The identification and chromatographic separation of a new highly analogous impurity of the active pharmaceutical ingredient icatibant.

Icatibant is a peptidomimetic drug serving as a bradykinin-receptor antagonist and is approved in Europe and the United States for the treatment of hereditary angioedema attacks. We have detected an impurity with a high structural similarity to icatibant in pharmaceutical dosage forms using an optimized chromatographic method based on reversed phase high performance liquid chromatography with UV detection. The abundance of the impurity was around 1% relative to the icatibant peak following storage at room temperature for 1 month, and raised up to ~16% upon temperature stressing at 100 °C. The impurity was isolated by fraction collection and further purified by solid phase extraction for structural identification. NMR and high resolution mass spectrometric analyses revealed that this impurity results from isomerization in the N-terminal single amino acid residue. The new impurity may warrant particular attention due to its exceptional similarity to the active ingredient icatibant.

Novel, efficient, facile, and comprehensive protocol for post-column amino acid analysis of icatibant acetate containing natural and unnatural amino acids using the QbD approach.

Qualitative and quantitative determination of amino acid composition using amino acid analysis (AAA) is an important quality attribute and considered an identity of therapeutic peptide drugs by the regulatory agencies. Although huge literature is available on pre- and post- column derivatization AAA methods, arriving at an appropriate hydrolysis protocol coupled with adequate separation of the derivatized/underivatized amino acids is always challenging. Towards achieving a facile and comprehensive protocol for AAA, the present work is geared towards developing a deeper understanding of the extent of hydrolysis of peptide, and the nature and stability of amino acids present in the peptide backbone. This defines the suitability of the method in meeting the end goals and the regulatory requirement. Analysis of historical data generated during the method optimization of AAA for icatibant acetate (ICT) using head space oven hydrolysis (HSOH) and microwave-assisted hydrolysis (MAH) methods helped in arriving at fast (< 1 h) and efficient hydrolysis (0.9-1.1 of theoretical residue) conditions. Better separations for the natural and unnatural amino acids were achieved using 3.45 ≤ pH ≤ 10.85, and a column oven gradient program. This approach was useful in meeting the method quality attributes [resolution (Rs) > 2.0; plate count (N) > 5600; and USP tailing factor < 1.2] with a target analytical method profile of relative amino acid mole ratios (RAAMR) in the range of 0.9-1.1 for Ser, Oic, Tic, Hyp, Ala (Thi), Gly and Pro, and between 2.7 and 3.3 for Arg. The developed method was validated as per the ICH guidelines and is precise, accurate, linear and robust.

New insights into the stereochemical requirements of the bradykinin B2 receptor antagonists binding.

Bradykinin (BK) is a member of the kinin family, released in response to inflammation, trauma, burns, shock, allergy and some cardiovascular diseases, provoking vasodilatation and increased vascular permeability among other effects. Their actions are mediated through at least two G-protein coupled receptors, B1 a receptor up-regulated during inflammation episodes or tissue trauma and B2 that is constitutively expressed in a variety of cell types. The goal of the present work is to carry out a structure-activity study of BK B2 antagonism, taking into account the stereochemical features of diverse non-peptide antagonists and the way these features translate into ligand anchoring points to complementary regions of the receptor, through the analysis of the respective ligand-receptor complex. For this purpose an atomistic model of the BK B2 receptor was built by homology modeling and subsequently refined embedded in a lipid bilayer by means of a 600 ns molecular dynamics trajectory. The average structure from the last hundred nanoseconds of the molecular dynamics trajectory was energy minimized and used as model of the receptor for docking studies. For this purpose, a set of compounds with antagonistic profile, covering maximal diversity were selected from the literature. Specifically, the set of compounds include Fasitibant, FR173657, Anatibant, WIN64338, Bradyzide, CHEMBL442294, and JSM10292. Molecules were docked into the BK B2 receptor model and the corresponding complexes analyzed to understand ligand-receptor interactions. The outcome of this study is summarized in a 3D pharmacophore that explains the observed structure-activity results and provides insight into the design of novel molecules with antagonistic profile. To prove the validity of the pharmacophore hypothesized a virtual screening process was also carried out. The pharmacophore was used as query to identify new hits using diverse databases of molecules. The results of this study revealed a set of new hits with structures not connected to the molecules used for pharmacophore development. A few of these structures were purchased and tested. The results of the binding studies show about a 33% success rate with a correlation between the number of pharmacophore points fulfilled and their antagonistic potency. Some of these structures are disclosed in the present work.

Design, synthesis and biological evaluation of multifunctional ligands targeting opioid and bradykinin 2 receptors.

We report here the design and synthesis of novel multifunctional ligands that act as (µ/δ) opioid agonists and bradykinin 2 receptor antagonists. These multifunctional ligands were designed to interact with the multiple receptors to show an enhanced analgesic effect, with no opioid-induced tolerance. We designed our multifunctional ligands based on the well-known second generation bradykinin 2 receptor antagonist Hoe 140 (DArg-Arg-Pro-Hyp-Gly-Thi-Ser-DTic-Oic-Arg-OH) and the opioid enkephalin analogues Tyr-DAla-Phe, Tyr-DAla-Gly-Phe and Tyr-Pro-Phe. We explored the conjugation of opioid pharmacophore to the Hoe 140 (DArg-Arg-Pro-Hyp-Gly-Thi-Ser-DTic-Oic-Arg-OH) in various positions with and without a linker. These bifunctional ligands showed very good binding affinity towards the both µ and δ opioid receptors. Among these bifunctional ligands 8, 11 and 12 showed excellent and balanced binding affinity at both µ and δ opioid receptors (0.5 nM, 2.0 nM; 0.3 nM, 2 nM; 2 nM and 3 nM), respectively. On the other hand these bifunctional ligands showed very weak and no binding affinity for rat brain bradykinin 2 receptors. Similarly, the Hoe 140 showed very low affinity (>10,000 nM and 9,000 nM) against [(3)H] BK binding in rat brain membranes and in HEK293 cells, respectively. In contrast, the Hoe 140 showed very good binding affinity in guinea pig ileum (0.43 nM) similar to that of previously reported. The bradykinin 2 receptors are known to be present in rat brain membrane, guinea pig ileum (GPI) and rabbit jugular vein. Previously the binding affinity of Hoe 140 for bradykinin 2 receptor was reported using guinea pig ileum. The above results suggest that the bradykinin 2 receptors present in rat brain membrane are a different sub type than the bradykinin 2 receptor present in guinea pig ileum (GPI).