Tumor cell invasion, a key step in the metastatic process, involves a complex series of correlated macromolecular interactions. These include interaction with, and movement through, collagen, most often type I and/or basement membrane (type IV) collagen. In general, invasion of the basement membrane is believed to be a critical step in the metastatic process. Human melanoma cells have been shown to bind distinct triple-helical regions within type IV collagen (1-5). Melanoma receptors for triple-helical collagen fall into one of two categories: members of the integrin heterodimeric protein family (␣ 1 ␤ 1 , ␣ 2 ␤ 1 , and ␣ 3 ␤ 1 integrins) or cell surface proteoglycans (such as CD44 and melanoma-associated proteoglycan/melanoma chondroitin sulfate proteoglycan (MPG/MCSP/NG2)).

Numerous approaches have been described for modifying biomaterials to incorporate extracellular matrix components. "Peptide-amphiphiles", whereby monoalkyl hydrocarbon chains are covalently linked to peptide sequences, have been shown previously to (a) form specific molecular architecture with enhanced stability and (b) promote cell adhesion, spreading, and signaling. The present study has examined the use of chimeric peptide-amphiphiles for inducing protein-like structures and peptide-amphiphile mixtures for enhancing surface bioactivity. The alpha-helical propensity of a 21 residue peptide, incorporating the SPARC(119-122) angiogenesis-inducing sequence and either unmodified or acylated with a C(6), C(10), C(14), C(16), C(18), C(18:1), or C(18:1-OH) monoalkyl hydrocarbon chain, has been examined. Peptide and peptide-amphiphile structures were characterized by circular dichroism and one- and two-dimensional NMR spectroscopic techniques. The 21 residue peptide alone does not form a distinct structure in solution, whereas N-terminal acylation by monoalkyl hydrocarbon chains results in the 21 residue peptide-amphiphile adopting a predominantly alpha-helical structure in solution. The thermal stability of the alpha-helix increases with increasing hydrocarbon chain length. The SPARC(119-122) peptide-amphiphiles were then screened for promotion of endothelial cell adhesion and spreading. The greatest activity was achieved by using a mixture of the alpha-helical SPARC(119-122) peptide-amphiphile, a triple-helical peptide-amphiphile incorporating the alpha2beta1 integrin binding site from type I collagen, and a pseudolipid. The pseudolipid is most likely required for a spatial distribution of the peptide-amphiphiles that allows for optimal cellular interactions. Overall, we have found that incorporation of bioactive sequences within peptide-amphiphiles results in the induction of an ordered structure of the bioactive sequence and that mixtures of peptide-amphiphiles can be used to promote endothelial cell behaviors comparable to extracellular matrix components.

Collagen is a multifunctional protein, serving as a structural scaffold and a modulator of cellular responses. Prior work has identified distinct regions from several collagen types that promote cell adhesion, spreading, migration, and signal transduction. One of these regions, alpha1(IV)1263-1277 from type IV collagen, mediates these responses via melanoma cell CD44-chondrotin sulfate proteoglycan receptors. In the study presented here, we have used a triple-helical model of alpha1(IV)1263-1277 to evaluate (a) conformational stability and (b) cellular responses based on single-site incorporation of trans-4-fluoro-L-proline (trans-Flp) or cis-4-fluoro-L-proline (cis-Flp) for trans-4-hydroxy-L-proline (trans-Hyp). The structural effects of cis-Flp and trans-Flp substitution were studied by circular dichroism and NMR spectroscopies. The peptide containing a single trans-Flp instead of trans-Hyp was slightly more thermally stable than the parent peptide (T(m) = 37 vs 34 degrees C), while the peptide containing cis-Flp was considerably less stable than the parent peptide (T(m) = 30 degrees C). Melanoma cell adhesion and spreading were examined under conditions where the trans-Hyp-, trans-Flp-, and cis-Flp-containing ligands were approximately 15, <10, and approximately 65% denatured, respectively. Adhesion to each of the three ligands was remarkably sensitive to the respective ligand conformation, with EC(50) values of approximately 2.5, approximately 0.35, and >5.0 microM for the trans-Hyp-, trans-Flp-, and cis-Flp-containing ligands, respectively. Melanoma cell spreading was quantitated over a ligand concentration range of 0.01-50 microM and, in a fashion similar to adhesion, was more extensive on the trans-Flp ligand than on the trans-Hyp ligand. Very low levels of spreading were observed with the cis-Flp-containing ligand at all concentrations tested. Melanoma cell adhesion to and spreading on the three ligands suggested the dramatic biological consequence of even subtle changes in relative triple-helical content. Such subtle changes may model those occurring in the basement membrane during the tumor cell invasion process, and thus provide mechanistic insight into this stage of metastasis.

Background-The objective of this study was to address the feasibility and the biological activity of orally administered human brain natriuretic peptide (hBNP). Proprietary technology has been developed in which short, amphiphilic oligomers are covalently attached to peptides. The conjugated peptides are intended to have an improved pharmacokinetic profile and to enable oral administration. We hypothesized that novel oral conjugated hBNP (CONJ-hBNP) increases plasma hBNP, activates cGMP, and reduces mean arterial pressure (MAP). Methods and Results-This randomized crossover-designed study tested the biological activity of oral CONJ-hBNP compared with oral native hBNP in normal conscious dogs. Measurements of MAP, plasma hBNP, and cGMP were made at baseline (BL) and repeated at 10, 30, 60, 120, 180, and 240 minutes after oral administration. Plasma hBNP was not detectable in dogs at BL. Plasma hBNP was detected after native hBNP and CONJ-HBNP administration. However, plasma hBNP concentration was significantly higher after CONJ-hBNP than after native hBNP administration (Pϭ0.0374 between groups). Plasma cGMP increased after CONJ-hBNP for 60 minutes (from 10.8Ϯ3 to 36.8Ϯ26 pmol/mL; PϽ0.05), whereas it did not change after native hBNP (Pϭ0.001 between groups). MAP decreased at 10 minutes and remained decreased for 60 minutes after CONJ-hBNP (from 113Ϯ8 to 101Ϯ12 mm Hg after 10 minutes to 97.5Ϯ10 mm Hg after 30 minutes to 99Ϯ13 mm Hg after 60 minutes) while remaining unchanged after native hBNP (Pϭ0.0387 between groups). BNP is an endogenous peptide produced by the heart as a nonactive 108 -amino acid hormone. [1][2][3] It is cleaved and activated into its 32-amino acid mature form by the transmembrane enzyme corin. 4 -6 BNP has natriuretic, diuretic, vasorelaxant, lusitropic, and antialdosterone properties, as well as direct and indirect antifibrotic actions. 7 BNP binds to the natriuretic peptide receptor-A (NPR-A), which is a membrane-bound receptor located on cardiomyocytes, vascular endothelium, smooth muscle, kidneys, and lungs, resulting in activation of its second messenger, cGMP. Conclusions-ThisWe recently reported that exogenous administration of BNP has favorable effects in experimental congestive heart failure (CHF). 8 Furthermore, recent studies have demonstrated the efficacy of intravenous administration of recombinant hBNP in decreasing cardiac filling pressures and improving symptoms in the setting of acute decompensated CHF. 9 -12 In experimental hypertension, administration of long-acting BNP synthesized as a fusion peptide with albumin sustained blood pressure-lowering actions, supporting a strategy for longer-term BNP therapy in cardiovascular dis- eases. 13 Although these are important advances for hypertension and CHF therapy, the current use of BNP is limited to acute intravenous administration. Recently, proprietary technology (Nobex) has been developed in which short, amphiphilic oligomers are covalently attached to peptides. In contrast to standard PEGylation technology, this technique u...

Echinocandins are a first-line therapy for candidemia and invasive candidiasis. They are generally safe with few drug interactions, but the stability and pharmacokinetic properties of currently approved echinocandins are such that each was developed for daily intravenous infusion. We sought to discover a novel echinocandin with properties that would enable more flexible dosing regimens, alternate routes of delivery, and expanded utility. Derivatives of known echinocandin scaffolds were generated, and an iterative process of design and screening led to the discovery of CD101, a novel echinocandin that has since demonstrated improved chemical stability and pharmacokinetics. Here, we report the structure-activity relationships (including preclinical efficacy and pharmacokinetic data) for the series of echinocandin analogs from which CD101 was selected. In a mouse model of disseminated candidiasis, the test compounds displayed clear dose responses and were generally associated with lower fungal burdens than that of anidulafungin. Single-dose pharmacokinetic studies in beagle dogs revealed a wide disparity in the half-lives and volumes of distribution, with one compound (now known as CD101) displaying a half-life that is nearly 5-fold longer than that of anidulafungin (53.1 h versus 11.6 h, respectively). In vitro activity data against panels of Candida spp. and Aspergillus spp. demonstrated that CD101 behaved similarly to approved echinocandins in terms of potency and spectrum of activity, suggesting that the improved efficacy observed in vivo for CD101 is a result of features beyond the antifungal potency inherent to the molecule. Factors that potentially contribute to the improved in vivo efficacy of CD101 are discussed.