Identification of Molecular Fragments in Equilibrium with Polysiloxane Ultrasmall Nanoparticles.

During recent decades, ultrasmall inorganic nanoparticles have attracted considerable interest due to their favorable biodistribution, pharmacokinetics and theranostic properties. In particular, AGuIX nanoparticles made of polysiloxane and gadolinium chelates were successfully translated to the clinics. In an aqueous medium, these nanoparticles are in dynamic equilibrium with polysiloxane fragments due to the hydrolysis of Si-O-Si bonds. Thanks to high-performance liquid chromatography coupled with electrospray ionization mass spectrometry, all these fragments were separated and identified.

Differential mechanisms of asparaginase resistance in B-type acute lymphoblastic leukemia and malignant natural killer cell lines.

Bacterial L-asparaginase (ASNase), hydrolyzing L-asparagine (Asn), is an important drug for treating patients with acute lymphoblastic leukaemia (ALL) and natural killer (NK) cell lymphoma. Although different native or pegylated ASNase-based chemotherapy are efficient, disease relapse is frequently observed, especially in adult patients. The neo-synthesis of Asn by asparagine synthetase (AsnS) following ASNase treatment, which involves the amino acid response and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathways, is believed to be the basis of ASNase-resistance mechanisms. However, AsnS expression has not emerged as an accurate predictive factor for ASNase susceptibility. The aim of this study was to identify possible ASNase sensitivity/resistance-related genes or pathways using a new asparaginase, namely a pegylated r-crisantaspase, with a focus on classic Asn-compensatory responses and cell death under conditions of Asn/L-glutamine limitation. We show that, for B-ALL cell lines, changes in the expression of apoptosis-regulatory genes (especially NFκB-related genes) are associated with ASNase susceptibility. The response of malignant NK cell lines to ASNase may depend on Asn-compensatory mechanisms and other cellular processes such as cleavage of BCL2A1, a prosurvival member of the Bcl-2 protein family. These results suggest that according to cellular context, factors other than AsnS can influence ASNase susceptibility.

The acylated (AG) to unacylated (UAG) ghrelin ratio in esterase inhibitor-treated blood is higher than previously described.

CONTEXT: The acylated/unacylated ghrelin (AG/UAG) ratio has been reported to range from 0·02 to 0·3, suggesting biologically relevant independent regulation of each ghrelin isoform. However, AG is deacylated to UAG by esterases in blood samples, and esterase inhibition is critical for their accurate measurement. Our hypothesis is that at least part of the variation in reported AG and UAG values is due to inconsistent sample preparation. DESIGN: A non-interventional study. Quantification with two different, commercially available, ELISA formats of AG and UAG in venous plasma stabilized or not with 4-(2-aminoethyl) benzenesulphonyl fluoride (AEBSF) and stored for 0-6 months at -20 or -80 °C. PARTICIPANTS: Healthy, non-obese, adults (n = 8; 4 women), age 26-42 yrs, after an overnight fast. MEASUREMENTS: AG and UAG stability following different methods of sample treatment and storage. RESULTS: Non-AEBSF plasma contained low AG and high UAG (>270 pg/ml) indicating rapid conversion of AG to UAG. However, AEBSF plasma, stored at -80 °C and measured at 0, 1, 3 and 6 months contained AG and UAG ranges of 12-350 and 17-170 pg/ml, respectively. Mean (SEM) AG/UAG ratios were 1·7(0·3), 1·2(0·2), 1·5(0·3) and 1·8(0·5) at each time point with no significant effect of storage period. CONCLUSIONS: AG and UAG levels measured in AEBSF-stabilized plasma indicate that the AG/UAG ratio is markedly higher than previously described and that UAG is a physiological component of the circulation. This highlights the importance of immediately stabilizing blood samples on collection for determination of both AG and UAG concentrations and provides a valuable tool for their measurement in physiological and interventional studies.

Pharmacology, immunogenicity, and efficacy of a novel pegylated recombinant Erwinia chrysanthemi-derived L-asparaginase.

Bacterial L-asparaginase (ASNase), hydrolyzing L-asparagine (Asn), is an indispensable component used in the treatment of acute lymphoblastic leukemia (ALL) and certain lymphoma entities. Native Erwinia chrysanthemi-derived ASNase (n-crisantaspase) has been approved as a second-line drug for treating patients exhibiting allergy syndromes to native and pegylated Escherichia coli-derived ASNase (EC-ASNase). However, it still induces hypersensitivity in at least 17 % of treated patients. In the present study, we investigated the pharmacological activity, immunogenicity and anti-leukemic activity of a new pegylated recombinant crisantaspase (PEG-r-crisantaspase). The results demonstrate that when compared to n-crisantaspase in vivo, PEG-r-crisantaspase maintains a complete depletion of plasma Asn for up to 72 h with a 50-fold lower dose. In mice receiving PEG-r-crisantaspase, specific antibodies against the enzyme were undetectable, indicating a lower immunogenicity of the pegylated enzyme. In vitro, PEG-r-crisantaspase exhibits similar cytotoxic effects (EC50 < 5 × 10(-4) U/mL for the most sensitive cell lines) to n-crisantaspase on various leukemia and lymphoma cells and was shown to be more efficient than EC-ASNase. Three repeated PEG-r-crisantaspase injections (2-20 U/Kg) prevented leukemia development in leukemia-bearing mice for 17 days and significantly prolonged animal survival to 7-12 days. Therefore, PEG-r-crisantaspase appears to be a promising drug candidate for ALL treatment and should be further explored in experimental and clinical trials.

In vitro and in vivo stability and pharmacokinetic profile of unacylated ghrelin (UAG) analogues.

Ghrelin, an endocrine hormone predominantly produced by the stomach, exists in acylated and unacylated forms in the circulation. Unacylated ghrelin (UAG), the more abundant form in blood, possesses similar, independent or opposite physiological actions as acylated ghrelin (AG). AZP502, a linear 8-amino acid peptide from the central region of UAG (UAG(6-13)), and its full (AZP531) and partially (AZP533) cyclised derivatives, exhibit the same pharmacological profile as UAG both in vitro and in vivo, independently of AG receptor binding. We investigated the stability of these three fragments in vitro in human blood samples and in vivo after subcutaneous and intravenous injection in rats and dogs using liquid chromatography-mass spectrometry. In both species, AZP502 is rapidly degraded generating two major metabolites. Partial cyclisation of AZP502 and acylation at its N-terminus (AZP533 peptide) improves its stability in human plasma in vitro. Full cyclisation of AZP502 (AZP531 peptide) also completely protects the peptide from peptidase degradation in vitro in human blood samples. Moreover this cyclisation strongly improves the stability and the bioavailability of this peptide in vivo in both dogs and rats (mean bioavailability of 10-15% and 85-95% for AZP502 and AZP531 respectively). Taken together these results support the rationale for developing AZP531 as a long-acting UAG analogue for subcutaneous injection for the treatment of type 2 diabetes mellitus and other metabolic disorders.

Des-acyl ghrelin fragments and analogues promote survival of pancreatic ß-cells and human pancreatic islets and prevent diabetes in streptozotocin-treated rats.

Des-acyl ghrelin, although devoid of binding to ghrelin receptor (GRLN), exerts many biological effects, including regulation of glucose and lipid metabolism. Indeed, des-acyl ghrelin promotes pancreatic ß-cell and human islet cell survival and prevents diabetes in streptozotocin (STZ) treated rats. We investigated whether des-acyl ghrelin fragments excluding serine(3), which is essential for binding to GRLN, would display similar actions. Among the different compounds tested, des-acyl ghrelin((6-13)) and des-acyl ghrelin((6-13)) with alanine substitutions or cyclization, but not with d-amino acid substitutions, showed the best survival effect, similar to des-acyl ghrelin. Des-acyl ghrelin((6-13)) even prevented diabetes in STZ-treated rats and protected human circulating angiogenic cells from oxidative stress and senescence, similar to des-acyl ghrelin. These results suggest that not only full-length des-acyl ghrelin but also short des-acyl ghrelin fragments have clear beneficial effects on several tissues in vitro and in vivo.

Analysis of catalytic carboxylate mutants E552Q and E1197Q suggests asymmetric ATP hydrolysis by the two nucleotide-binding domains of P-glycoprotein.

In the nucleotide-binding domains (NBDs) of ABC transporters, such as mouse Mdr3 P-glycoprotein (P-gp), an invariant carboxylate residue (E552 in NBD1; E1197 in NBD2) immediately follows the Walker B motif (hyd(4)DE/D). Removal of the negative charge in mutants E552Q and E1197Q abolishes drug-stimulated ATPase activity measured by P(i) release. Surprisingly, drug-stimulated trapping of 8-azido-[alpha-(32)P]ATP is still observed in the mutants in both the presence and absence of the transition-state analogue vanadate (V(i)), and ADP can be recovered from the trapped enzymes. The E552Q and E1197Q mutants show characteristics similar to those of the wild-type (WT) enzyme with respect to 8-azido-[alpha-(32)P]ATP binding and 8-azido-[alpha-(32)P]nucleotide trapping, with the latter being both Mg(2+) and temperature dependent. Importantly, drug-stimulated nucleotide trapping in E552Q is stimulated by V(i) and resembles the WT enzyme, while it is almost completely V(i) insensitive in E1197Q. Similar nucleotide trapping properties are observed when aluminum fluoride or beryllium fluoride is used as an alternate transition-state analogue. Partial proteolytic cleavage of photolabeled enzymes indicates that, in the absence of V(i), nucleotide trapping occurs exclusively at the mutant NBD, whereas in the presence of V(i), nucleotide trapping occurs at both NBDs. Together, these results suggest that there is single-site turnover occurring in the E552Q and E1197Q mutants and that ADP release from the mutant site, or another catalytic step, is impaired in these mutants. Furthermore, our results support a model in which the two NBDs of P-gp are not functionally equivalent.

Structural and functional asymmetry of the nucleotide-binding domains of P-glycoprotein investigated by attenuated total reflection Fourier transform infrared spectroscopy.

The dynamic changes occurring during the catalytic cycle of MDR3 P-glycoprotein (Pgp) and the role of each nucleotide-binding domain (NBD) in the transport process were investigated using attenuated total reflection Fourier transform infrared spectroscopy. For this purpose, wild-type Pgp and two mutations of homologous residues in each NBD were studied. On the one hand, we demonstrate here that, during its catalytic cycle, Pgp does not undergo secondary structure changes, but only modifications in its stability and accessibility to the external environment. On the other hand, amide H/D exchange kinetics demonstrate that homologous mutations in the two NBDs affect, in a different way, the dynamic properties of Pgp and also the dynamic changes occurring during ATP hydrolysis. These observations led to the conclusion that the NBDs have an asymmetric structure and different functions in the catalytic cycle of Pgp. Our data suggest that the release of drug from the membrane into the extracellular environment is due to decreased stability and/or increased accessibility to the external medium of the membrane-embedded drug-binding site(s). NBD1 would play an important role in this first restructuring of the membrane-embedded domains. NBD2 would be directly implicated in the subsequent restructuring of the membrane-embedded binding sites by which they recover their initial stability and accessibility to the membrane. It is proposed that this restructuring step would allow the binding and transport of another molecule of substrate.