A Novel Angiotensin-(1-7) Glycosylated Mas Receptor Agonist for Treating Vascular Cognitive Impairment and Inflammation-Related Memory Dysfunction.

Increasing evidence indicates that decreased brain blood flow, increased reactive oxygen species (ROS) production, and proinflammatory mechanisms accelerate neurodegenerative disease progression such as that seen in vascular contributions to cognitive impairment and dementia (VCID) and Alzheimer's disease and related dementias. There is a critical clinical need for safe and effective therapies for the treatment and prevention of cognitive impairment known to occur in patients with VCID and chronic inflammatory diseases such as heart failure (HF), hypertension, and diabetes. This study used our mouse model of VCID/HF to test our novel glycosylated angiotensin-(1-7) peptide Ang-1-6-O-Ser-Glc-NH2 (PNA5) as a therapy to treat VCID and to investigate circulating inflammatory biomarkers that may be involved. We demonstrate that PNA5 has greater brain penetration compared with the native angiotensin-(1-7) peptide. Moreover, after treatment with 1.0/mg/kg, s.c., for 21 days, PNA5 exhibits up to 10 days of sustained cognitive protective effects in our VCID/HF mice that last beyond the peptide half-life. PNA5 reversed object recognition impairment in VCID/HF mice and rescued spatial memory impairment. PNA5 activation of the Mas receptor results in a dose-dependent inhibition of ROS in human endothelial cells. Last, PNA5 treatment decreased VCID/HF-induced activation of brain microglia/macrophages and inhibited circulating tumor necrosis factor α, interleukin (IL)-7, and granulocyte cell-stimulating factor serum levels while increasing that of the anti-inflammatory cytokine IL-10. These results suggest that PNA5 is an excellent candidate and "first-in-class" therapy for treating VCID and other inflammation-related brain diseases.

A Single Dose-Escalation Study to Evaluate the Safety and Pharmacokinetics of Orally Administered Des-Aspartate Angiotensin I in Healthy Subjects.

Des-aspartate-angiotensin I (DAA-I) is an endogenous angiotensin peptide and a prototype angiotensin receptor agonist (ARA). It acts on the angiotensin AT1 receptor and antagonises the deleterious actions of angiotensin II. DAA-I attenuates animal models of human disease in which angiotensin II has been implicated, such as cardiac hypertrophy, neointima formation, arteriosclerosis, renal failure, post-infarction injuries, diabetes, viral infection, chemical-induced inflammation, heat stroke, cancer, and gamma radiation lethality. DAA-I crosses Caco-2 cells and is effective at sub-nanomolar concentrations. These two properties are responsible for its oral efficacy. A single dose-escalation study was conducted to evaluate the safety, tolerability and pharmacokinetics of orally administered DAA-I in 18 healthy subjects. DAA-I was safe and well tolerated by the subjects, who were administered either 0.08, 0.70 or 1.50 mg/kg of the compound. The heart rate and systolic and diastolic blood pressures determined at each post-dose measurement remained within the clinically acceptable range. Across all cohorts, DAA-I had no substantial effect on blood pressures compared with placebo. Electrocardiographs (ECGs) were normal, and none of the subjects complained of chest discomfort. All clinical laboratory tests obtained before and after DAA-I and placebo treatment were normal. Pharmacokinetic analysis over a 12-h period following DAA-I administration did not show any increase of its level beyond basal concentration. This is in line with studies showing that intravenously administered DAA-I is rapidly metabolized and has a short half-life. We postulate that, during its short systemic sojourn, DAA-I exerts its actions via biased agonism on the angiotensin AT1 receptor.The ClinicalTrial.gov assignment number for this study is NCT02666196.

Toxicological and toxicokinetic analysis of angiotensin (1-7) in two species.

The objectives of this study were to determine the potential systemic and local toxicity, as well as evaluate the toxicokinetic (TK) profile of angiotensin (1-7) [A(1-7)] when administered daily via subcutaneous injection for 28 days to Sprague-Dawley rats and Beagle dogs. A(1-7) is a member of the renin-angiotensin system and has undergone clinical evaluation for the treatment of chemotherapy-induced myelosuppression. In this present study, A(1-7) was given at 10 mg/(kg day) for 28 days to rats and canines. At day 27, blood was harvested to evaluate the TK parameters. On day 28, systemic toxicology was evaluated. Following A(1-7) administration for 27 days, no plasma A(1-7) accumulation was detected in canines; however, increased A(1-7) plasma concentrations were detected in rats. Despite the accumulation observed in rats, no detectable toxicity was found following A(1-7) administration for 28 days. The TK analysis of A(1-7) revealed a plasma half-life of 20-30 min in both rats and canines. The time to maximum plasma concentration was found to be 15 and 26.25 min in rats and canines, respectively. This study shows that subcutaneous administration of A(1-7) at 10 mg/(kg day) for 28 days did not lead to any detectable toxicities in either rats or canines.

Phase I and pharmacokinetic study of angiotensin-(1-7), an endogenous antiangiogenic hormone.

PURPOSE: Angiotensin-(1-7) [Ang-(1-7)] is an endogenous peptide hormone of the renin-angiotensin system with antiproliferative and antiangiogenic properties. The primary objective of this study was to establish the recommended phase II dose of Ang-(1-7) for treating patients with advanced cancer. Secondary objectives were to assess toxicities, pharmacokinetics, clinical activity, and plasma biomarkers. EXPERIMENTAL DESIGN: Patients with advanced solid tumors refractory to standard therapy were treated with escalating doses of Ang-(1-7) in cohorts of three patients. Ang-(1-7) was administered by s.c. injection once daily for 5 days on a 3-week cycle. Tumor measurements were done every two cycles and treatment was continued until disease progression or unacceptable toxicity. RESULTS: Eighteen patients were enrolled. Dose-limiting toxicities encountered at the 700 microg/kg dose included stroke (grade 4) and reversible cranial neuropathy (grade 3). Other toxicities were generally mild. One patient developed a 19% reduction in tumor measurements. Three additional patients showed clinical benefit with stabilization of disease lasting more than 3 months. On day 1, Ang-(1-7) administration led to a decrease in plasma placental growth factor (PlGF) levels in patients with clinical benefit (P = 0.04) but not in patients without clinical benefit (P = 0.25). On day 5, PlGF levels remained lower in patients with clinical benefit compared with patients without clinical benefit (P = 0.04). CONCLUSIONS: Ang-(1-7) is a first-in-class antiangiogenic drug with activity for treating cancer that is linked to reduction of plasma PlGF levels. The recommended phase II dose is 400 microg/kg for this administration schedule.

Angiotensin-(1-7): pharmacological properties and pharmacotherapeutic perspectives.

Therapeutic modulation of the renin-angiotensin system is not complete without taking into consideration the beneficial effects of angiotensin-(1-7) in cardiovascular pathology. Various pharmacological pathways are already exploited to involve this heptapeptide in therapy as both inhibitors of angiotensin-converting enzyme and angiotensin II type 1 receptor blockers increase its levels. These drugs and administered angiotensin-(1-7) elicit various common effects, and some effects of the drugs are partially mediated by angiotensin-(1-7). The pharmacodynamic profile of angiotensin-(1-7) is rather complex, and in vitro and in vivo studies demonstrated a wide palette of effects for angiotensin-(1-7), some of them potentially beneficial for cardiovascular disease. Using various animal models to study cardiovascular physiology and disease it was shown that angiotensin-(1-7) has antihypertensive, antihypertrophic, antifibrotic and antithrombotic properties, all properties that may prove beneficial in a clinical setting. We also observed a novel action of angiotensin-(1-7), namely its capacity to stimulate the proliferation of endothelial progenitor cells. Access of angiotensin-(1-7) to the clinic, however, is restricted due to its unfavorable pharmacokinetic properties. In order to benefit of the therapeutic potential of angiotensin-(1-7) it is crucial to increase its half-life, either by using more stable analogues, which are now under development, or specific delivery methods. We here review the pharmacological characteristics and therapeutic potential of angiotensin-(1-7), implementing the experimental strategies taken to exploit the pharmacological mechanism of this heptapeptide in a clinical setting, and present our contribution to this field of research.

A novel approach based on nanotechnology for investigating the chronic actions of short-lived peptides in specific sites of the brain.

This review presents a novel experimental approach for investigating the chronic actions of short-lived peptides in specific sites of the brain. This method combines the advantages of three different techniques: liposome encapsulation, site-specific microinjection and telemetry. First, liposomes can be designed to remain located at the injection site for a long period of time, where they protect encapsulated peptide from rapid degradation and act as a sustained-release system. Secondly, microinjection allows the administration of peptides in specific sites of the brain with minimal side effects. Finally, using telemetry, it is possible to register physiological parameters and their circadian variations in undisturbed free-moving animals for several days. Angiotensin-(1-7) and angiotensin II were used as peptide models, in order to validate the proposed method. Following the unilateral microinjection of the liposome-encapsulated peptides into the rostral ventrolateral medulla (RVLM) of Wistar rats, long-lasting cardiovascular actions were elicited, for several days. Importantly, new physiological actions of angiotensin-(1-7) at the RVLM were unmasked: modulation of the circadian rhythms of blood pressure and heart rate. It is felt that this method can be applied to a wide variety of short-lived bioactive peptides and should encounter numerous applications in the field of neurosciences.

Phase I/II dose escalation study of angiotensin 1-7 [A(1-7)] administered before and after chemotherapy in patients with newly diagnosed breast cancer.

PURPOSE: Multilineage cytopenias occur following myelosuppressive chemotherapy. Most hematopoietic agents differentiate along a single lineage and fail to prevent progressive cytopenias. Angiotensin 1-7 [A(1-7)] is a hematopoietic agent that stimulates the proliferation of multipotential and differentiated progenitor cells in cultured bone marrow and human cord blood. The purpose of this study was to determine the optimal biologic dose and the maximum tolerated dose of A(1-7). EXPERIMENTAL DESIGN: This study determined the safety and activity of A(1-7) following chemotherapy in patients with breast cancer. Toxicity was assessed by administering A(1-7) daily for 7 days followed by a 7-day washout prior to the first cycle of chemotherapy. Beginning 2 days after chemotherapy and continuing daily for at least 10 days, fifteen patients received five different A(1-7) doses and five patients received filgrastim as a comparator group over three cycles of chemotherapy. RESULTS: No dose-limiting toxicity was observed following A(1-7). The frequency of adverse events was slightly lower in A(1-7) than in filgrastim patients. No patient required a chemotherapy modification due to hematologic toxicity. There was an apparent differential dose-response sensitivity of the various lineages to A(1-7). At a dose of 100 microg/kg, A(1-7) reduced the frequency of grade 2-4 thrombocytopenia, anemia, and grade 3-4 lymphopenia as compared to filgrastim. CONCLUSION: These data suggest that A(1-7) may be beneficial in attenuating multilineage cytopenias following chemotherapy at a dose of 100 mug/kg per day.

Prejunctional AT(1) receptor subtype-dependent modification of neurotransmitter releases in canine isolated splenic arteries.

1. The regulation by angiotensin II (Ang II) formed locally on nerve-stimulated purinergic and adrenergic components of double-peaked vasoconstrictions in the canine splenic artery and Ang II receptor subtypes involved were investigated. 2. The perfusion of the precursor angiotensin I (Ang I, 0.1-1 nm) did not affect the vasoconstrictor responses to noradrenaline (NA, 0.03-1 nmol) and adenosine 5'-triphosphate (ATP, 0.03-1 micromol). The second component vasoconstrictor response to nerve stimulation was dose dependently potentiated by Ang I (0.1-1 nm). The first peaked constriction was slightly, but insignificantly increased. The potentiating effects of Ang I were abolished by KRH-594 (10 nm), a selective AT(1) receptor antagonist, but not by PD 123319 (1-10 nm), an AT(2) receptor antagonist. KRH-594 (10 nm) or PD 123319 (10 nm) never affected the vasoconstrictions to either NA or ATP. 3. The treatment with KRH-594 (1-10 nm) produced a greater inhibition on the second peaked response than the first one, although both of them were dose dependently inhibited. PD 123319 (1-10 nm) did not affect the vasoconstrictor responses induced by nerve stimulation. 4. Inhibition of angiotensin-converting enzyme with 10 nm enalaprilat reduced the second peaked response, having no significant inhibition on the first peaked response. A higher dose of enalaprilat (100 nm) produced a greater inhibition of the second peak than the first one. It reduced the second peak by approximately 65%, while the first peak was decreased approximately 35%. After treatment with enalaprilat, Ang I (1 nm) failed to enhance the neuronal vascular response. Enalaprilat at doses used did not affect the vasoconstrictions to either NA or ATP. 5. The present results indicate that endogenously generated Ang II may produce a more marked potentiation of adrenergic transmission than purinergic transmission via activation of prejunctional AT(1) receptors.

Multiple ion isolation applications in FT-ICR MS: exact-mass MSn internal calibration and purification/interrogation of protein-drug complexes.

Two new applications using multiple ion isolations in the cell of a Fourier transform-ion cyclotron resonance mass spectrometer equipped with an electrospray ionization source are described. A procedure that uses multiple ion isolations of an analyte and calibrants for internal calibration at each stage in a MSn experiment, under high-resolution exact-mass conditions, for structural characterization/elucidation of angiotensin I and rapamycin is illustrated. Fragment ion mass accuracies < 1.0 ppm are demonstrated and routinely achieved. Purification of a mixture is illustrated by isolating multiple charge states of a protein-drug complex from residual protein for further MSn studies to elucidate the site of covalent drug bonding using IRMPD for a mixture of epidermal growth factor receptor (EGFr) protein and EGFr-drug complex. The procedure developed for multiple ion isolations is referred to as multi-CHEF, multiple correlated harmonic excitation fields, in which tailored waveforms are used to notch out multiple mass regions of a spectrum with minimal off-resonance excitation.

Vmax. activation of pp60c-src tyrosine kinase from neuroblastoma neuro-2A.

A kinetic analysis of the tyrosine-specific protein kinase of pp60c-src from the C1300 mouse neuroblastoma cell line Neuro-2A and pp60c-src expressed in fibroblasts was carried out to determine the nature of the increased specific activity of the neuroblastoma enzyme. In immune-complex kinase assays with ATP-Mn2+ and the tyrosine-containing peptide angiotensin I as phosphoacceptor substrate, pp60c-src from the neuroblastoma cell line was characterized by a maximum velocity (Vmax.) that was 7-15-fold greater than the Vmax. of pp60c-src from fibroblasts. The neuroblastoma enzyme exhibited Km values for ATP (16 +/- 3 microM) and angiotensin I (6.8 +/- 2.6 mM) that were similar to Km values for ATP (25 +/- 3 microM) and angiotensin I (6.5 +/- 1.7 mM) of pp60c-src from fibroblasts. pp60v-src expressed in Rous-sarcoma-virus-transformed cells exhibited an ATP Km value (25 +/- 4 microM) and an angiotensin I Km value (6.6 +/- 0.5 mM) that approximated the values determined for pp60c-src in neuroblastoma cells and fibroblasts. These results indicate that the pp60c-src kinase from neuroblastoma cells has a higher turnover number than pp60c-src kinase from fibroblasts, and that the neural form of the enzyme would be expected to exhibit increased catalytic activity at the saturating concentrations of ATP that are found intracellularly.