Improved pharmacokinetics and bone tissue accumulation of Angiotensin-(1-7) peptide through bisphosphonate conjugation.

The renin-angiotensin system (RAS) has a central role in renal and cardiovascular homeostasis. Angiotensin-(1-7) (Ang1-7), one of the RAS active peptides, exerts beneficial effects through different mechanisms. These biological actions suggest that Ang1-7 is an effective therapeutic agent for treating various diseases associated with activated RAS. However, its short half-life and poor pharmacokinetics restrict its therapeutic utility. Our laboratory has successfully synthesized and characterized an Ang1-7 conjugate (Ang Conj.) with a prolonged half-life and improved pharmacokinetics profile. The Ang Conj. has been prepared by PEGylation of Ang1-7 and conjugation with a bisphosphonate using solid-phase peptide synthesis and characterized by HPLC and mass spectrometer. The compound's stability has been tested in different storage conditions. The bone binding capacity was evaluated using a hydroxyapatite assay. Pharmacokinetic and tissue distribution studies were performed using iodinated peptides in rats. Ang Conj. was synthesized with > 90% purity. Bone mineral affinity testing showed Ang Conj. exhibited significantly higher bone mineral affinity than Ang1-7. The Ang Conj. remained stable for more than a month using all tested storage conditions. The Ang Conj. demonstrated higher affinity to bone, a longer half-life, and better bioavailability when compared with the native peptide. These results support that conjugation of Ang1-7 with bisphosphonate enables it to utilize bone as a reservoir for the sustained delivery of Ang1-7 to maintain therapeutic plasma levels. High chemical stability and about five to tenfold prolongation of Ang Conj. plasma half-life after administrations into rats proves the effectiveness of our approach.

Stabilization of Angiotensin-(1-7) by key substitution with a cyclic non-natural amino acid.

Angiotensin-(1-7) [Ang-(1-7)], a heptapeptide hormone of the renin-angiotensin-aldosterone system, is a promising candidate as a treatment for cancer that reflects its anti-proliferative and anti-angiogenic properties. However, the peptide's therapeutic potential is limited by the short half-life and low bioavailability resulting from rapid enzymatic metabolism by peptidases including angiotensin-converting enzyme (ACE) and dipeptidyl peptidase 3 (DPP 3). We report the facile assembly of three novel Ang-(1-7) analogues by solid-phase peptide synthesis which incorporates the cyclic non-natural δ-amino acid ACCA. The analogues containing the ACCA substitution at the site of ACE cleavage exhibit complete resistance to human ACE, while substitution at the DDP 3 cleavage site provided stability against DPP 3 hydrolysis. Furthermore, the analogues retain the anti-proliferative properties of Ang-(1-7) against the 4T1 and HT-1080 cancer cell lines. These results suggest that ACCA-substituted Ang-(1-7) analogues which show resistance against proteolytic degradation by peptidases known to hydrolyze the native heptapeptide may be novel therapeutics in the treatment of cancer.

Angiotensin II analogues. 13. Role of the hydroxyl group of position 4 tyrosine in pressor activity.

In order to determine the features of the phenolic ring in position 4 of [Asn1,Ile5]angiotensin II that contribute to pressor activity, analogues with selected aromatic substituents were synthesized by the solid-phase method. They showed pressor activities in the rat: [Asn1,Phe(4-NH2)4]AII, 24%; [Asn1,Phe(4-NO2)4AII, 0.1%; [Asn1,Tyr(3,5-Me2)4]AII, 2.2%; [Asn1,D-Tyr(3,5-Me2)4]AII, 1.4%. These results indicate that the activity contributed by the aromatic character of the phenyl ring in the side chain of position 4 is enhanced by a group in the para position that may function as a proton donor in hydrogen-bond formation. Bulky substituents ortho to this hydrogen-bonding group decrease activity by steric interference with hydrogen-bond formation. Bulky groups than cannot act as hydrogen donors in the para position of the aromatic ring drastically decrease the activating effect of the aromatic ring on pressor activity.

Synthesis and pharmacology of a prohormone angiotensin antagonist.

A prohormone angiotensin antagonist, Sarcosyl1-Alanyl8-angiotensin I (Sar1-Ala8-A-I) was synthesized and its pharmacological properties were evaluated in three biological systems. It was found to be a good inhibitor in vivo in the rat blood pressure assay, somewhat less active in guinea pig ileum and a relatively weak antagonist in rat uterus. In vivo the inhibitory effect was greatly attenuated by the presence of the converting enzyme inhibitor BPP5alpha.

Organ selective angiotensin antagonists: sarcosyl1-cysteinyl(S-methyl)8-angiotensin I.

An angiotensin antagonist, Sarcosyl1-Cysteinyl(S-Methyl)8-angiotensin I [Sar1-Cys(Me)8-ANG I] was synthesized and its pharmacological properties evaluated in vivo (rat blood pressure assay) and in vitro (rabbit aortic strips, guinea-pig ileum and rat uterus assays). It was found to be an extremely potent angiotensin II (ANG II) antagonist in the rat pressor assay (dose ratio for ANG II of 1300 during infusion of 5.0 micrograms/kg/min Sar1-Cys(Me)8-ANG I) and a moderately effective antagonist in guinea-pig ileum (pA2 congruent to 8.2). Moderate antagonism was also seen in the rabbit aortic strip preparation (pA2 congruent to 8.1) while the analog was inactive in the rat uterus assay. In each of the preparations where antagonist activity was observed there was evidence of non-competitive antagonism. Most striking was the inability of extremely high doses ( up to 125 micrograms ANG II/kg) of ANG II to overcome the Sar1-Cys(Me)8-ANG I blockade. In both the rat pressor and guinea-pig ileum assays the Sar1-Cys(Me)8-ANG I antagonism is completely abolished in the presence of the converting enzyme inhibitor SQ14225 (Captopril-Squibb). Organ selectivity of this analog is discussed in terms of the inherent activity of the active principle (i.e. the Sar1-Cys(Me)8-angiotensin II [Sar1-Cys(Me)8-ANG II] released by the action of converting enzyme) and the availability of converting enzyme in each bioassay.

Angiotensin-(1-7): a peptide hormone with anti-cancer activity.

The development of peptides as therapeutic agents has progressed such that these small molecules of less than fifty amino acids are currently in use for the treatment of a variety of pathologies. This review focuses on the pre-clinical studies and clinical trials assessing the anti-cancer properties of angiotensin-(1-7) [Ang-(1-7)], an endogenous heptapeptide hormone of the renin-angiotensin system. Ang-(1-7) mediates biological responses by activating mas, a unique G protein- coupled receptor, thereby providing specific targeted actions when used as a therapeutic agent. Studies in in vitro as well as in vivo mouse models demonstrated that the heptapeptide hormone reduced proliferation of human cancer cells and xenograft tumors. This attenuation was concomitant with decreased angiogenesis, cancer associated fibrosis, osteoclastogenesis, tumor-induced inflammation and metastasis as well as altered regulation of growth promoting cellular signaling pathways. In three clinical trials, Ang-(1-7) was well tolerated with limited toxic or quality-of-life side effects and showed clinical benefit in cancer patients with solid tumors. Taken together, these studies suggest that Ang-(1-7) may serve as a first-in-class peptide chemotherapeutic agent, reducing cancer growth and metastases by pleiotrophic mechanisms as well as targeting the tumor microenvironment.

Synthesis of [beta-(4-pyridyl-1-oxide)-L-alanine4]-angiotensin I as a potential suicide substrate for protein-tyrosine kinases.

An asymmetric synthesis of [beta-(4-pyridyl-1-oxide)-L-alanine4]-angiotensin I (1a), which is a potential suicide substrate (mechanism-based inhibitor) for protein-tyrosine kinases, has been performed. Deprotonation of 6 with n-butyllithium in THF gave the anion 7, which was alkylated with 4-(chloromethyl)pyridine-1-oxide to afford intermediate 9 as a crystalline solid. Hydrolysis of 9 afforded a mixture of 11 and 12 in a ratio of 96:4 as estimated by conversion to the diastereomeric dipeptides 13 and 14 followed by HPLC analysis. The 96:4 mixture of 11 and 12 was used in the solid phase synthesis of the target angiotensin analog 1a and its diastereomer 1b, which were separated and tested for inhibitory activity against two thymocyte protein-tyrosine kinases: p40 and p56lck. Neither peptide displayed significant inhibitory activity toward p40 and both served as weak competitive inhibitors of p56lck.

Inhibition of angiotensin-converting enzyme by angiotensin I analogue peptide inhibitors. A kinetic study.

Angiotensin I analogues with a phosphonic acid group replacing the C-terminal carboxyl group were shown to be competitive inhibitors of angiotensin-converting enzyme. This new class of inhibitors was used to study the binding requirements of the angiotensin I-like ligands to the enzyme's active site. These studies indicate that angiotensin-converting enzyme recognizes at least five amino acid residues at the C-terminus of the peptide. The effect of pH on the binding of the most potent inhibitor peptide was compared to Captopril. The two inhibitors showed similar Ki-pH profiles despite their structural differences. Chloride enhanced the binding of the peptide inhibitor at both pH 9.0 and pH 6.5. At pH 9.0 the inhibitor peptide and the anion bind randomly to the enzyme, while at pH 6.5 the mechanism is ordered. In the latter case, the anion binds first to the enzyme.

Angiotensin-(1-7). One step forward?

Angiotensin-(1-7) [Ang-(1-7)] is a paracrine hormone of the renin-angiotensin system (RAS). It counterbalances the negative actions of angiotensin II (Ang II) acting in the cardiovascular system, kidneys and central nervous system, and is responsible for blood pressure regulation and antiproliferative effects. Current data strongly suggest the existence of a specific receptor for this peptide. The concentration of Ang-(1-7) increases significantly during the administration of RAS blockers. One may suggest the involvement of this peptide in a beneficial effect of these drugs.