Category: Preclinical Development
Purpose: The renin-angiotensin (Ang) system (RAS) is one of the major regulators of human physiology and specifically it has a central role in renal and cardiovascular homeostasis. Its systemic actions include the regulation of blood pressure, fluid, and electrolyte balance.1 Constitutively, the RAS consists of two opposing arms. The first arm is constituted of the angiotensin converting enzyme (AEC)/Ang II peptide/angiotensin type 1 receptor, which is responsible for physiologic and pathologic effects.2 The second arm is protective and composed of the ACE2/Ang 1-7/Mas receptor, which results in anti-inflammatory, antifibrotic, anti-proliferative, and vasodilatory effects.3 The activation of the RAS plays an important role in the pathophysiology of various inflammatory diseases. The RAS has been studied as a target for therapeutic intervention in various pathological conditions. Prior to the discovery of ACE2, all interventional approaches for targeting the RAS were focused on ACE and AngII, such as ACE inhibition or AngII receptor blocking. However, the importance of the ACE2/Ang 1-7/Mas receptor arm in the regulation of the RAS has become evident from the results of recent studies.3,4 As one of the RAS active peptides, Ang1-7 exerts beneficial and protective effects through different mechanisms. Due to these biological actions, Ang1-7 serves as a basis for the development of new therapeutic agent for the treatment of numerous diseases.5 However, the peptide’s therapeutic potential is severely hampered by the short half-life and low bioavailability due to rapid degradation by peptidases. Several approaches have been taken for addressing this issue such as cyclization,6 substitution with non-natural amino acid and complexation with β-cyclodexterin7 which provided with some level of progress. In a novel approach by using a bone targeting moiety attached to Ang peptide through a spacer, we aimed at improvement of its stability and prolongation of circulation half-life. At the same time by targeting it to the bone, we propose to utilize the skeletal bone of the body as a reservoir for extended delivery of active Ang 1-7 peptide.
Methods: The bone seeking bisphosphonate (BP)-mediated conjugates have be prepared by PEGylating and conjugation of Ang 1-7 peptide with a spacer and bone targeting moiety (BP) using Solid Phase Peptide Synthesis. After confirmation of the conjugate formation using MOLDI –ToF mass spectrophotometer, the resulting conjugate (Ang 1-7 conjugate) was separated using an analytical revers-phase HPLC and eluted major peak attributed to the conjugate was collected. Stability of the compound in different mediums and storage conditions has been tested. The bone binding capacity was tested by Surface Plasmon Resonance using a custom coated hydroxyapatite chips. Biodistribution and pharmacokinetic (PK) studies of I-125- Ang 1-7 conjugate and native peptide were done by intravenous (i.v.) and subcutaneous (s.c.) routes administration into healthy rats.
Results: Ang 1-7 Conjugate was synthesized successfully, isolated by HPLC and confirmed by MALDI-TOF. (Fig.1). The representative HPLC-UV chromatogram of Ang1-7 Conjugate shows a single peak with purity more than 90% purity (Fig.2). Bone mineral affinity test showed Ang 1-7 conjugate exhibited significantly higher bone mineral affinity over Ang 1-7. The Ang 1-7 conjugate remained stable for at least 2 months in solution and 1 month in plasma at -20 °C. Biodistribution and PK studies showed significantly increased bone retention and prolonged plasma half-life for Ang1-7 conjugate compared to the Ang 1-7. The tissue distribution of peptides was conducted on bone heart, liver, kidney, lung, etc. The conjugate demonstrated higher affinity to bone, resulted in sustained plasma concentration and longer half-life when compared with native peptides (Fig.3).
Conclusion: These results support that the conjugation of Ang 1-7 with BP was able to target the bone and utilize it as reservoir for sustaining therapeutic plasma level of the active peptide. Higher stability in solution and 10-fold prolongation of plasma half-life of Ang 1-7 conjugate after i.v. administration indicates the effectiveness of our approach for improvement of peptide chemical and biological stability. 15-fold increase in half-life after s.c. injection support the notion of bone targeting and loading of conjugate on the bone which results in longer mean resistance time compared with native peptide. Overall, these results emphasize on the potential applicability of Ang 1-7 conjugate in different diseases which activated RAS. Through administration of Ang1-7 conjugate, the plasma and tissue concentration of active peptide Ang 1-7 increases which in turn by acting on Mas receptor it can restore the altered balance between protective and harmful arms of the activated RAS. This approach has additional benefit for any disease condition which involves bone such as arthritis, bone cancers or any cancer with bone metastasis.
Protected under provisional patent application.