H-HomoArg(Boc)2-OH

Aim: The aim of this study was to evaluate biodegradable cationic surfactants based on lysine. Methods: Lysine was esterified with cholesterol, oleyl alcohol and 1-decanol resulting in cholesteryl lysinate (CL), oleyl lysinate (OL) and decyl lysinate (DL). Esters were investigated regarding their log Dn-octanol/water, critical micelle concentration (CMC) and biodegradability. Hemolytic potential of CL, OL, DL and the already established hexadecyl lysinate (HL) was determined and complexes with insulin (INS) were formed by hydrophobic ion pairing (HIP). Lipophilic characteristics of ion-pairs were examined by analyzing their log Pn-butanol/water. Results: Successful synthesis of CL, OL and DL was confirmed by IR, NMR and MS. Log D analysis revealed amphiphilic properties for the esters and a CMC of 0.01 mM, 2.0 mM and 6.0 mM was found for CL, OL and DL, respectively. Biodegradability was proven, as over 99% of OL and DL were degraded by isolated enzymes within 30 min and after 3 h 97% of CL was cleaved by membrane bound enzymes. OL as well as DL displayed no hemolytic effect and for CL cytotoxicity was significantly reduced in comparison to HL. INS/CL complex exhibited highest lipophilicity. Conclusion: Cholesterol-amino acid based surfactants seem to be promising agents for HIP.

Working hypothesis: It was the hypothesis of this study that esters of arginine (Arg) with medium and long chain aliphatic alcohols are biodegradable and less cytotoxic than well-established cationic surfactants being used for hydrophobic ion pairing (HIP) with hydrophilic macromolecular drugs. Experiments: Arg was linked to nonan-1-ol and hexadecan-1-ol (C9 and C16) via an ester linkage. The newly formed Arg-nonyl ester (ANE) and Arg-hexadecanoyl ester (AHE) surfactants were evaluated regarding critical micelle concentration (CMC) using pyrene fluorescent method, cytotoxicity on human colorectal adenocarcinoma-derived cells (Caco-2) and biodegradability at the concentrations of 2.5 and 5 mg/mL using 2500 Nα-benzoyl-l-arginine ethyl ester hydrochloride (BAEE) units/mL of trypsin. Furthermore, in order to evaluate their potential for HIP, heparin and daptomycin were used as model polysaccharide and peptide drugs, respectively. Findings: Chemical structures of ANE and AHE surfactants were confirmed by FTIR, 1H NMR, and LC-MS. CMC of ANE was 7.5 mM and CMC of AHE was 2 mM. Arg-surfactants were not cytotoxic below their CMC. At CMC and above CMC, ANE was significantly (P < 0.05) more cytotoxic than AHE. ANE in both concentrations was degraded ˃98% within 48 h. The degradation of AHE at lower concentration was ˃97% and about 50% at higher concentration. Arg-surfactants were able to efficiently precipitate heparin and daptomycin from corresponding aqueous solutions. Conclusion: Arg-surfactants being biodegradable and less toxic seems to be a promising alternative to well-established cationic surfactants for the formation of hydrophobic ion pairs (HIPs) with hydrophilic macromolecular drugs.

Hydrophobic ion pairing is a promising strategy to raise the lipophilic character of therapeutic peptides and proteins. In past studies, docusate, an all-purpose surfactant with a dialkyl sulfosuccinate structure, showed highest potential as hydrophobic counterion. Being originally not purposed for hydrophobic ion pairing, it is likely still far away from the perfect counterion. Thus, within this study, docusate analogues with various linear and branched alkyl residues were synthesized to derive systematic insights into which hydrophobic tail is most advantageous for hydrophobic ion pairing, as well as to identify lead counterions that form complexes with superior hydrophobicity. The successful synthesis of the target compounds was confirmed by FT-IR, 1H-NMR, and 13C-NMR. In a screening with the model protein hemoglobin, monostearyl sulfosuccinate, dioleyl sulfosuccinate, and bis(isotridecyl) sulfosuccinate were identified as lead counterions. Their potential was further evaluated with the peptides and proteins vancomycin, insulin, and horseradish peroxidase. Dioleyl sulfosuccinate and bis(isotridecyl) sulfosuccinate significantly increased the hydrophobicity of the tested peptides and proteins determined as logP or lipophilicity determined as solubility in 1-octanol, respectively, in comparison to the gold standard docusate. Dioleyl sulfosuccinate provided an up to 8.3-fold higher partition coefficient and up to 26.5-fold higher solubility in 1-octanol than docusate, whereas bis(isotridecyl) sulfosuccinate resulted in an up to 6.7-fold improvement in the partition coefficient and up to 44.0-fold higher solubility in 1-octanol. The conjugation of highly lipophilic alkyl tails to the polar sulfosuccinate head group allows the design of promising counterions for hydrophobic ion pairing. STATEMENT OF SIGNIFICANCE: Hydrophobic ion pairing enables efficient incorporation of hydrophilic molecules into lipid-based formulations by forming complexes with hydrophobic counterions. Docusate, a sulfosuccinate with two branched alkyl tails, has shown highest potential as anionic hydrophobic counterion. As it was originally not purposed for hydrophobic ion pairing, its structure is likely still far away from the perfect counterion. To improve its properties, analogues of docusate with various alkyl tails were synthesized in the present study. The investigation of different alkyl residues allowed to derive systematic insights into which tail structures are most favorable for hydrophobic ion pairing. Moreover, the lead counterions dioleyl sulfosuccinate and bis(isotridecyl) sulfosuccinate bearing highly lipophilic alkyl tails provided a significant improvement in the hydrophobicity of the resulting complexes.