Extrahepatic in vitro metabolism of peptides; comparison of human kidney and intestinal S9 fraction, human plasma and proximal tubule cells, using cyclosporine A, leuprorelin, and cetrorelix as model compounds.

Peptide therapeutics showcase number of advantages compared to the traditional small molecule drugs, e,g. they usually have higher affinity to target and lower toxicity profiles. Endogenous peptides are mostly cleared from the body through renal clearance or proteolytic hydrolysis. As a part of drug discovery, metabolite identification is an important part in their development to identify metabolic hot spots and to further improve their stability. As the catabolism of the peptides and peptide-like drugs is often considered to be extrahepatic, the use of in vitro systems derived from these organs might be beneficial. In this study, multiple extrahepatic metabolic systems were evaluated for the applicability for peptide metabolism studies. Three peptide drugs (leuprorelin, cetrorelix, cyclosporin) were incubated in kidney and intestinal S9 fraction ( ± NADPH), fresh plasma (anticoagulants EDTA and heparin separately), and plated proximal tubule cells. Additionally, leuprorelin was also incubated with human kidney microsomes and cytosol to further investigate the NADPH-dependent metabolism detected in kidney S9 fraction. Both substrate disappearance and metabolite formation were monitored, using UPLC/HR-MS analysis of the collected samples.Overall, the largest number of metabolites was formed in the incubation with kidney S9 fraction, followed by intestinal S9, while incubations with proximal tubule cells produced lower number of metabolites All investigated peptides were stable in plasma and only a few metabolites were detected, likely because the studied peptide drugs have been optimized to be stable in plasma. Leuprorelin showed NADPH-dependent metabolite formation in kidney S9 fraction, while the metabolism of cetrorelix was more NADPH independent. As expected, formation of cytochrome P450 (CYP) catalyzed metabolism of cyclosporine was not observed with the employed extrahepatic systems. The NADPH-dependent metabolism of leuprorelin was detected also in the incubation with kidney cytosol, but not with kidney microsomes, and was thus not caused by CYPs or FMOs, but with cytosolic NADPH-dependent drug metabolizing enzymes. These enzymes could, in principle, activate the amide bond via reductive or oxidative metabolism outside the amide bond. The identity of the involved drug metabolizing enzymes in this process is still unknown.

Hepatic in vitro metabolism of peptides; Comparison of human liver S9, hepatocytes and Upcyte hepatocytes with cyclosporine A, leuprorelin, desmopressin and cetrorelix as model compounds.

The number of approved peptide therapeutics has increased significantly in recent years. Peptide therapeutics have many advances over small molecule drugs, such as higher affinity to target and lower toxicity profiles. Although peptide-like drugs are mainly metabolized/catabolized in the body for smaller peptides and amino acids, metabolite identification still has an essential part of in their development, especially if their structure contains modified amino acids, and also to identify the metabolic soft spots enabling modification to more stable sequence. The use of human derived in vitro systems is an important tool when investigating metabolism of peptide drugs, and comparison of results by various hepatic systems was investigated here. Peptides were incubated in several different in vitro human liver-derived subcellular and cellular incubation systems, i.e. liver S9 fraction, suspended cryo-preserved human primary hepatocytes and plated Upcyte hepatocytes. Samples were collected at different time points and analysed by UPLC/HR-MS-method developed for the purpose. Both substrate disappearance and metabolite formation were monitored, and the systems were compared. S9 fraction formed the highest number of metabolites for leuprorelin and cetrorelix, while for desmopressin and cyclosporin, primary hepatocytes and liver S9 produced similar metabolite profiles. Interestingly, not only cyclosporin, but also leuprorelin and cetrorelix showed metabolites whose formation was CYP (NADPH) dependent in liver S9. For leuprorelin and cetrorelix, the metabolites that showed NADPH dependency with liver S9, were not detected with hepatocytes, even though for leuprorelin these reactions played a major role in liver S9. The hydrolytic metabolic reactions were very similar between liver S9 and hepatocytes, i.e. the metabolite profiles in hepatocytes matched better with liver S9 profiles without NADPH, which may be caused by cell uptake rate limitation with hepatocytes, or then hydrolytic processes are more stressed in peptide metabolism with hepatocytes, in comparison to CYP-mediated processes.

Pharmacokinetics of intra-articular vitamin D analogue calcipotriol in sheep and metabolism in human synovial and mesenchymal stromal cells.

Calcipotriol (MC903) is a side chain analogue of the biologically active 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Due to its anti-inflammatory and anti-proliferative effects on stromal cells, calcipotriol is a promising candidate for the local treatment of arthritis. In this preliminary work, we studied the pharmacokinetics and safety of calcipotriol after an IV (0.1 mg/kg given to one sheep) and intra-articular dose (0.054 mg/kg, 0.216 mg/kg and 0.560 mg/kg given to three sheep). The terminal half-life of calcipotriol was approximately 1 h after an IV dose. After intra-articular dosing, the systemic absorption was between 1 and 13% during the observed 24 h. Hypercalcemia or other clinical adverse effects did not occur in any animal during the study, and no macroscopic or microscopic alterations were seen in the synovium of the calcipotriol-injected knees compared to the vehicle knees. The in vitro metabolism of calcipotriol was analyzed with LC-MS from human synovial and mesenchymal stromal cell cultures. Both cell types were able to metabolize calcipotriol with MC1080 and MC1046 as the main metabolites. CYP24A1 transcripts were strongly induced by a 48-hour calcipotriol exposure in mesenchymal stromal cells, but not consistently in synovial stromal cells, as determined by RT-qPCR. Calcipotriol proved to be safe after a single intra-articular dose with applied concentrations, and it is metabolized by the cells of the joint. Slow dissolution of calcipotriol crystals in the joint can extend the pharmaceutical impact on the synovium, cartilage and subcortical bone.

Intestinal microbiome as a risk factor for urinary tract infections in children.

As urinary tract infection (UTI) pathogens originate from the gut, we hypothesized that the gut environment reflected by intestinal microbiome influences the risk of UTI. Our prospective case-control study compared the intestinal microbiomes of 37 children with a febrile UTI with those of 69 healthy children. We sequenced the regions of the bacterial 16S rRNA gene and used the LefSe algorithm to calculate the size of the linear discriminant analysis (LDA) effect. We measured fecal lactoferrin and iron concentrations and quantitative PCR for Escherichia coli. At the phylum level, there were no significant differences. At the genus level, Enterobacter was more abundant in UTI patients with an LDA score > 3 (log 10), while Peptostreptococcaceae were more abundant in healthy subjects with an LDA score > 3 (log 10). In total, 20 OTUs with significantly different abundances were observed. Previous use of antimicrobials did not associate with intestinal microbiome. The relative abundance of E. coli was 1.9% in UTI patients and 0.5% in controls (95% CI of the difference-0.8 to 3.6%). The mean concentration of E.coli in quantitative PCR was 0.14 ng/µl in the patients and 0.08 ng/µl in the controls (95% CI of the difference-0.04 to 0.16). Fecal iron and lactoferrin concentrations were similar between the groups. At the family and genus level, we noted several differences in the intestinal microbiome between children with UTI and healthy children, which may imply that the gut environment is linked with the risk of UTI in children.