Hepatic arterial infusion of irinotecan and EmboCept® S results in high tumor concentration of SN-38 in a rat model of colorectal liver metastases.

Intraarterial chemotherapy for colorectal liver metastases (CRLM) can be applied alone or together with embolization particles. It remains unclear whether different types of embolization particles lead to higher intratumoral drug concentration. Herein, we quantified the concentrations of CPT-11 and its active metabolite SN-38 in plasma, liver and tumor tissue after hepatic arterial infusion (HAI) of irinotecan, with or without further application of embolization particles, in a rat model of CRLM. Animals underwent either systemic application of irinotecan, or HAI with or without the embolization particles Embocept® S and Tandem™. Four hours after treatment concentrations of CPT-11 and SN-38 were analyzed in plasma, tumor and liver samples by high-performance liquid chromatography. Additionally, DNA-damage and apoptosis were analyzed immunohistochemically. Tumor tissue concentrations of SN-38 were significantly increased after HAI with irinotecan and EmboCept® S compared to the other groups. The number of apoptotic cells was significantly higher after both HAI with irinotecan and EmboCept® S or Tandem™ loaded with irinotecan compared to the control group. HAI with irinotecan and EmboCept® S resulted in an increased SN-38 tumor concentration. Both HAI with irinotecan and EmboCept® S or Tandem™ loaded with irinotecan were highly effective with regard to apoptosis.

In vivo characterization of a new type of biodegradable starch microsphere for transarterial embolization.

Transarterial embolization is an established minimally invasive treatment for solid tumors. Unintended inflammation, foreign body reactions and ischemia-triggered neoangiogenesis are clinical drawbacks of permanent embolic materials. The aim of the current study was to characterize a new type of biodegradable starch microsphere with regard to angiographic and histopathological features such as patterns of acute arterial occlusion as well as induction of tissue necrosis, microsphere biodegradation, and inflammation and foreign body reactions during follow-up. Key characteristics of both biodegradable prototypes (L1 and L2; prototype groups) were as follows: microspheres are biodegradable by serum α-amylase, produced from chemically crosslinked potato starch to different extents, in a diameter range of ∼300-800 µm, differing in size distribution and featuring a microsphere deformation of ∼1%. In vivo transarterial embolization with L1 and L2, while applying clinical standard techniques, was performed and compared with clinically established permanent microspheres (Embosphere®500-700 and Embosphere®700-900; control groups). Twenty-four pig kidneys were embolized with the different embolic materials by following the study protocol, and there were no technical failures or complications. Parenchymal necrosis with interstitial calcification was observed in all kidneys independent of the type of embolic material used. Compared with the permanent embolic materials, biodegradable microspheres showed complete (L1) or partial (L2) biodegradation within one week after transarterial embolization, and induced a comparable (L1) or a lower (L2) degree of arterial wall necrosis and a lower degree of inflammation and foreign body reactions. In conclusion, the presented new type of biodegradable microsphere is promising, and could be further evaluated in terms of clinical translation.

Biological Role of Paenilarvins, Iturin-Like Lipopeptide Secondary Metabolites Produced by the Honey Bee Pathogen Paenibacillus larvae.

The Gram-positive bacterium Paenibacillus larvae (P. larvae) is the causative agent of a deadly honey bee brood disease called American Foulbrood (AFB). AFB is a notifiable epizootic in most countries and, hence, P. larvae is of considerable relevance for veterinarians and apiculturists alike. Over the last decade, much progress has been made in the understanding of the (patho)biology of P. larvae. Recently, several non-ribosomally produced peptides (NRP) and peptide/polyketide (NRP/PK) hybrids produced by P. larvae were identified. Among these NRPs were iturin-like lipopeptides, the paenilarvins A-C. Iturins are known to exhibit strong anti-fungal activity; for some iturins, cytotoxic activity towards mammalian erythrocytes and human cancer cell lines are described. We here present our results on the analysis of the natural function of the paenilarvins during pathogenesis of P. larvae infections. We demonstrated production of paenilarvins in infected larvae. However, we could neither demonstrate cytotoxicity of paenilarvins towards cultured insect cells nor towards larvae in feeding assays. Accordingly, exposure bioassays performed with larvae infected by wild-type P. larvae and a knockout mutant of P. larvae lacking production of paenilarvins did not substantiate a role for the paenilarvins as virulence factor. Further experiments are necessary to analyze the relevance of the paenilarvins' anti-fungal activity for P. larvae infections in the presence of fungal competitors in the larval midgut or cadaver.

Towards Biocontained Cell Factories: An Evolutionarily Adapted Escherichia coli Strain Produces a New-to-nature Bioactive Lantibiotic Containing Thienopyrrole-Alanine.

Genetic code engineering that enables reassignment of genetic codons to non-canonical amino acids (ncAAs) is a powerful strategy for enhancing ribosomally synthesized peptides and proteins with functions not commonly found in Nature. Here we report the expression of a ribosomally synthesized and post-translationally modified peptide (RiPP), the 32-mer lantibiotic lichenicidin with a canonical tryptophan (Trp) residue replaced by the ncAA L-ß-(thieno[3,2-b]pyrrolyl)alanine ([3,2]Tpa) which does not sustain cell growth in the culture. We have demonstrated that cellular toxicity of [3,2]Tpa for the production of the new-to-nature bioactive congener of lichenicidin in the host Escherichia coli can be alleviated by using an evolutionarily adapted host strain MT21 which not only tolerates [3,2]Tpa but also uses it as a proteome-wide synthetic building block. This work underscores the feasibility of the biocontainment concept and establishes a general framework for design and large scale production of RiPPs with evolutionarily adapted host strains.

Engineering the heterologous expression of lanthipeptides in Escherichia coli by multigene assembly.

Lantibiotics are an important class of ribosomally synthesised peptide antibiotics with a remarkable pharmacological potential. Structural variants of lantibiotics generated by peptide engineering in vivo are an important aspect for improving the peptide's efficacy, stability and bioavailability as well as production titre, which severely impacts the potential exploitation in pharmaceutical applications. Therefore, expression systems are needed which allow for a robust genetic access for ample mutagenesis experiments. Based on previous heterologous expression of the two-component lanthipeptide lichenicidin (Bliα and Bliß) in Escherichia coli BLic5, we now employ a multigene assembly strategy for recombinant lantibiotic peptide production in the Gram-negative host. Two E. coli high copy plasmids for separate and increased expression of a two-component lantibiotic were cloned and tested for expression. From these E. coli HP expression strains, an up to 100 times increased expression was found compared with Bacillus licheniformis I89 and E. coli BLic5. Total expression yields reach 4 mg L(-1) for Bliα and 6 mg L(-1) for Bliß. The expression system developed in this study constitutes an important cornerstone for future in vivo peptide engineering studies and is of significance for potential applications aiming at higher production titres of ribosomally synthesised, post translationally modified peptides.

Site-directed and global incorporation of orthogonal and isostructural noncanonical amino acids into the ribosomal lasso peptide capistruin.

Expansion of the structural diversity of peptide antibiotics was performed through two different methods. Supplementation-based incorporation (SPI) and stop-codon suppression (SCS) approaches were used for co-translational incorporation of isostructural and orthogonal noncanonical amino acids (ncAAs) into the lasso peptide capistruin. Two ncAAs were employed for the SPI method and five for the SCS method; each of them probing the incorporation of ncAAs in strategic positions of the molecule. Evaluation of the assembly by HR-ESI-MS proved more successful for the SCS method. Bio-orthogonal chemistry was used for post-biosynthetic modification of capistruin congener Cap_Alk10 containing the ncAA Alk (Nε-Alloc-L-lysine) instead of Ala. A second-generation Hoveyda-Grubbs catalyst was used for an in vitro metathesis reaction with Cap_Alk10 and an allyl alcohol, which offers options for post-biosynthetic modifications. The use of synthetic biology allows for the in vivo production of new peptide-based antibiotics from an expanded amino acid repertoire.

Congeneric lantibiotics from ribosomal in vivo peptide synthesis with noncanonical amino acids.

Expanded repetoire: Synthetic amino acids translated into propeptides dramatically increase the chemical diversity of the two-component lantibiotic lichenicidin. This opens new routes towards novel and unique peptide antibiotic sequences, which could display features important for medical applications.