Maximising absorption of peptide and protein drugs for oral administration has been an ongoing challenge in medicinal chemistry for many years. Poor absorption is mainly due to low membrane permeability and enzymatic degradation of peptides among the multitude of gastrointestinal barriers.
The main objectives of this thesis were:
1) to synthesise a library of peptides that were chemically modified by lipid and/or carbohydrate conjugation. These peptides were Glu-Trp (IM862), core peptide (CP: a T-cell function inhibitor), luteinising hormone-releasing hormone (LHRH) and A2 (ryanodine receptor (RyR) channel activator);
2) to examine the degree of permeability enhancement and the transport mechanisms of these peptides using monolayers of human intestinal epithelial (Caco-2) cells in vitro;
3) to develop LC-MS or LC-MS/MS analytical methods to quantify peptides
and their glycolipid conjugates;
4) to study the pharmacokinetics and to improve the oral bioavailability of Glu-Trp, CP and their lipoamino acid conjugates by dosing rats intravenously and orally;
5) to explore the influence of lipoamino acid and/or carbohydrate conjugation on the pharmacological activities of these peptides.
All peptides were synthesised using stepwise Boc or Fmoc chemistry. Conjugation of lipoamino acid and carbohydrate to LHRH and Glu-Trp peptide reduced the extent of enzymatic degradation and enhanced their permeability through the intestinal epithelium. Caco-2 results also indicated that peptides in the Glu-Trp library were all passively diffused, and the carbohydrate-coupled peptides were not actively transported by sodium-dependent glucose transporters (SGLTl). In vivo studies in rats confirmed the Glu-Trp and its lipoamino acid conjugates were not well absorbed orally.
For peptides in the CP library, all modified derivatives exhibited higher membrane permeability than CP in Caco-2 studies. The concentration of peptides in samples obtained from these assays was determined by LC/MS analyses. For quantification of CP and its lipoamino acid conjugate in plasma samples, an LC/MS-MS method was developed to study their pharmacokinetics. Intravenous pharmacokinetics in rats was described. CP was not detectable in plasma obtained from orally dosed rats whereas its lipoamino acid conjugate displayed an oral bioavailability of - 1 % . This indicated that lipoamino acid could improve absorption of peptides and increase oral bioavailability.
Lipoamino acid was conjugated to peptide A2 to examine the effects on peptide permeability, structure and the effects on Ca^"^ release from sarcoplasmic reticulum (SR). The lipid conjugation increased the apparent permeability of the peptide A2 across the intestinal
epithelium by 20-fold. Furthermore, these lipidic peptides were more effective in enhancing Ca^"^ release from isolated SR than their unconjugated counterparts. The results showed that the lipoamino acid conjugated A2 peptides can be used as high affinity probes for skeletal RyR activity.
Finally, in the course of further investigating the membrane penetration properties of CP and lipidic CP, they were discovered to possess transfection abilities in delivering oligonucleotides into cells that were similar to commercial transfection agents. This was demonstrated by their ability to complex with antisense ODN, transfected human retinal pigment epithelial cells, and to effectively decrease the production of the protein human vascular endothelial growth factor (hVEGF). These results suggested peptide CP and lipidic CP might be useful as transfection agents and in antisense therapeutics for the treatment of ocular neovascularisation.