Systemic Delivery of hGhrelin Derivative by Lyophilizate for Dry Powder Inhalation System in Monkeys

Ghrelin is the peptide that increases the hunger sensation and food intake and is expected to be clinically applied for treatment of diseases such as cachexia and anorexia nervosa. In the clinical application of ghrelin, injections are problematic in that they are invasive and inconvenient. Thus, we aimed to develop a formulation that can eliminate the need for injections and can be applied clinically. We prepared formulations of an hGhrelin derivative, in which the octanoyl group essential for expression of activity is modified to avoid rapid des-acylation, using lyophilizate for a dry powder inhalation (LDPI) system. The formulation of hGhrelin derivative was optimized by the addition of phenylalanine, of which the fine particle fraction of 5 µm or less was 41.7 ± 3.8%. We also performed pharmacokinetic/pharmacodynamic tests in monkeys using the optimum formulation that can be applied clinically. The absolute bioavailability of inhaled hGhrelin derivative with respect to that intravenously injected was 16.9 ± 2.6%. An increase in growth hormone was shown as an effect of the inhaled hGhrelin derivative similar to intravenous injection. The LDPI formulation can deliver the hGhrelin derivative systemically, and it is expected to be applied clinically as a substitute for injections.

Deciphering the enigmatic role of the amidotransferase LipL inBacillus subtilislipoic acid utilization

Lipoate is an essential cofactor for key enzymes of oxidative and one-carbon metabolism. It is covalently attached to E2 subunits of dehydrogenase (DH) complexes and the GcvH subunit of the glycine cleavage system.Bacillus subtilispossess two protein lipoylation pathways: biosynthesis and scavenging. The former requires octanoylation of GcvH, amidotransfer of the octanoate to E2s, and insertion of sulfur atoms. Lipoate scavenging is mediated by a lipoate ligase (LplJ), that catalizes a classical two-step ATP-dependent reaction. Although these pathways were thought to be redundant, a ΔlipLmutant, unable to transfer the octanoyl group from GcvH to the E2s during lipoate synthesis, showed growth defects in minimal media even when supplemented with this cofactor, despite the presence of a functional LplJ. In this study we demonstrated that LipL is essential to modify E2 subunits of branched chain ketoacid and pyruvate DH during lipoate scavenging. LipL must be functional and it is not forming a complex with LplJ, which suggests that these enzymes might be acting sequentially. We also show that the E2 subunit of oxoglutarate DH is a good donor for LipL amidotransfer reaction. The essential role of LipL during lipoate utilization relies on the strict substrate specificity of LplJ, determined by charge complementarity between the ligase and the lipoylable subunits. LplJ does not recognize E2 subunits without a negatively charged residue in key positions of the target protein, and thus LipL is required to transfer the lipoate to them. This model of lipoate scavenging seems widespread among Gram-positive bacteria.

Fusariumins B-D, Three New Alkylamine Octanoylated Derivatives from Fusarium sp

Three new alkylamine octanoylated derivatives, fusariumins B-D (1-3), and four known compounds 4-7 were isolated from the culture broth of the fungus Fusarium sp. Compounds 1-3 maintaining an unusual tetradecan-2-amine or tetradecan-3-amine moiety with an octanoyl group were characterized based on HRMS, 1D and 2D NMR spectroscopic data.