Application of biomimetic HPLC to estimate lipophilicity, protein and phospholipid binding of potential peptide therapeutics

Abstract

<jats:p>&lt;p class="ADMETabstracttext"&gt;Peptide therapeutics are new modalities offering several challenges to drug discovery. They are generally less stable and permeable in vivo. The characterization of their lipophilicity cannot be carried out using the traditional in silico or wet octanol/water partition coefficients. The prediction of their in vivo distribution and permeability is also challenging. In this paper, it is demonstrated that the biomimetic properties such as lipophilicity, protein and phospholipid binding can be easily assessed by HPLC using chemically bonded protein and immobilized artificial membrane (IAM) stationary phases. The obtained properties for a set of potential therapeutic peptides with 3 to 33 amino acids have been analysed and it was found that similar characteristics of the properties could be observed as for small molecule drugs. The albumin binding showed correlation with their measured lipophilicity on the C-18 stationary phase with acidic peptides showing stronger than expected albumin binding. The (IAM) chromatography revealed peptide membrane affinity, which was stronger for positively charged peptides (containing arginine) and showed correlation to the alpha-1-acid glycoprotein (AGP) binding, which was also stronger for positively charged compounds. The in vivo volume of distribution and drug efficiency of the peptides have been estimated using the models developed for small molecules. One of the candidate linear peptides has been assessed in various cellular and in vivo assays and the results have confirmed the estimated cell partition and brain to plasma ratio. It can be demonstrated, that up to 21 amino acids, the peaks of the peptides obtained on the protein phase were symmetrical and narrow. The interaction of larger peptides with the protein stationary phases resulted in wide peaks showing multiple equilibrium processes with slow kinetics during chromatography. The larger peptides showed narrow and symmetrical peaks on the IAM column enabling the quantification of peptide - cell membrane interactions.&lt;/p&gt;</jats:p>

In vitro biomimetic HPLC and in vivo characterisation of GM6, an endogenous regulator peptide drug candidate for amyotrophic lateral sclerosis

“…The models developed for small molecules [13] have been applied to estimate the in vivo PK profile of GM6 with special emphasis on the expected free tissue concentration. GM6 has been found to have good drug-like properties with an estimated good cellular accumulation and tissue partition as has been described previously [14].…”

“…In order to be able to estimate the in vivo distribution, cell penetration, cell membrane affinity and plasma protein binding biomimetic HPLC characterization of GM6 were carried out using the methods described earlier [13,14,19]. The in vivo distribution behaviour of GM6 has been estimated on C-18, IAM, HSA and AGP stationary phases based on the measured generic gradient retention times described above.…”

Membrane Active Peptides and Their Biophysical Characterization

“…Biomimetic high-performance liquid chromatography (HPLC) offers an alternative method for estimating in vivo peptide stability, peptide lipophilicity, and peptide affinity for the phospholipid membranes. It is based on measuring biomimetic properties using chemically bonded protein and immobilized artificial membranes as stationary phases [ 274 ].…”

GM604 regulates developmental neurogenesis pathways and the expression of genes associated with amyotrophic lateral sclerosis

“…The peptide drug GM6 is a 6 amino acid analog of the MNTF1 active site that is able to cross the blood-brain barrier [ 9 ] and appears to retain functional activity of the full-length protein [ 16 ]. GM6 was shown to have good drug-like properties based upon pharmacokinetic profiling, with an estimated volume of distribution of 7.8 L/kg and minimum effective concentration of 1.6 mg/kg in humans [ 10 , 17 ]. Although the half-life of GM6 in human blood appears short (15 min), the drug partitions readily into tissues where the half-life was estimated to be 5.8 h, with an expected brain to plasma concentration ratio of 1.65 [ 10 ].…”