| Summary: | Small Cell Lung Cancer (SCLC) is an aggressive form of cancer accounting for 25% of lung cancer deaths worldwide. Treatment relies on combination chemotherapy (etoposide and cisplatin or carboplatin) with or without radiation therapy. However, disease relapse and resistance occurs quickly, prompting unmet need for alternative treatment options. One such option is the use of broad-spectrum antagonists, known as Substance P (SP) analogues. Historically, these analogues have not succeeded clinically due to low potency and bioavailability. In this project, novel SP analogues were developed to address these shortfalls. A chemical strategy was designed to synthesise novel short peptides including DMePhe-DTrp-Phe-DTrp-Leu-NH2 (25) as the new lead. Fmoc and Boc D-Trp derivatives with indole nitrogen having substituents (methyl, ethyl, propyl, butyl, pentyl, propargyl, benzyl and tert-prenyl) were made and characterised by 1H and 13C NMR spectroscopy and mass spectrometry (MS). These building blocks were incorporated into the first series of peptides, substituting the D-Trp residue located near the C-terminal of 25, via solid and/or liquid phase procedures. Final products were purified by RP-HPLC to >90% purity and structures verified by MS and/or 1H NMR. Cell viability assays were conducted to evaluate cytotoxicity against two SCLC cell lines: H69 (chemo-naive) and DMS79 (from a patient after treatment). The IC50 values for the D-Trp residue modified peptides were < 5 μM. One of the earliest candidates to emerge from this work was DMePhe-DTrp-Phe-DTrp(N-tert-prenyl)-Leu-NH2 (33). Subsequently, the most potent peptide was the one bearing D-Trp(N-butyl) (29) with IC50 values of 1.0 μM (H69) and 1.4 μM (DMS79), compared to the lead 25 with IC50 values of 30.7 μM (H69) and 23.0 μM (DMS79). A second series of peptides were produced to optimise 29 by incorporating a D-Trp(N-butyl) residue. The study focused on peptides by (a) modifying the N-terminal D-Trp residue, (b) modifying both D-Trp residues, (c) changing the C-terminal amide to free carboxylic acid, and (d) adding a charged amino acid (arginine) or removing a hydrophobic amino acid (leucine) to additionally aid in solubility. The most potent candidate was found to bear dual D-Trp(N-butyl) residues (35) with IC50 value of 0.6 μM (H69) and 2.3 μM (DMS79). Peptides 29 and 35 were at least 26 times more potent than SP antagonist G (SPG, previously subjected to a Phase I clinical trial), as revealed by in vitro screening in this project. Both sequences induced apoptosis as evident from fluorescence staining. Flow cytometric analysis of 29 with the DMS79 cell line showed that the level of late apoptotic cells rose from 36% at 2 μM to 96% at 6 μM, compared to 25 that exhibited no effect. Efficacy of peptide 33 was separately evaluated in vivo using DMS79 xenografts. A low dose (1.5 mg/kg) was found to reduce tumour growth by ~ 30% (p < 0.05) at day 7, relative to the control group. Higher doses could not be used due to limited aqueous solubility. Furthermore, these peptides were shown to have improved stability. Exposed to neat mouse plasma for 48 hours, 29 and 35 remained intact by 68.5% and 81.0%, respectively, compared to 59.0% for 25 and 35.9% for 33. Complete metabolic stability of 29 and 35 was observed after 3 hours incubation in mouse S9 liver fraction. Aqueous solubility issues were overcome in feasibility studies incorporating 29 into liposomes for future in vivo efficacy testing. Finally, due to the high potency and stability of 29, a liposomal formulation of it may have a profound effect in in vivo efficacy studies against chemo-resistant SCLC.
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