Peroral Amphotericin B Polymer Nanoparticles Lead to Comparable or Superior In Vivo Antifungal Activity to That of Intravenous Ambisome? or Fungizone?

Background

Despite advances in the treatment, the morbidity and mortality rate associated with invasive aspergillosis remains unacceptably high (70–90%) in immunocompromised patients. Amphotericin B (AMB), a polyene antibiotic with broad spectrum antifungal activity appears to be a choice of treatment but is available only as an intravenous formulation; development of an oral formulation would be beneficial as well as economical.

Methodology

Poly(lactide-co-glycolode) (PLGA) nanoparticles encapsulating AMB (AMB-NPs) were developed for oral administration. The AMB-NPs were 113±20 nm in size with ∼70% entrapment efficiency at 30% AMB w/w of polymer. The in vivo therapeutic efficacy of oral AMB-NPs was evaluated in neutropenic murine models of disseminated and invasive pulmonary aspergillosis. AMB-NPs exhibited comparable or superior efficacy to that of Ambisome® or Fungizone™ administered parenterally indicating potential of NPs as carrier for oral delivery.

Conclusions

The present investigation describes an efficient way of producing AMB-NPs with higher AMB pay-load and entrapment efficiency employing DMSO as solvent and ethanol as non-solvent. The developed oral formulation was highly efficacious in murine models of disseminated aspergillosis as well as an invasive pulmonary aspergillosis, which is refractory to treatment with IP Fungizone™and responds only modestly to AmBisome®.     

Dimeric and trimeric proanthocyanidins possessing a doubly linked structure from Pavetta owariensis

Pavetannin A-2, a new A-type proanthocyanidin, along with the trimers cinnamtannin B-1, pavetannin B-1, B-2, B-3, B-5 and B-6 have been isolated in their free phenolic form from the stem bark of Pavetta owariensis. Spectral data and partial acid-catalysed degradation established their structures as ent-epicatechin-(4→8,2→O→7)-catechin, epicatechin-(4β→8,2β→O→7)-epicatechin-(4→8)-epicatechin, epicatechin-(4β→8,2β→O→7)-epicatechin-(4β→8)-ent-epicatechin, epicatechin-(4β→8,2β→O→7)-epicatechin-(4β→8)-epicatechin, epicatechin-(4β→6,2β→O→7)-epicatechin-(4β→8)-epicatechin, epicatechin-(4β→6,2β→O→7)-catechin-(4β→8)-epicatechin, epicatechin-(4β→8,2β→O→7)-epicatechin-(4→8)-catechin, respectively.     

Six new C21 steroidal glycosides from Asclepias curassavica L

Six new C₂₁ steroidal glycosides, named curassavosides A–F (3–8), were obtained from the aerial parts of Asclepias curassavica (Asclepiadaceae), along with two known oxypregnanes, 12-O-benzoyldeacylmetaplexigenin (1) and 12-O-benzoylsarcostin (2). By spectroscopic methods, the structures of the six new compounds were determined as 12-O-benzoyldeacylmetaplexigenin 3-O-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (3), 12-O-benzoylsarcostin 3-O-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (4), sarcostin 3-O-β-d-oleandropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (5), sarcostin 3-O-β-d-oleandropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-digitoxopyranoside (6), 12-O-benzoyldeacylmetaplexigenin 3-O-β-d-glucopyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (7), and 12-O-benzoylsarcostin 3-O-β-d-glucopyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-canaropyranosyl-(1 → 4)-β-d-oleandropyranosyl-(1 → 4)-β-d-digitoxopyranoside (8), respectively. All compounds (1–8) were tested for in vitro cytotoxicity; only compound 3 showed weak inhibitory activity against Raji and AGZY cell lines.     

Enzymatic transglycosylation of xylose using a glycosynthase

The application of the hyperactive glycosynthase derived from Agrobacterium sp. β-glucosidase (AbgE358G-2F6) to the synthesis of xylo-oligosaccharides by using -d-xylopyranosyl fluoride as donor represents the first successful use of glycosynthase technology for xylosyl transfer. Transfer to p-nitrophenyl β-d-glucopyranoside yields di- and trisaccharide products with β-(1→4) linkages in 63% and 35% yields, respectively. By contrast, transfer to p-nitrophenyl β-d-xylopyranoside yielded the β-(1→3) linked disaccharide and β-d-Xyl-(1→4)-β-d-Xyl-(1→3)-β-d-Xyl-pNP as major products in 42% and 30% yields, respectively. Transfer of xylose to β-d-Xyl-(1→4)-β-d-Xyl-pNP yielded the β-(1→4) linked trisaccharide in 98% yield, thereby indicating that transfers to xylo-disaccharides occur with formation of β-(1→4) bonds. Xylosylation of carbamate-protected deoxyxylonojirimycin produced a mixture of di- and tri-‘saccharide’ products in modest yields.     

The structures of six urinary oligosaccharides that are characteristic for a patient with morquio syndrome type b

Morquio syndrome type B is an inherited, lysosomal storage disease characterised by a marked deficiency in acid β-d-galactosidase, while the 2-acetamido-2-deoxy-β-d-galactose 6-sulphate sulphatase activity is normal. Urinary oligosaccharides were studied in order to evaluate the effect of the diminished β-d-galactosidase activity on the catabolism of glycoconjugates and to compare their structures with those excreted by patients with GM₁-gangliosidosis. The following oligosaccharides were isolated: β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→6)-β-d-Manp-(1→4)- d-GlcpNAc (1), β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→6)-[α-d-Manp- (1→3)]-β-d-Manp-(1→4)-d-GlcpNAc (2a), β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)- α-d-Manp-(1→3)-[α-d-Manp-(1→6)]-β-d-Manp-(1→4)-d-GlcpNAc (2b), β-d-Galp- (1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→3)-[β-d-Galp-(1→4)-β-d-GlcpNAc-(1→ 2)-α-d-Manp-(1→6)]-β-d-Manp-(1→4)-d-GlcpNAc (3), β-d-Galp-(1→4)-β-d-Glcp- NAc-(1→2)-α-d-Manp-(1→3)-{β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-[β-d-Galp- (1→4)-β-d-GlcpNAc-(1→6)]-α-d-Manp-(1→6)}-β-d-Manp-(1→4)-d-GlcpNAc (4), β-d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→3)-[β-d-GlcpNAc-(1→4)]-[β- d-Galp-(1→4)-β-d-GlcpNAc-(1→2)-α-d-Manp-(1→6)]-β-d-Manp-(1→4)-d-Glcp- NAc (5). Significant differences between Morquio syndrome type B and GM₁-gangliosidosis have been observed, with regard to the excretion rate and the specific structures of urinary oligosaccharides. Compounds 2a, 2b, and 5 are novel members of the series of oligosaccharides isolated from the urine of patients with inherited, lysosomal storage diseases.     

Isoflavonoids from the roots of Erythrina poeppigiana

Five isoflavonoids, 7,4′-dihydroxy-2′-methoxy-8-(γ,γ-dimethylallyl)isoflav-3-ene, 4′-hydroxy-2′-methoxy-6″,6″-dimethylpyran[2″,3″:7,8]isoflav-3-ene, 5,7,4′-trihydroxy-2′-methoxy-5′-(γ,γ-dimethylallyl)isoflavanone, 5,4′-dihydroxy-7,2′-dimethoxy-5′-(γ,γ-dimethylallyl)isoflavanone and 3,9-dihydroxy-4-(γ,γ-dimethylallyl)pterocarpene as well as six known compounds were isolated from the roots of Erythrina poeppigiana. Their structures were established on the basis of spectroscopic evidence.     

Inhibition of human and rat 11beta-hydroxysteroid dehydrogenase type 1 by 18beta-glycyrrhetinic acid derivatives

11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) plays an important role in regulating the cortisol availability to bind to corticosteroid receptors within specific tissue. Recent advances in understanding the molecular mechanisms of metabolic syndrome indicate that elevation of cortisol levels within specific tissues through the action of 11β-HSD1 could contribute to the pathogenesis of this disease. Therefore, selective inhibitors of 11β-HSD1 have been investigated as potential treatments for metabolic diseases, such as diabetes mellitus type 2 or obesity. Here we report the discovery and synthesis of some 18β-glycyrrhetinic acid (18β-GA) derivatives (2–5) and their inhibitory activities against rat hepatic11β-HSD1 and rat renal 11β-HSD2. Once the selectivity over the rat type 2 enzyme was established, these compounds’ ability to inhibit human 11β-HSD1 was also evaluated using both radioimmunoassay (RIA) and homogeneous time resolved fluorescence (HTRF) methods. The 11-modified 18β-GA derivatives 2 and 3 with apparent selectivity for rat 11β-HSD1 showed a high percentage inhibition for human microsomal 11β-HSD1 at 10 μM and exhibited IC₅₀ values of 400 and 1100 nM, respectively. The side chain modified 18β-GA derivatives 4 and 5, although showing selectivity for rat 11β-HSD1 inhibited human microsomal 11β-HSD1 with IC₅₀ values in the low micromolar range.     

Saponins from Albizzia lucida.

Three main saponins were isolated from the seeds of Albizzia lucida. Their structures were established by spectral analyses and chemical and enzymatic transformations as 3-O-[β- -xylopyranosyl(1→2)-- -arabinopyranosyl (1→6)] [β- -glucopyranosyl (1→2)] β- -glucopyranosyl echinocystic acid; 3-O-[- -arabinopyranosyl (1→6)][β- -glucopyranosyl (1→2)]-β- -glucopyranosyl echinocystic acid and 3-O-[β- -xylopyranosyl (1→2)-β- -fucopyranosyl (1→6)-2-acetamido-2-deoxy-β- -glucopyranosyl echinocystic acid, characterized as its methyl ester.     

Fourier transform infrared spectroscopy used to evidence the prevention of beta-sheet formation of amyloid beta(1-40) peptide by a short amyloid fragment

Reflectance Fourier transform infrared (FT-IR) microspectroscopy was applied to study the prevention of β-sheet formation of amyloid β (Aβ)(1–40) peptide by co-incubation with a hexapeptide containing a KLVFF sequence (Aβ(15–20) fragment). Second-derivative spectral analysis was used to locate the position of the overlapping components of the amide I band of Aβ peptide and assigned them to different secondary components. The result indicates that each intact sample of Aβ(15–20) fragment or Aβ(1–40) peptide previously incubated in distilled water at 37 °C transformed their secondary structure from 1649 (1651) or 1653 cm⁻¹ to 1624 cm⁻¹, suggesting the transformation from -helix and/or random coil structures to β-sheet structure. By co-incubating both samples with different molar ratio in distilled water at 37 °C, the structural transformation was not found for Aβ(1–40) peptide after 24 h-incubation. But the β-sheet formation of Aβ(1–40) peptide after 48 h-incubation was evidenced from the appearance of the IR peak at 1626 cm⁻¹ by adding a little amount of Aβ(15–20) fragment. There was no β-sheet formation of Aβ(1–40) peptide after addition with much amount of Aβ(15–20) fragment, however, suggesting the higher amount of Aβ(15–20) fragment used might inhibit the β-sheet formation of Aβ(1–40) peptide. The more Aβ(15–20) fragment used made the more stable structure of Aβ(1–40) peptide and the less β-sheet formation of Aβ(1–40) peptide. The study indicates that the reflectance FT-IR microspectroscopy can easily evidence the prevention of β-sheet formation of Aβ(1–40) peptide by a short amyloid fragment.     

Proteolytic modification of membrane-associated phospholipase C-β by μ-calpain enhances its activation by G-protein βγ subunits in human platelets

Membrane-associated phosphoinositide-phospholipase C (PI-PLC)-β (150 kDa) and its truncated forms (100 kDa and 45 kDa) were purified from human platelets. The 100 kDa PI-PLC-β was found to be activated to a greater extent by brain G-protein βγ subunits compared to the intact 150 kDa enzyme. Furthermore, treatment with μ-calpain of the intact PI-PLC-β (150 kDa) caused a marked augmentation of its activation by βγ subunits. This enhanced PLC activation by βγ subunits was due to truncation by μ-calpain, producing a 100 kDa PI-PLC, but not by another protease,thrombin.     

Distinct Roles of Glycogen Synthase Kinase (GSK)-3�� and GSK-3�� in Mediating Cardiomyocyte Differentiation in Murine Bone Marrow-derived Mesenchymal Stem Cells

The signaling mechanisms facilitating cardiomyocyte (CM) differentiation from bone marrow (BM)-derived mesenchymal stem cells (MSCs) are not well understood. 5-Azacytidine (5-Aza), a DNA demethylating agent, induces expression of cardiac-specific genes, such as Nkx2.5 and α-MHC, in mouse BM-derived MSCs. 5-Aza treatment caused significant up-regulation of glycogen synthase kinase (GSK)-3β and down-regulation of β-catenin, whereas it stimulated GSK-3α expression only modestly. The promoter region of GSK-3β was heavily methylated in control MSCs, but was demethylated by 5-Aza. Although overexpression of GSK-3β potently induced CM differentiation, that of GSK-3α induced markers of neuronal and chondrocyte differentiation. GSK-3 inhibitors, including LiCl, SB 216743, and BIO, abolished 5-Aza-induced up-regulation of CM-specific genes, suggesting that GSK-3 is necessary and sufficient for CM differentiation in MSCs. Although specific knockdown of endogenous GSK-3β abolished 5-Aza-induced expression of cardiac specific genes, surprisingly, that of GSK-3α facilitated CM differentiation in MSCs. Although GSK-3β is found in both the cytosol and nucleus in MSCs, GSK-3α is localized primarily in the nucleus. Nuclear-specific overexpression of GSK-3β failed to stimulate CM differentiation. Down-regulation of β-catenin mediates GSK-3β-induced CM differentiation in MSCs, whereas up-regulation of c-Jun plays an important role in mediating CM differentiation induced by GSK-3α knockdown. These results suggest that GSK-3α and GSK-3β have distinct roles in regulating CM differentiation in BM-derived MSCs. GSK-3β in the cytosol induces CM differentiation of MSCs through down-regulation of β-catenin. In contrast, GSK-3α in the nucleus inhibits CM differentiation through down-regulation of c-Jun.     

A spirostane hexaglycoside from Agave cantala fruits.

A new steroidal glycoside, agaveside D, isolated from the fruits of Agave cantata was characterized as 3β-{- -rhamnopyranosyl-(1→2), β- -glycopyranosyl-(1→3)-β- -glucopyranosyl[β- -xylopyransoyl-(1→4)-- -rhamnopyranosyl-(1→2)]-β- -glucopyranosyl}-25R-5- spirostane on the basis of chemical degradation and spectrometry.     

Heterobimetallic chloro bridgedcomplexes of (η:η−C10H16)-ruthenium(IV) with palladium(II), platinum(II), rhodium(III), iridium(III), copper(I) and rhodium(I)

Metathesis of [(η³:η³−C₁₀H₁₆)Ru(Cl) (μ−Cl)]₂ (1) with [R₃P) (Cl)M(μ-Cl)]₂ (M = Pd, Pt), [Me₂NCH₂C₆H₄Pd(μ-Cl)]₂ and [(OC)₂Rh(μ-Cl)]₂ affords the heterobimetallic chloro bridged complexes (η³:η³-C₁₀H₁₆) (Cl)Ru(μ-Cl)₂M(PR₃)(Cl) (M = Pd, Pt), (η³:η³-C₁₀H₁₆) (Cl)Ru(μ-Cl)₂PdC₆H₄CH₂NMe₂ and (η³:η³-C₁₀H₁₆) (Cl)Ru(μ-Cl)₂Rh(CO)₂, respectively. Complex 1 reacts with [Cp^*M(Cl) (μ-Cl)]₂ (M = Rh, Ir), [p-cymene Ru(Cl) (μ-Cl]₂ and [(Cy₃P)Cu(μ-Cl)]₂ to give an equilibrium of the heterobimetallic complexes and of educts. The structures of (η³:η³-C₁₀H₁₆)Ru(μ-Cl)₂Pd(PR₃) (Cl) (R = Et, Bu) and of one diastereoisomer of (η³:η³-C₁₀H₁₆)Ru(μ-Cl)₂IrCp^*(Cl) were determined by X-ray diffraction.     

Chaperone activities of bovine and camel beta-caseins: Importance of their surface hydrophobicity in protection against alcohol dehydrogenase aggregation

β-Casein (β-CN) showing properties of intrinsically unstructured proteins (IUP) displays many similarities with molecular chaperones and shows anti-aggregation activity in vitro. Chaperone activities of bovine and camel β-CN were studied using alcohol dehydrogenase (ADH) as a substrate. To obtain an adequate relevant information about the chaperone capacities of studied caseins, three different physical parameters including chaperone constant (k_c, μM⁻¹), thermal aggregation constant (k_T, °C⁻¹) and aggregation rate constant (k_t, min⁻¹) were measured. Bovine β-CN displays greater chaperone activity than camel β-CN. Fluorescence studies of 8-anilino-1-naphthalenesulfonic acid (ANS) binding demonstrated that bovine β-CN is doted with larger effective hydrophobic surfaces at all studied temperatures than camel β-CN. Greater relative hydrophobicity of bovine β-CN than camel β-CN may be a factor responsible for stronger interactions of bovine β-CN with the aggregation-prone pre denatured molecular species of the substrate ADH, which resulted in greater chaperone activity of bovine β-CN.     

Oxidation of 1,4-alkanediols into γ-lactones via γ-lactols using Rhodococcus erythropolis as biocatalyst

Whole cells of Rhodococcus erythropolis DSM 44534 grown on ethanol, (R)- and (S)-1,2-propanediol were used for biotransformation of racemic 1,4-alkanediols into γ-lactones. The cells oxidized 1,4-decanediol (1a) and 1,4-nonanediol (2a) into the corresponding γ-lactones 5-hexyl-dihydro-2(3H)-furanone (γ-decalactone, 1c) and 5-pentyl-dihydro-2(3H)-furanone (γ-nonalactone, 2c), respectively, with an EE(R) of 40–75%. The transient formation of the γ-lactols 5-hexyl-tetrahydro-2-furanol (γ-decalactol, 1b) and 5-pentyl-tetrahydro-2-furanol (γ-nonalactol, 2b) as intermediates was observed by GC–MS. 1,4-Pentanediol (3a) was transformed into 5-methyl-dihydro-2(3H)-furanone (γ-valerolactone, 3c) whereas (R)- and (S)-2-methyl-1,4-butanediol (4a) was converted to the methyl-substituted γ-butyrolactones 4-methyl-dihydro-2(3H)-furanone (4c₁) and 3-methyl-dihydro-2(3H)-furanone (4c₂) in a ratio of 80:20 with a yield of 55%. Also cis-2-buten-1,4-diol (5a) was transformed resulting in the formation of 2(5H)-furanone (γ-crotonolactone, 5c). At the higher pH values of 8.8 the yield of lactone formed was improved; however, the enatiomeric excesses were slightly higher at the lower pH of 5.2.     

��3 Integrin in Cardiac Fibroblast Is Critical for Extracellular Matrix Accumulation during Pressure Overload Hypertrophy in Mouse

The adhesion receptor β3 integrin regulates diverse cellular functions in various tissues. As β3 integrin has been implicated in extracellular matrix (ECM) remodeling, we sought to explore the role of β3 integrin in cardiac fibrosis by using wild type (WT) and β3 integrin null (β3−/−) mice for in vivo pressure overload (PO) and in vitro primary cardiac fibroblast phenotypic studies. Compared to WT mice, β3−/− mice upon pressure overload hypertrophy for 4 wk by transverse aortic constriction (TAC) showed a substantially reduced accumulation of interstitial fibronectin and collagen. Moreover, pressure overloaded LV from β3−/− mice exhibited reduced levels of both fibroblast proliferation and fibroblast-specific protein-1 (FSP1) expression in early time points of PO. To test if the observed impairment of ECM accumulation in β3−/− mice was due to compromised cardiac fibroblast function, we analyzed primary cardiac fibroblasts from WT and β3−/− mice for adhesion to ECM proteins, cell spreading, proliferation, and migration in response to platelet derived growth factor-BB (PDGF, a growth factor known to promote fibrosis) stimulation. Our results showed that β3−/− cardiac fibroblasts exhibited a significant reduction in cell-matrix adhesion, cell spreading, proliferation and migration. In addition, the activation of PDGF receptor associated tyrosine kinase and non-receptor tyrosine kinase Pyk2, upon PDGF stimulation were impaired in β3−/− cells. Adenoviral expression of a dominant negative form of Pyk2 (Y402F) resulted in reduced accumulation of fibronectin. These results indicate that β3 integrin-mediated Pyk2 signaling in cardiac fibroblasts plays a critical role in PO-induced cardiac fibrosis.     

Some observations on the measurement of estradiol-17β in human plasma by radioimmunoassay

The use of specific and non-specific antisera for estradiol-17β (E₂17β) were compared in the radioimmunoassay of the steroid. The effects of various “blank” mateirials on the standard curve and on the accuracy of recovery of E₂17β added to plasma before and after chromatography on LH-20 Sephadex were examined. It was concluded that the use of the specific antiserum (anti-6-oxoE₂17β -6-(O-carboxymethyl)oxime-bovine serum albumin(antiE₂17β-6-BSA) was an improvement on the non-specific serum anti-E₂17β-17-hemisuccinyl-bovine serum albumin (antiE₂ 17β-17-BSA) following chromatography of extracts. However, although a precise result could be obtained with the anti-E₂17β-6-BSA without the Chromatographic step, recovery of E₂17β added to plasma was only possible if the step was included.

The cross-reactivity of estrone (E₁)with E₂17β using anti-E₂17β-17-BSA as defined by Abraham (J. Clin. Endocr. , 866 (1969) was examined under conditions of constant and of changing E₁:E₂17β ratio.     

Application of a Novel Method of Double APAAP Staining With Subsequent Quantitative Image Analysis to the Examination of Integrin Expression in Undifferentiated-type Gastric Carcinomas

In undifferentiated-type gastric carcinoma (UGC), recognition of cancer cells is not easy, which has hampered its precise phenotypic analysis. To examine alterations of the integrin phenotype during the progression of UGC, we used double alkaline phosphatase anti-alkaline phosphatase staining and computer-aided image analyses for the expression of α1, α2, α3, α5, α6, αV, β1, and β4 integrin subunits and αVβ3, αVβ5, and αVβ6 integrins in cytokeratin-positive cells in the mucosal, the submucosal, and the deeper parts of 10 early and 17 advanced UGCs, their non-neoplastic counterparts, and 9 lymph node (LN) metastases. We revealed declining expression of epithelial integrin subunits (α2, α3, α6, β4) and increasing expression of mesenchymal integrin subunits (α1, α5) as the tumor invaded deeper, reflecting gradual epithelial-to-mesenchymal transition of the integrin phenotype during tumor invasion. Enhanced expression of the αV integrin subunit and αVβ3 and αVβ5 integrins correlated with tumor invasion, and that of αVβ6 integrins with LN metastasis. Our results have demonstrated that the method we introduced is suitable for analysis of dynamic alterations of the integrin repertoire in UGC progression. (J Histochem Cytochem 57:1183–1193, 2009)