Synthesis and Evaluation of Polymyxins Bearing Reductively Labile Disulfide-Linked Lipids

Polymyxins are a class of lipopeptide anti-infective agents with potent and specific activity against Gram-negative bacteria. While toxicity concerns associated with polymyxin B and E (colistin) have historically limited their clinical application, today they are increasingly used as last-resort antibiotics given the rise of multidrug-resistant Gram-negative pathogens. The adverse side effects of polymyxins are well known, particularly as related to their nephrotoxicity. Here, we describe the synthesis and evaluation of a novel series of polymyxin analogues, aimed at reducing their nephrotoxic effects. Using a semisynthetic approach, we explored modifications of the exocyclic part of the polymyxin scaffold, namely, the terminal amino acid and lipophilic tail. By incorporating a reductively labile disulfide linkage in the lipid tail, we obtained novel polymyxins that exhibit potent antibacterial activity on par with polymyxin B but with reduced toxicity toward human renal proximal tubular epithelial cells

Synthesis and Evaluation of Polymyxins Bearing Reductively Labile Disulfide-Linked Lipids

Polymyxins are a class of lipopeptide anti-infective agents with potent and specific activity against Gram-negative bacteria. While toxicity concerns associated with polymyxin B and E (colistin) have historically limited their clinical application, today they are increasingly used as last-resort antibiotics given the rise of multidrug-resistant Gram-negative pathogens. The adverse side effects of polymyxins are well known, particularly as related to their nephrotoxicity. Here, we describe the synthesis and evaluation of a novel series of polymyxin analogues, aimed at reducing their nephrotoxic effects. Using a semisynthetic approach, we explored modifications of the exocyclic part of the polymyxin scaffold, namely, the terminal amino acid and lipophilic tail. By incorporating a reductively labile disulfide linkage in the lipid tail, we obtained novel polymyxins that exhibit potent antibacterial activity on par with polymyxin B but with reduced toxicity toward human renal proximal tubular epithelial cells

The Functional Implications of Common Genetic Variation in <i>CYP3A5</i> and <i>ABCB1</i> in Human Proximal Tubule Cells

<i>Background:</i> Calcineurin inhibitors (CNIs) are the primary immunosuppressive drugs used in solid organ transplantation but are associated with the development of histological lesions leading to kidney failure. CNIs are metabolized by CYP3A and excreted by not only P-glycoprotein (P-gp) (<i>ABCB1</i>) in the gut and liver, but also by proximal tubule cells (PTCs) in the kidney. Multiple studies have demonstrated the importance of genetic variation in <i>CYP3A5</i> and <i>ABCB1</i> for CNI disposition and nephrotoxicity. The present study was designed to study the functional implication of variation in these two genes in human PTCs. <i>Methods</i>: A technique was developed to culture cells from renal tissue obtained from renal graft recipients by routine kidney biopsy. Primary cells were immortalized, subcloned, and then characterized for specific PTC markers (AQP1, CD13, brush border morphology) and donor <i>CYP3A5</i>(<i>rs776746</i>)/<i>ABCB1</i>(<i>rs1045642</i>) genotype. We then selected specific sets of confirmed conditionally immortalized PTCs (ciPTC) according to different combinations of the aforementioned genetic variants. Quantitative real-time polymerase chain reaction, Western blot, and immunohistochemistry were performed for studying <i>CYP3A5</i> and <i>ABCB1</i> expression. <i>CYP3A5</i> activity was assessed by differential midazolam (MDZ) hydroxylation and P-gp (<i>ABCB1</i> product) activity by a calcein efflux assay. Differential drug metabolism between cell lines was assessed by tacrolimus disappearance over 24 h. <i>Results:</i> Cell lines were generated from 27 out of 38 tissue samples. On the basis of genotype and PTC biomarkers, 11 subclones were selected. <i>In vitro</i> PTC morphology with brush border microvilli was confirmed. <i>CYP3A5*1</i> carriers had increased 1-OH/4-OH MDZ formation versus homozygous <i>*3</i> carriers (mean: 2.36 (95% CI:1.11–3.40) vs 0.88 (95% CI:0.48–1.27); <i>p</i> < 0.05). P-gp activity was confirmed by calcein accumulation (mean 38.6%; 95% CI:32.8–44.4%), which was higher in cell lines with the <i>ABCB1 3435TT</i> than the <i>3435CC</i>/<i>CT</i> genotype (46.2% vs 35.5%; 95% CI:28.7–42.2%). Tacrolimus disappearance was about two-fold higher in cell lines with the combined <i>CYP3A5*1</i>/<i>ABCB1 3435TT</i> genotype versus other genotype combinations. <i>Conclusion:</i> Biopsy-derived and immortalized human PTC cell lines demonstrate functional expression of genes involved in CNI metabolism. Differences in functional expression were detected according to common genetic variants in <i>CYP3A5</i> and <i>ABCB1</i>. The studied genetic variants had a significant impact on <i>in vitro</i> tacrolimus metabolism. In particular, ciPTC with the combined <i>CYP3A5*1/ABCB1 3435TT</i> genotype demonstrated higher tacrolimus disappearance versus ciPTCs with a different pharmacogenetic profile. This <i>in vitro</i> model stresses the importance of the incorporation of pharmacogenetic variation in studies involved in (renal) drug disposition

Flow chart of study selection.

<p>The number of studies in each phase is indicated between brackets.</p

Effect of IPC on serum creatinine after renal IRI.

<p>Left side favours control (renal IRI only), right side favours IPC. An overall beneficial effect of IPC on serum creatinine was observed (1.54 [1.16, 1.93]). Data presented as SMD and 95% CI.</p

Accumulation of uremic solutes.

<p>500 MHz ¹H-NMR spectrum of plasma (ultrafiltrate) from (<b>A</b>) a healthy control and (<b>B</b>) a CKD patient. Insets show 5 regions of interest in greater detail. Metabolite abnormalities: Creatinine (1 and 2), 1-methylhistidine (3), 3-methylhistidine (4), <i>myo</i>-Inositol (5), trimethylamine <i>N</i>-oxide (6), dimethyl sulphone (7), <i>N,N</i>-dimethylglycine (8) and 2-hydroxyisobutyric acid (9).</p

Optimized Metabolomic Approach to Identify Uremic Solutes in Plasma of Stage 3–4 Chronic Kidney Disease Patients

<div><p>Background</p><p>Chronic kidney disease (CKD) is characterized by the progressive accumulation of various potential toxic solutes. Furthermore, uremic plasma is a complex mixture hampering accurate determination of uremic toxin levels and the identification of novel uremic solutes.</p><p>Methods</p><p>In this study, we applied ¹H-nuclear magnetic resonance (NMR) spectroscopy, following three distinct deproteinization strategies, to determine differences in the plasma metabolic status of stage 3–4 CKD patients and healthy controls. Moreover, the human renal proximal tubule cell line (ciPTEC) was used to study the influence of newly indentified uremic solutes on renal phenotype and functionality.</p><p>Results</p><p>Protein removal via ultrafiltration and acetonitrile precipitation are complementary techniques and both are required to obtain a clear metabolome profile. This new approach, revealed that a total of 14 metabolites were elevated in uremic plasma. In addition to confirming the retention of several previously identified uremic toxins, including p-cresyl sulphate, two novel uremic retentions solutes were detected, namely dimethyl sulphone (DMSO₂) and 2-hydroxyisobutyric acid (2-HIBA). Our results show that these metabolites accumulate in non-dialysis CKD patients from 9±7 µM (control) to 51±29 µM and from 7 (0–9) µM (control) to 32±15 µM, respectively. Furthermore, exposure of ciPTEC to clinically relevant concentrations of both solutes resulted in an increased protein expression of the mesenchymal marker vimentin with more than 10% (p<0.05). Moreover, the loss of epithelial characteristics significantly correlated with a loss of glucuronidation activity (Pearson r = −0.63; p<0.05). In addition, both solutes did not affect cell viability nor mitochondrial activity.</p><p>Conclusions</p><p>This study demonstrates the importance of sample preparation techniques in the identification of uremic retention solutes using ¹H-NMR spectroscopy, and provide insight into the negative impact of DMSO₂ and 2-HIBA on ciPTEC, which could aid in understanding the progressive nature of renal disease.</p></div

Effect of IPC on BUN after renal IRI.

<p>Left side favours control (renal IRI only), right side favours IPC. An overall beneficial effect of IPC on BUN was observed (1.42 [0.97, 1.87]). Data presented as SMD and 95% CI.</p

Relative retention of uremic solutes in stage 3–4 CKD patients.

<p>The M/N index is the ratio of the mean uremic concentration (M) found in the present study to the normal concentration (N) measured in healthy controls reported in literature.</p

¹H resonance assignments and plasma concentrations of uremic solutes in stage 3–4 CKD patients.

<p>Values are shown as mean (C_u) ± SD or range (µM) and maximal uremic concentration (C_max,). ND, not detected; NA, not applicable.</p>a<p>Numbers correspond to peaks in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071199#pone-0071199-g003" target="_blank">Fig. 3</a>.</p>b<p>Hypothetical C_max calculated as C_max = C_u +2 SD, as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071199#pone.0071199-Mutsaers1" target="_blank">[2]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071199#pone.0071199-Vanholder1" target="_blank">[3]</a>.</p>c<p>Data obtained from the Human Metabolome Database (<a href="http://www.hmdb.ca" target="_blank">www.hmdb.ca</a>) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0071199#pone.0071199-Wishart1" target="_blank">[42]</a>.</p>d<p>Only detected in one patient.</p