Structure-Activity Relationships of a Series of Echinocandins and the Discovery of CD101, a Highly Stable and Soluble Echinocandin with Distinctive Pharmacokinetic Properties.

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.

Chronic actions of a novel oral B-type natriuretic peptide conjugate in normal dogs and acute actions in angiotensin II-mediated hypertension.

BACKGROUND: We previously reported the feasibility of an acute, orally delivered, newly developed, conjugated form of human B-type natriuretic peptide (hBNP) in normal animals. The objective of the present study was to extend our findings and to define the chronic actions of an advanced oral conjugated hBNP (hBNP-054) administered for 6 days on sodium excretion and blood pressure. We also sought to establish the ability of this new conjugate to acutely activate cGMP and to reduce blood pressure in an experimental model of angiotensin II (ANG II) -mediated hypertension. METHODS AND RESULTS: First, we developed additional novel conjugated forms of oral hBNP that were superior to our previously reported hBNP-021 in reducing blood pressure in 6 normal dogs. We then tested the new conjugate, hBNP-054, chronically in 2 normal dogs to assess its biological actions as a blood pressure-lowering agent and as a natriuretic factor. Second, we investigated the effects of acute oral hBNP-054 or vehicle in 6 dogs that received continuous infusion of ANG II to induce hypertension. After baseline determination of mean blood pressure (MAP) and blood collection for plasma hBNP and cGMP, all dogs received continuous ANG II infusion (20 ng . kg(-1) . min(-1), 1 mL/min) for 4 hours. After 30 minutes of ANG II, dogs received oral hBNP-054 (400 microg/kg) or vehicle in a random crossover fashion with a 1-week interval between dosing. Blood sampling and MAP measurements were repeated 30 minutes after ANG II administration and 10, 30, 60, 120, 180, and 240 minutes after oral administration of hBNP-054 or vehicle. In the chronic study in normal dogs, oral hBNP-054 effectively reduced MAP for 6 days and induced a significant increase in 24-hour sodium excretion. hBNP was not present in the plasma at baseline in any dogs, and it was not detected at any time in the vehicle group. However, hBNP was detected throughout the duration of the study after oral hBNP-054, with a peak concentration at 30 minutes of 1060+/-818 pg/mL. In the acute study, after ANG II administration, plasma cGMP was not activated after vehicle, whereas it was significantly increased after oral hBNP-054 (P=0.01 between the 2 groups). Importantly, MAP was significantly increased after ANG II throughout the acute study protocol. However, although no changes occurred in MAP after vehicle administration, oral hBNP-054 reduced MAP for >2 hours (from 138+/-1 mm Hg after ANG II to 124+/-2 mm Hg at 30 minutes, 124+/-2 mm Hg at 1 hour, and 130+/-5 mm Hg at 2 hours after oral hBNP-054; P<0.001). CONCLUSIONS: This study reports for the first time that a novel conjugated oral hBNP possesses blood pressure-lowering and natriuretic actions over a 6-day period in normal dogs. Furthermore, hBNP-054 activates cGMP and reduces MAP in a model of acute hypertension. These findings advance the concept that orally administered chronic BNP is a potential therapeutic strategy for cardiovascular diseases such as hypertension.

Amphiphilic conjugates of human brain natriuretic peptide designed for oral delivery: in vitro activity screening.

Congestive heart failure (CHF) is a complex syndrome involving altered neurohormonal levels and impaired cardiac and renal function. In recent years, intravenous administration of exogenous human brain-type natriuretic peptide (hBNP) has become an important therapy in treating patients with acutely decompensated CHF. However, reports during the past year suggest that hBNP could play a prominent role in the chronic treatment of CHF patients as well. We are currently developing conjugates of hBNP suitable for oral delivery to provide a patient-friendly treatment option for chronic heart failure patients. In this report, we present in vitro activity results obtained from hBNP conjugates featuring a variety of rationally designed amphiphilic oligomers. Mapping studies revealed that the hydrophobic/hydrophilic balance of the oligomer impacted the regioselectivity of conjugation. Additionally, the regiochemistry and extent of conjugation had a significant impact on activity. Many monoconjugates retained activity comparable to native peptide and are currently under evaluation in subsequent in vivo screens.

Oral human brain natriuretic peptide activates cyclic guanosine 3',5'-monophosphate and decreases mean arterial pressure.

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 (P=0.0374 between groups). Plasma cGMP increased after CONJ-hBNP for 60 minutes (from 10.8+/-3 to 36.8+/-26 pmol/mL; P<0.05), whereas it did not change after native hBNP (P=0.001 between groups). MAP decreased at 10 minutes and remained decreased for 60 minutes after CONJ-hBNP (from 113+/-8 to 101+/-12 mm Hg after 10 minutes to 97.5+/-10 mm Hg after 30 minutes to 99+/-13 mm Hg after 60 minutes) while remaining unchanged after native hBNP (P=0.0387 between groups). CONCLUSIONS: This study reports for the first time that novel conjugated oral BNP activates cGMP and significantly reduces MAP, thus implying an efficacious coupling of CONJ-hBNP to the natriuretic receptor-A. These data advance a new concept of orally administered chronic BNP therapy for cardiovascular diseases.

Characterization of peptide-amphiphiles possessing cellular activation sequences.

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.

Melanoma cell CD44 interaction with the alpha 1(IV)1263-1277 region from basement membrane collagen is modulated by ligand glycosylation.

Invasion of the basement membrane is believed to be a critical step in the metastatic process. Melanoma cells have been shown previously to bind distinct triple-helical regions within basement membrane (type IV) collagen. Additionally, tumor cell binding sites within type IV collagen contain glycosylated hydroxylysine residues. In the present study, we have utilized triple-helical models of the type IV collagen alpha1(IV)1263-1277 sequence to (a) determine the melanoma cell receptor for this ligand and (b) analyze the results of single-site glycosylation on melanoma cell recognition. Receptor identification was achieved by a combination of methods, including (a) cell adhesion and spreading assays using triple-helical alpha1(IV)1263-1277 and an Asp(1266)Abu variant, (b) inhibition of cell adhesion and spreading assays, and (c) triple-helical alpha1(IV)1263-1277 affinity chromatography with whole cell lysates and glycosaminoglycans. Triple-helical alpha1(IV)1263-1277 was bound by melanoma cell CD44/chondroitin sulfate proteoglycan receptors and not by the collagen-binding integrins or melanoma-associated proteoglycan. Melanoma cell adhesion to and spreading on the triple-helical alpha1(IV)1263-1277 sequence was then compared for glycosylated (replacement of Lys(1265) with Hyl(O-beta-d-galactopyranosyl)) versus non-glycosylated ligand. Glycosylation was found to strongly modulate both activities, as adhesion and spreading were dramatically decreased due to the presence of galactose. CD44/chondroitin sulfate proteoglycan did not bind to glycosylated alpha1(IV)1263-1277. Overall, this study (a) is the first demonstration of the prophylactic effects of glycosylation on tumor cell interaction with the basement membrane, (b) provides a rare example of an apparent unfavorable interaction between carbohydrates, and (c) suggests that sugars may mask "cryptic sites" accessible to tumor cells with cell surface or secreted glycosidase activities.

Modulation of triple-helical stability and subsequent melanoma cellular responses by single-site substitution of fluoroproline derivatives.

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.