Population Pharmacokinetics and Exposure-Response Analyses to Guide Dosing of Icatibant in Pediatric Patients With Hereditary Angioedema.

Elevated bradykinin levels are responsible for the development of clinical symptoms in patients with hereditary angioedema (HAE). Icatibant is a bradykinin type 2 receptor antagonist indicated for the acute treatment of HAE attacks. A population modeling and simulation approach was used to examine sources of variability impacting icatibant pharmacokinetics (PK) and provide guidance on icatibant dosing in pediatric patients with HAE. An exposure-response analysis was performed for the time to onset of symptom relief (TOSR). Data from 141 adults (133 healthy, 8 with HAE) who received subcutaneous icatibant 30 mg and 31 pediatric patients with HAE who received 0.4 mg/kg (capped at 30 mg) were included in the analysis. Icatibant PK was described by a 2-compartment model with linear elimination. Complete absorption of icatibant was expected within 1 hour of dosing. The apparent clearance and central volume of distribution were 15.4 L/h and 20.4 L, respectively. Icatibant PK was mainly dependent on body weight. The mean TOSR was very short (1.38 hours). A flat exposure-response was observed, confirming that the relationship plateaued at the level of exposure observed in pediatric patients. Simulations confirmed that weight band-based dosing regimens (10 mg [12-25 kg], 15 mg [26-40 kg], 20 mg [41-50 kg], 25 mg [51-65 kg], and 30 mg [>65 kg]) resulted in exposure similar to the 0.4-mg/kg dose. This analysis showed that icatibant undergoes rapid absorption, reaches levels required for therapeutic response, and promptly relieves HAE symptoms. A weight band-based dosing regimen is appropriate in pediatric patients with HAE.

Efficacy, pharmacokinetics, and safety of icatibant for the treatment of Japanese patients with an acute attack of hereditary angioedema: A phase 3 open-label study.

BACKGROUND: Hereditary angioedema (HAE) is a genetic disease characterized by recurrent swelling episodes affecting the skin, gastrointestinal mucosa, and upper respiratory tract. METHODS: A phase 3, single-arm, open-label study was performed to evaluate a selective bradykinin B2 receptor antagonist, icatibant, for the treatment of acute attacks in Japanese patients with HAE Type I or II. After the onset of an acute attack, icatibant 30 mg was administered by the patient or a healthcare professional via subcutaneous injection in the abdomen. RESULTS: Eight patients who had an attack affecting the skin (n = 4), abdomen (n = 3), or larynx (n = 1) were treated with icatibant (3 of the injections were self-administered). The median time to onset of symptom relief was 1.75 h (95% confidence interval, 1.00-2.50), and all patients had symptom relief within 5 h after administration. The time to maximum plasma concentration of icatibant was 1.79 h, and the maximum plasma concentration was 405 ng/ml. Seven patients experienced an injection site reaction, and 3 patients had adverse events (2 patients had a worsening or repeat HAE attack 29.0 and 18.3 h after icatibant administration, respectively, and 1 had headache). CONCLUSIONS: Although the number of patients is small, the efficacy and tolerability of icatibant for acute attacks were demonstrated in Japanese patients with HAE, regardless of self-administration or administration by healthcare professional.

[EFFICACY, PHARMACOKINETICS, AND SAFETY OF ICATIBANT FOR THE TREATMENT OF JAPANESE PATIENTS WITH AN ACUTE ATTACK OF HEREDITARY ANGIOEDEMA: A PHASE 3 OPEN-LABEL STUDY].

BACKGROUND: Hereditary angioedema (HAE) is characterized by paroxysmal edema of the skin, gastrointestinal mucosa, and upper respiratory tract. PURPOSE: This study investigated icatibant, a selective bradykinin B2 receptor antagonist, as treatment for Japanese patients with an acute HAE attack. METHODS: This was an open-label, single-arm, Phase 3 study of Japanese adults with HAE type I or II. Icatibant (30 mg) was administered (by a healthcare professional [HCP] or self-administered) as a subcutaneous injection in the abdomen. RESULTS: Eight patients (4 cutaneous, 3 abdominal, 1 laryngeal) were treated with icatibant (all single injection; 3 self-administered, 5 HCP-administered). The median time to onset of symptom relief was 1.75 hours (95% confidence interval, 1.00 to 2.50); all patients had onset of relief within 5 hours. The estimated time to maximum icatibant concentration in the circulation was 1.79 hours and the maximum concentration was 405 ng/mL. There were 3 patients who experienced 3 adverse events (2 HAE attacks and 1 headache); 7 patients experienced an injection site reaction. CONCLUSION: Although our study was limited by the small number of patients, we found that icatibant was an effective and well-tolerated treatment for Japanese patients with acute HAE attacks, regardless of whether it was administered by a HCP or self-administered.

Neuroprotection against apoptosis of SK-N-MC cells using RMP-7- and lactoferrin-grafted liposomes carrying quercetin.

A drug delivery system of quercetin (QU)-encapsulated liposomes (LS) grafted with RMP-7, a bradykinin analog, and lactoferrin (Lf) was developed to permeate the blood-brain barrier (BBB) and rescue degenerated neurons, acting as an Alzheimer's disease (AD) pharmacotherapy. This colloidal formulation of QU-encapsulated LS grafted with RMP-7 and Lf (RMP-7-Lf-QU-LS) was used to traverse human brain microvascular endothelial cells (HBMECs) regulated by human astrocytes (HAs) and to treat SK-N-MC cells after an insult with cytotoxic ß-amyloid (Aß) fibrils. We found that surface RMP-7 and Lf enhanced the ability of QU to cross the BBB without inducing strong toxicity and damaging the tight junction. In addition, RMP-7-Lf-QU-LS significantly reduced Aß-induced neurotoxicity and improved the viability of SK-N-MC cells. Compared with free QU, RMP-7-Lf-QU-LS could also significantly inhibit the expression of phosphorylated c-Jun N terminal kinase, phosphorylated p38, and phosphorylated tau protein at serine 202 by SK-N-MC cells, indicating an important role of RMP-7, Lf, and LS in protecting neurons against apoptosis. RMP-7-Lf-QU-LS is a promising carrier targeting the BBB to prevent Aß-insulted neurodegeneration and may have potential in managing AD in future clinical applications.

Involvement of bradykinin B2 and muscarinic receptors in the prolonged diuretic and antihypertensive properties of Echinodorus grandiflorus (Cham. & Schltdl.) Micheli.

BACKGROUND: Although Echinodorus grandiflorus (Cham. & Schltr.) Michel are used in Brazilian folk medicine as a diuretic drug, to date, no study has evaluated the mechanisms involved in this activity after prolonged administration in rats. AIM OF THE STUDY: Evaluate the possible mechanisms involved in the prolonged diuretic activity of ethanol soluble fraction obtained from Echinodorus grandiflorus (ES-EG) and to assess its relationship with hypotensive and antihypertensive activity using normotensive rats and those with renovascular hypertension (2K1C). METHODS: The diuretic effects of ES-EG (30-300 mg/kg; p.o.) were compared with hydrochlorothiazide in a repeated-dose treatment for 7 days. The urinary volume and sodium, potassium, chloride, bicarbonate contents, conductivity, pH and density were estimated in sample collected in 24 h for 7 days. Plasma sodium, potassium, total protein, urea, creatinine, aldosterone, vasopressin, nitrite, acetylcholinesterase concentration and angiotensin converting enzyme (ACE) activity were measured in samples collected at the end of the experimental period (seventh day). Using pharmacological antagonists or inhibitors, the involvement of bradykinin, prostaglandin, acetylcholine and nitric oxide (NO) in ES-EG-induced diuresis was determined. In addition, activities of erythrocytary carbonic anhydrase and renal Na+/K+/ATPase were evaluated in vitro. RESULTS: ES-EG increased diuresis similarly to hydrochlorothiazide and also presented HCO3-sparing effects and increased serum nitrite levels. Moreover, the intraduodenal administration of ES-EG induces significant hypotensive and antihypertensive effects in 2K1C rats. Previous treatment with HOE-140, indometacin and atropine fully avoided the diuretic effect of ES-EG, and including L-NAME pre-administration, it prevented the hypotensive and hypertensive activity induced by ES-EG. In addition, the association between HOE-140 and atropine or indometacin and L-NAME fully inhibited the hypotensive and antihypertensive effects of ES-EG. The 7-day treatment with ES-EG resulted in increased plasma nitrite levels. All other parameters were not affected by treatment with ES-EG. CONCLUSIONS: Our results suggest that the mechanisms through which Echinodorus grandiflorus extracts induce prolonged diuresis and reduce blood pressure in normotensive and 2K1C rats are mainly related to activation of muscarinic and bradykinin receptors with direct effects on prostaglandins and nitric oxide pathways.

[Role of Bradikynin in the Mechanism of Ischemic Preconditioning of the Heart. Prospects of Bradykinin Application in Cardiosurgical Praxis].

Bradykinin level is increased in myocardium in response to short-term ischemia/reperfusion that is one of the evidences of its trigger role in ischemic preconditioning (IP). Pharmacological induced increase of endogenous bradykinin and kallidin-like peptide levels in myocardium enhances cardiac tolerance to impact of ischemia/reperfusion. Experiments with genetically modified mice indicate that kinins are involved in preconditioning but they are not the only trigger of IP. The B2-receptor blocking abolishes antiarrhythmic, infarct reducing effects ofpreconditioning, eliminates IP-induced cardiac tolerance to oxidative stress. Exogenous bradykinin mimics inotropic and cardioprotective effects of IP but does not mimic antiarrhythmic effect of preconditioning. The intracoronary or intravenous bradykinin infusion enhances human heart resistance to ischemia/reperfusion. Implementation of the cardioprotective effect of IP is provided by the activation of multiple signaling pathways that involve: B2-receptor, calcitonin gene-related peptide, NO-synthase, guanylyl cyclase, cGMP, protein kinase G, mitochondrial KATP channels, reactive oxygen species, kinases C, ERK andAkt. To increase of the human heart tolerance to ischemia/reperfusion is necessary to develop B2-receptor agonists devoid hypotensive and pro-inflammatory properties.

Pharmacokinetics of single and repeat doses of icatibant.

PURPOSE: Icatibant is a bradykinin-2 receptor antagonist approved to treat acute hereditary angioedema attacks in adults. To-date, no thorough investigation of the clinical pharmacokinetic (PK) parameters of icatibant and its primary metabolites has been reported. Here we present the PK results of two phase I clinical studies of icatibant in healthy human volunteers. METHODS: Single- and multiple-dose plasma pharmacokinetics of icatibant were characterized in healthy volunteers. Icatibant concentration-time profiles and PK parameters were derived after a single 30- or 90-mg dose or three 30-mg doses given at 6-hour intervals. RESULTS: Maximal plasma concentrations for the 30-mg (979 ± 262 ng/mL) and 90-mg doses (2,719 ± 666 ng/mL) were achieved at <1 hour postdose. The total plasma icatibant exposure for the 30- and 90-mg doses was 2,191 ± 565 and 6,736 ± 1,230 h · ng/mL, respectively, with elimination half-life values of 1.48 ± 0.35 and 2.00 ± 0.57 hours, respectively. CONCLUSIONS: Single 30- and 90-mg subcutaneous administration of icatibant exhibited dose-proportional PK with no appreciable accumulation upon repeated 30-mg doses administered at 6-hour intervals.

Preclinical pharmacology, metabolic stability, pharmacokinetics and toxicology of the peptidic kinin B1 receptor antagonist R-954.

We previously showed that R-954 (AcOrn[Oic(2),(αMe)Phe(5),dßNal(7),Ile(8)]desArg(9)-bradykinin) is a potent, selective and stable peptide antagonist of the inducible GPCR kinin B1 receptor. This compound shows potential applications for the treatment of several diseases, including cancer and neurological disturbances of diabetes. To enable clinical translation, more information regarding its pharmacological, pharmacokinetics (PK) and toxicological properties at preclinical stage is warranted. This was the principal objective of the present study. Herein, specificity of R-954 was characterized in binding studies on 133 human molecular targets to reveal minor cross-reactivities against the angiotensin AT2 and the bombesin receptors (110- and 330-fold lower affinity than for B1R, respectively). The pharmacokinetic of R-954 was studied in both normal and streptozotocin-diabetic anaesthetized rats providing half-lives of 1.9-2.7h. R-954 does not appear to be metabolized in the rat circulation and in several rat tissue homogenates, as the kidney, lung and liver. It appears to be excreted as parent drug in the bile (21%) and in urine. A preliminary toxicological profile of R-954 was obtained in rats under various administration routes. R-954 appears to be well tolerated. Overall, these results indicate that R-954 exhibits favorable preclinical pharmacological/PK characteristics and encouraging safety profiles, suitable for early studies in humans.

Activation of TRPV1 by capsaicin induces functional kinin B(1) receptor in rat spinal cord microglia.

BACKGROUND: The kinin B(1) receptor (B(1)R) is upregulated by pro-inflammatory cytokines and oxydative stress, which are enhanced by transient receptor potential vanilloid subtype 1 (TRPV1) activation. To examine the link between TRPV1 and B(1)R in inflammatory pain, this study aimed to determine the ability of TRPV1 to regulate microglial B(1)R expression in the spinal cord dorsal horn, and the underlying mechanism. METHODS: B(1)R expression (mRNA, protein and binding sites) was measured in cervical, thoracic and lumbar spinal cord in response to TRPV1 activation by systemic capsaicin (1-50 mg/kg, s.c) in rats pre-treated with TRPV1 antagonists (capsazepine or SB-366791), the antioxidant N-acetyl-L-cysteine (NAC), or vehicle. B(1)R function was assessed using a tail-flick test after intrathecal (i.t.) injection of a selective B(1)R agonist (des-Arg(9)-BK), and its microglial localization was investigated by confocal microscopy with the selective fluorescent B(1)R agonist, [Nα-bodipy]-des-Arg(9)-BK. The effect of i.t. capsaicin (1 µg/site) was also investigated. RESULTS: Capsaicin (10 to 50 mg/kg, s.c.) enhanced time-dependently (0-24h) B(1)R mRNA levels in the lumbar spinal cord; this effect was prevented by capsazepine (10 mg/kg, i.p.; 10 µg/site, i.t.) and SB-366791 (1 mg/kg, i.p.; 30 µg/site, i.t.). Increases of B(1)R mRNA were correlated with IL-1ß mRNA levels, and they were significantly less in cervical and thoracic spinal cord. Intrathecal capsaicin (1 µg/site) also enhanced B(1)R mRNA in lumbar spinal cord. NAC (1 g/kg/d × 7 days) prevented B(1)R up-regulation, superoxide anion production and NF-kB activation induced by capsaicin (15 mg/kg). Des-Arg(9)-BK (9.6 nmol/site, i.t.) decreased by 25-30% the nociceptive threshold at 1 min post-injection in capsaicin-treated rats (10-50 mg/kg) while it was without effect in control rats. Des-Arg(9)-BK-induced thermal hyperalgesia was blocked by capsazepine, SB-366791 and by antagonists/inhibitors of B(1)R (SSR240612, 10 mg/kg, p.o.), glutamate NMDA receptor (DL-AP5, 10 µg/site, i.t.), substance P NK-1 receptor (RP-67580, 10 µg/site, i.t.) and nitric oxide synthase (L-NNA, 10 µg/site, i.t.). The B(1)R fluorescent agonist was co-localized with an immunomarker of microglia (Iba-1) in spinal cord dorsal horn of capsaicin-treated rats. CONCLUSION: This study highlights a new mechanism for B(1)R induction via TRPV1 activation and establishes a link between these two pro-nociceptive receptors in inflammatory pain.

Management of acute attacks of hereditary angioedema: potential role of icatibant.

Icatibant (Firazyr(®)) is a novel subcutaneous treatment recently licensed in the European Union for acute hereditary angioedema. Hereditary angioedema, resulting from inherited partial C1 inhibitor deficiency, is a disabling condition characterized by intermittent episodes of bradykinin-mediated angioedema. Icatibant blocks bradykinin B2 receptors, attenutating the episode. Randomized double-blind, placebo-controlled trials of icatibant, showed significant superiority over oral tranexamic acid in 74 European patients and a trend to improvement in a similar US trial comparing icatibant with placebo in 55 patients. Outcomes for several endpoints did not reach significance in the US trial, perhaps because of low participant numbers and confounding factors: a further trial is planned. Open label studies have shown benefit in multiple treatments for attacks at all sites. Approximately 10% of patients require a second dose for re-emergent symptoms, usually 10 to 27 hours after the initial treatment. Its subcutaneous route of administration, good tolerability and novel mode of action make icatibant a promising addition to the limited repertoire of treatments for hereditary angioedema.

Inhibition of type 1 diabetic hyperalgesia in streptozotocin-induced Wistar versus spontaneous gene-prone BB/Worchester rats: efficacy of a selective bradykinin B1 receptor antagonist.

Insulin-dependent type 1 diabetes (T1D) is linked to a series of complications, including painful diabetic neuropathy (PDN). Several neurovascular systems are activated in T1D, including the inducible bradykinin (BK) B1 receptor (BKB1-R) subtype. We assessed and compared the efficacy profile of a selective BKB1-R antagonist on hyperalgesia in 2 models of T1D: streptozotocin (STZ) chemically induced diabetic Wistar rats and spontaneous BioBreeding/Worchester diabetic-prone (BB/Wor-DP) rats. Nociception was measured using the hot plate test to determine thermal hyperalgesia. STZ diabetic rats developed maximal hyperalgesia (35% decrease in their hot plate reaction time) within a week and remained in such condition and degree for up to 4 weeks postinjection. BB/Wor-DP rats also developed hyperalgesia over time that preceded hyperglycemia, starting at the age of 6 weeks (9% decrease in the hot plate reaction time) and stabilizing over the age of 16 to 24 weeks to a maximum (60% decrease in the hot plate reaction time). Single, acute subcutaneous administration of the selective BKB1-R antagonist induced significant time- and dose-dependent attenuation of hyperalgesia in both STZ diabetic and BB/Wor-DP rats. Thus, selective antagonism of the inducible BKB1-R subtype may constitute a novel and potential therapeutic approach for the treatment of PDN.

Angiotensin-converting enzyme inhibition increases basal vascular tissue plasminogen activator release in women but not in men.

OBJECTIVE: Angiotensin-converting enzyme inhibition (ACEI) increases vascular tissue plasminogen activator (t-PA) release through endogenous bradykinin (BK). We tested the hypothesis that gender influences the effect of ACEI on t-PA release. METHODS AND RESULTS: We measured the effect of intra-arterial enalaprilat (0.33 microg/min per 100 mL forearm volume) on forearm blood flow (FBF) and net t-PA release before and during BK (25 to 400 ng/min) and methacholine (3.2 to 12.8 microg/min) in premenopausal women, postmenopausal women not using hormone replacement, young men, and older men. Baseline net t-PA release was similar among groups. Enalaprilat increased basal t-PA release in premenopausal (from 0.9+/-1.0 to 5.1+/-1.7 ng/min per 100 mL, P=0.023) and postmenopausal women (from -3.9+/-2.2 to 3.9+/-1.1 ng/min per 100 mL, P=0.010) but not in young or older men (P=0.028 men versus women). Enalaprilat potentiated the effect of exogenous BK on FBF similarly in all groups. However, during enalaprilat, BK-stimulated t-PA release was greatest in premenopausal women (339.9+/-86.4 ng/min per 100 mL at the 100 ng/min dose, P<0.05 versus any other group), intermediate in postmenopausal women (243.8+/-51.1 ng/min per 100 mL, P<0.05 versus either male group), and least in young (111.9+/-19.2 ng/min/100 mL) and older men (103.4+/-27.6 ng/min/100 mL). CONCLUSIONS: ACEI enhances basal t-PA release in women, independent of menopausal status, but not in men. During ACEI, both gender and menopausal status affect BK stimulated t-PA release.

Fate of bradykinin on the rat liver when administered by the venous or arterial route.

BACKGROUND AND AIM: Bradykinin (BK) infused into the portal vein elicits a hypertensive response via the B2 receptor (B2R) and is efficiently hydrolyzed by the liver. Our purpose was to characterize the mechanism of interaction between BK and the liver. METHOD: BK, HOE-140 (a B2R antagonist), des-R(9)-BK (a B1R agonist) and enzyme inhibitors were used in monovascular or bivascular perfusions and in isolated liver cell assays. RESULTS: Des-R(9)-BK did not elicit a portal hypertensive response (PHR); BK infused into the hepatic artery elicited a calcium-dependent PHR and a calcium-independent arterial hypertensive response (HAHR), with the latter being almost abolished by naproxen. BK has a predominant distribution in the extracellular space and an average hepatic extraction of 8% in the steady state. Hydrolysis products of infused BK (R(1)-F(5) and R(1)-P(7)) did not elicit PHR. Angiotensin converting enzyme (ACE) is concentrated in the perivenous region and B2R in the periportal region. Microphysiometry showed that BK (and not a B1 agonist) interacts with stellate cells and the endothelial sinusoidal/Kupffer cell fraction. This effect was inhibited by the B2R antagonist. CONCLUSIONS: Events can be summarized as: the hypertensive action of BK on sinusoidal cells of the periportal region is followed by its hydrolysis by ACE which is primarily present in the perivenous region; there is no functional B1R in the normal liver; BK induces HAHR via eicosanoid release and PHR by a distinct pathway on the B2R. Our data suggest that BK may participate in the modulation of sinusoidal microvasculature tonus both in the portal and the arterial routes.

Expression of kinin B1 receptors in the spinal cord of streptozotocin-diabetic rat.

Previous studies have reported cardiovascular and nociceptive responses after intrathecal injection of kinin B1 receptor (B1R) agonists in the model of streptozotocin (STZ)-diabetic rat (diabetic). The aim of this study was to measure the early up-regulation of B1R binding sites and mRNA in the thoracic spinal cord of diabetic and control rats. Data show significant increases of specific B1R binding sites in the dorsal horn of diabetic rats 2 days (+315%), 7 days (+303%) and 21 days (+181%) after STZ treatment. Levels of mRNA were significantly increased (+68%) at 2 and 7 days but not at 21 days. These data bring the first molecular evidence for an early up-regulation of B1R in the spinal cord of diabetic rat.

Icatibant: HOE 140, JE 049, JE049.

Icatibant [HOE 140, JE 049] is a potent, specific and selective peptidomimetic bradykinin beta2-receptor antagonist. It has a modified peptide structure, and is the first bradykinin receptor antagonist to act on the guinea-pig trachea without demonstrating agonist effects. Icatibant was originated by Hoechst Marion Roussel (now Sanofi-Aventis). Jerini is seeking a partner for development and marketing of icatibant for the treatment of refractory ascites in liver cirrhosis, angioedema and burns. In August 2004, Aventis merged with Sanofi-Synthelabo to form Sanofi-Aventis.Icatibant has shown an excellent safety profile in phase I studies. In December 2003, Jerini demonstrated positive results in the phase IIa study. Results obtained were statistically significant and clinically relevant. At the BIO 2004 International Annual Convention (BIO-2004) [San Francisco, CA, USA; 6-9 June 2004], Jerini reported plans to initiate phase IIb trials in this indication in the second half of 2004. Positive results from an icatibant formulation comparative study, in patients with acute attacks of hereditary angioedema, were announced in August 2004; IV and SC formulations showed no difference in efficacy and safety. It was announced in September 2004 by Jerini that a pivotal study, known as For Angioedema Subcutaneous Treatment (FAST) 1, had been initiated in the US and Canada. The protocol of a European study, to be known as FAST 2, is to be submitted to the authorities in September 2004. Jerini expects to launch the product in 2006. The US FDA granted icatibant, for the treatment of hereditary angioedema, fast-track designation in July 2004. In January 2003, the European Agency for the Evaluation of Medicinal Products granted icatibant orphan drug status in Europe for the treatment of angioedema. In November 2003, Jerini announced that effective December 2003, icatibant had orphan drug status in the US for the same indication.

The relationship between flow-mediated brachial artery vasodilation and coronary vasomotor responses to bradykinin: comparison with those to acetylcholine.

Flow-mediated dilation (FMD) of brachial artery provides a noninvasive assessment of coronary endothelial dysfunction. Acetylcholine (ACh) has been used as an agent for estimating coronary endothelial function. In contrast to ACh, there is no evidence for a relationship between FMD and coronary vasodilation to bradykinin (BK). The aim of this study was to compare the flow-mediated vasodilation of brachial artery with coronary vasomotor responses to intracoronary ACh or BK in patients with an angiographically normal left anterior descending coronary artery. Ninety-one patients underwent the cardiac catheterization examination with coronary endothelial function testing and the brachial ultrasound study. BK (0.2, 0.6, 2.0 microg/min) and ACh (3, 10, 30 microg/min) were administered into the left coronary artery in a stepwise manner. Coronary blood flow was evaluated by the Doppler flow velocity measurement. Coronary diameters were measured by the quantitative coronary angiography. The assessment of endothelial function in the brachial artery was made in response to reactive hyperemia with high-resolution ultrasound. Bradykinin induced dose-dependent increases in epicardial coronary diameter and blood flow. There was a significant positive correlation between FMD- and BK-induced vasodilations of epicardial coronary arteries (0.2 microg/min: r = 0.30; 0.6 microg/min: r = 0.42; 2.0 microg/min: r = 0.44, P < 0.01, respectively) and resistance coronary arteries (0.2 microg/min: r = 0.40; 0.6 microg/min: r = 0.56; 2.0 microg/min: r = 0.59, P < 0.0001, respectively). FMD correlated with ACh-induced vasomotions of resistance but not epicardial coronary arteries. No correlation was seen between nitroglycerin-induced brachial artery vasodilation and BK-induced coronary vasodilation. The endothelial dysfunction of peripheral arteries correlated well with that of the coronary arteries especially vasomotor responses to BK.

The binding specificity of OppA determines the selectivity of the oligopeptide ATP-binding cassette transporter.

The purification and functional reconstitution of a five-component oligopeptide ATP-binding cassette transporter with a remarkably wide substrate specificity are described. High-affinity peptide uptake was dependent on liganded substrate-binding protein OppA, which interacts with the translocator OppBCDF with higher affinity than unliganded OppA. Transport screening with combinatorial peptide libraries revealed that (i) the Opp transporter is not selective with respect to amino acid side chains of the transported peptides; (ii) any peptide that can bind to OppA is transported via Opp, including very long peptides up to 35 residues long; and (iii) the binding specificity of OppA largely determines the overall transport selectivity.

The mutation Ser511Asn leads to N-glycosylation and increases the cleavage of high molecular weight kininogen in rats genetically susceptible to inflammation.

Crohn disease is immunologically mediated and characterized by intestinal and systemic chronic inflammation. In a rat model, injection of peptidoglycan-polysaccharide complexes into the intestinal wall induced chronic inflammation in Lewis but neither Fischer nor Buffalo rats, indicating a differential genetic susceptibility. Proteolysis of plasma high molecular weight kininogen (HK) yielding bradykinin and cleaved HK (HKa) was faster in Lewis than in Fischer or Buffalo rat plasma. A single point mutation at nucleotide 1586 was found translating from Ser511 (Buffalo and Fisher) to Asn511 (Lewis). The latter defines an Asn-Xaa-Thr consensus sequence for N-glycosylation. We expressed these domains in Escherichia coli and found no differences in the rate of cleavage by purified kallikrein in the 3 strains in the absence of N-glycosylation. We then expressed these domains in Chinese hamster ovary (CHO) cells, which are capable of glycosylation, and found an increased rate of cleavage of Lewis HK. The Lewis mutation is associated with N-glycosylation as evidenced by a more rapid migration after treatment with N-glycosidase F. When CHO cells were cultured in the presence of tunicamycin, the kallikrein-induced cleavage rate of Lewis HK was not increased. This molecular alteration might be one contributing factor resulting in chronic inflammation in Lewis rats.