bound to the human, rat and guinea-pig recombinant B2 receptor expressed in CHO cells giving K(i) values of 0.67 nM, 1.74 nM and 1.37 nM, respectively. It also bound to the native BK B2 receptor from human umbilical vein (HUV), rat uterus (RU) and guinea-pig ileum (GPI) giving K(i) values of 0.89 nM, 0.28 nM and 0.98 nM, respectively. It inhibited BK-induced IP1, IP2 and IP3 formation in INT407 cells yielding pK(B) values of 8.5, 8.6 and 8.7, respectively. In isolated organs experiments, LF 16-0687 behaved as a competitive antagonist of BK-mediated contractions giving pA2 values of 9.1 in HUV, 7.7 in RU and 9.1 in GPI. Binding and functional studies performed over 40 different receptors revealed that LF 16-0687 was selective for the BK B2 receptor. A continuous intravenous infusion of LF 16-0687 antagonized in a dose-dependent manner and with a rapid onset of action BK-induced hypotensive response. Subcutaneous administration of LF 16-0687 at 1.1 micromol/kg to rats markedly reduced BK-induced edema of the stomach (- 69%), duodenum (-65%) and pancreas (-56%).