PET-CT imaging with [(18)F]-gefitinib to measure Abcb1a/1b (P-gp) and Abcg2 (Bcrp1) mediated drug-drug interactions at the murine blood-brain barrier.

INTRODUCTION: The efflux transporters P-glycoprotein (P-gp, ABCB1) and breast cancer resistance protein (BCRP, ABCG2) are expressed at the blood-brain barrier (BBB), and can limit the access of a wide range of drugs to the brain. In this study we developed a PET-CT imaging method for non-invasive, quantitative analysis of the effect of ABCB1 and ABCG2 on brain penetration of the anti-cancer drug gefitinib, and demonstrated the applicability of this method for identification and quantification of potential modulators of ABCB1 and ABCB2 using the dual inhibitor elacridar. METHODS: In vitro cellular accumulation studies with [(14)C]-gefitinib were conducted in LLC-PK1, MDCKII, and the corresponding ABCB1/Abcb1a and ABCG2/Abcg2 overexpressing cell lines. Subsequently, in vivo brain penetration of [(18)F]-gefitinib was quantified by PET-CT imaging studies in wild-type, Abcg2(-/-), Abcb1a/1b(-/-), and Abcb1a/1b;Abcg2(-/-) mice. RESULTS: In vitro studies showed that [(14)C]-gefitinib is a substrate of the human ABCB1 and ABCG2 transporters. After i.v. administration of [(18)F]-gefitinib (1mg/kg), PET-CT imaging showed 2.3-fold increased brain levels of [(18)F]-gefitinib in Abcb1a/1b;Abcg2(-/-) mice, compared to wild-type. Levels in single knockout animals were not different from wild-type, showing that Abcb1a/1b and Abcg2 together limit access of [(18)F]-gefitinib to the brain. Furthermore, enhanced brain accumulation of [(18)F]-gefitinib after administration of the ABCB1 and ABCG2 inhibitor elacridar (10 mg/kg) could be quantified with PET-CT imaging. CONCLUSIONS: PET-CT imaging with [(18)F]-gefitinib is a powerful tool to non-invasively assess potential ABCB1- and ABCG2-mediated drug-drug interactions (DDIs) in vivo. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: This minimally-invasive, [(18)F]-based PET-CT imaging method shows the interplay of ABCB1 and ABCG2 at the BBB in vivo. The method may be applied in the future to assess ABCB1 and ABCG2 activity at the BBB in humans, and for personalized treatment with drugs that are substrates of ABCB1 and/or ABCG2.

Validation of interventional fiber optic spectroscopy with MR spectroscopy, MAS-NMR spectroscopy, high-performance thin-layer chromatography, and histopathology for accurate hepatic fat quantification.

OBJECTIVES: To validate near-infrared (NIR)-based optical spectroscopy measurements of hepatic fat content using a minimally invasive needle-like probe with integrated optical fibers, enabling real-time feedback during percutaneous interventions. The results were compared with magnetic resonance spectroscopy (MRS) as validation and with histopathology, being the clinical gold standard. Additionally, ex vivo magic angle spinning nuclear magnetic resonance spectroscopy and high-performance thin-layer chromatography were performed for comparison. MATERIALS AND METHODS: Ten mice were used for the study, of which half received a regular chow diet and the other half received a high-fat diet to induce obesity and hepatosteatosis. The mice were imaged with a clinical 3-Tesla MR to select a region of interest within the right and left lobes of the liver, where MRS measurements were acquired in vivo. Subsequently, optical spectra were measured ex vivo at the surface of the liver at 6 different positions immediately after resection. Additionally, hepatic fat was determined by magic angle spinning nuclear magnetic resonance spectroscopy and high-performance thin-layer chromatography. Histopathologic analyses were performed and used as the reference standard. Pearson correlation and linear regression analyses were performed to assess the correlation of the various techniques with NIR. A 1-way analysis of variance including post hoc Tukey multiple comparison tests was used to study the difference in fat estimation between the various techniques. RESULTS: For both the mice groups, the estimated fat fractions by the various techniques were significantly similar (P = 0.072 and 0.627 for chow diet and high-fat diet group, respectively). The Pearson correlation value between NIR and the other techniques for fat determination showed the same strong linear correlation (P above 0.990; P < 0.001), whereas for histopathologic analyses, which is a rather qualitative measure, the Pearson correlation value was slightly lower (P = 0.925, P < 0.001) . Linear regression coefficient computed to compare NIR with the other techniques resulted in values close to unity with MRS having the narrowest confidence interval (r = 0.935, 95% confidence interval: 0.860-1.009), demonstrating highly correlating results between NIR and MRS. CONCLUSIONS: NIR spectroscopy measurements from a needle-like probe with integrated optical fibers for sensing at the tip of the needle can quickly and accurately determine hepatic fat content during an interventional procedure and might therefore be a promising novel diagnosing tool in the clinic.

Automated synthesis of [18F]gefitinib on a modular system.

In recent years, [(18)F]gefitinib PET has successfully been employed for a number of applications ranging from oncology to in vivo studies of drug transporter proteins. We here report a reliable, automated procedure for routine synthesis of this radiotracer on an Eckert and Ziegler modular system. The 3-step radiosynthesis followed by preparative HPLC-purification provided [(18)F]gefitinib in 17.2±3.3% (n=22) overall decay-corrected radiochemical yield with radiochemical purity >99% in a total synthesis time of about 2.5h.

Evaluation of a 99mTc-labeled AnnexinA5 variant for non-invasive SPECT imaging of cell death in liver, spleen and prostate.

PURPOSE: We investigate radio-labeling and pharmacokinetics of a new AnnexinA5 variant (HYNIC-cys-AnxA5) and then assess its utility for the non-invasive detection of cell death in liver, spleen and prostate. METHODS: AnnexinA5 binds to phosphatidylserine expressed on the surface of apoptotic and necrotic cells. Contrary to other AnnexinA5 variants, the new cys-AnxA5 allows for site-specific conjugation of a hydrazinonicotinamide-maleimide moiety and subsequent radio-labeling with (99m)Tc at a position not involved in the AnxA5-phosphatidylserine interaction. Distribution of (99m)Tc-HYNIC-cys-AnxA5 was studied in rats, both invasively and via SPECT/CT. Cycloheximide was used to induce cell death in liver and spleen, whereas apoptosis in the prostate was induced by castration. RESULTS: HYNIC-cys-AnxA5 was efficiently and reproducibly labeled with (99m)Tc. Blood clearance of radioactivity after iv-injection was adequately described by a two-compartment model, the renal cortex representing the main site of accumulation. Cycloheximide treatment resulted in increased accumulation of intravenous-injected (99m)Tc-HYNIC-cys-AnxA5 in liver and spleen over controls, which correlated well with TUNEL staining for cell death in corresponding tissue sections. However, the increase in TUNEL-positive prostate epithelial cells observed following castration was not paralleled by greater (99m)Tc-HYNIC-cys-AnxA5 accumulation. CONCLUSION: (99m)Tc-HYNIC-cys-AnxA5 appears a suitable tracer for assessment of cell death in liver and spleen, but not prostate.

A highly active adipyl-cephalosporin acylase obtained via rational randomization.

There is strong interest in creating an enzyme that can deacylate natural cephalosporins such as cephalosporin C in order to efficiently acquire the starting compound for the industrial production of semisynthetic cephalosporin antibiotics. In this study, the active site of the glutaryl acylase from Pseudomonas SY-77 was randomized rationally. Several mutations that were found in previous studies to enhance the activity of the enzyme towards adipyl-7-aminodesacetoxycephalosporanic acid (ADCA) and cephalosporin C have now been combined, and libraries have been made in which random amino acid substitutions at these positions are joined. The mutants were expressed in a leucine-deficient Escherichia coli strain and subjected to growth selection with adipyl-leucine or amino-adipyl-leucine as sole leucine source. The mutants growing on these media were selected and purified, and their hydrolysis activities towards adipyl-7-ADCA and cephalosporin C were tested. Several mutants with highly improved activities towards the desired substrates were found in these rationally randomized libraries. The best mutant was selected from a library of totally randomized residues: 178, 266, and 375. This mutant comprises two mutations, Y178F + F375H, which synergistically improve the catalytic efficiency towards adipyl-7-ADCA 36-fold. The activity of this mutant towards adipyl-7-ADCA is 50% of the activity of the wild-type enzyme towards the preferred substrate glutaryl-7-aminocephalosporanic acid, and therefore the characteristics of this mutant approach those needed for industrial application.

Quorum quenching by an N-acyl-homoserine lactone acylase from Pseudomonas aeruginosa PAO1.

The virulence of the opportunistic human pathogen Pseudomonas aeruginosa PAO1 is controlled by an N-acyl-homoserine lactone (AHL)-dependent quorum-sensing system. During functional analysis of putative acylase genes in the P. aeruginosa PAO1 genome, the PA2385 gene was found to encode an acylase that removes the fatty acid side chain from the homoserine lactone (HSL) nucleus of AHL-dependent quorum-sensing signal molecules. Analysis showed that the posttranslational processing of the acylase and the hydrolysis reaction type are similar to those of the beta-lactam acylases, strongly suggesting that the PA2385 protein is a member of the N-terminal nucleophile hydrolase superfamily. In a bioassay, the purified acylase was shown to degrade AHLs with side chains ranging in length from 11 to 14 carbons at physiologically relevant low concentrations. The substituent at the 3' position of the side chain did not affect activity, indicating broad-range AHL quorum-quenching activity. Of the two main AHL signal molecules of P. aeruginosa PAO1, N-butanoyl-l-homoserine lactone (C4-HSL) and N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL), only 3-oxo-C12-HSL is degraded by the enzyme. Addition of the purified protein to P. aeruginosa PAO1 cultures completely inhibited accumulation of 3-oxo-C12-HSL and production of the signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone and reduced production of the virulence factors elastase and pyocyanin. Similar results were obtained when the PA2385 gene was overexpressed in P. aeruginosa. These results demonstrate that the protein has in situ quorum-quenching activity. The quorum-quenching AHL acylase may enable P. aeruginosa PAO1 to modulate its own quorum-sensing-dependent pathogenic potential and, moreover, offers possibilities for novel antipseudomonal therapies.

Improved beta-lactam acylases and their use as industrial biocatalysts.

Whereas the beta-lactam acylases are traditionally used for the hydrolytic processing of penicillin G and cephalosporin C, new and mutated acylases can be used for the hydrolysis of alternative fermentation products as well as for the synthesis of semisynthetic beta-lactam antibiotics. Three-dimensional structural analyses and site-directed mutagenesis studies have increased the understanding of the catalytic mechanism of these enzymes. The yield of hydrolysis and synthesis has been greatly improved by process design, including immobilization of the enzyme and the use of alternative reaction media. Significant advances have also been made in the resolution of racemic mixtures by means of stereoselective acylation/hydrolysis using beta-lactam acylases.

Mutational analysis of a key residue in the substrate specificity of a cephalosporin acylase.

beta-Lactam acylases are crucial for the synthesis of semisynthetic cephalosporins and penicillins. Unfortunately, there are no cephalosporin acylases known that can efficiently hydrolyse the amino-adipic side chain of Cephalosporin C. In a previous directed evolution experiment, residue Asn266 of the glutaryl acylase from Pseudomonas SY-77 was identified as being important for substrate specificity. In order to explore the function of this residue in substrate specificity, we performed a complete mutational analysis of position 266. Codons for all amino acids were introduced in the gene, 16 proteins that could be functionally expressed in Escherichia coli were purified to homogeneity and their catalytic parameters were determined. The mutant enzymes displayed a broad spectrum of affinities and activities, pointing to the flexibility of the enzyme at this position. Mutants in which Asn266 was changed into Phe, Gln, Trp and Tyr displayed up to twofold better catalytic efficiency (k(cat)/K(m))than the wild-type enzyme when adipyl-7-aminodesacetoxycephalosporanic acid (adipyl-7-ADCA) was used as substrate, due to a decreased K(m). Only mutants SY-77(N266H) and SY-77(N266M) showed an improvement of both catalytic parameters, resulting in 10- and 15-times higher catalytic efficiency with adipyl-7-ADCA, respectively. Remarkably, the catalytic activity (k(cat)) of SY-77(N266M) when using adipyl-7-ADCA as substrate was as high as when glutaryl-7-aminocephalosporanic acid (glutaryl-7-ACA) was used, and approaches commercially interesting activity. SY-77(N266Q), SY-77(N266H) and SY-77(N266M) mutants showed a modest improvement in hydrolysing Cephalosporin C. Since these mutants also have a good catalytic efficiency when adipyl-7-ADCA is used and are still active towards glutaryl-7-ACA, they can be regarded as broad substrate acylases. These results demonstrate that the combination of directed evolution for the identification of important positions, together with saturation mutagenesis for finding the optimal amino acid, is a very effective method for finding improved biocatalysts.

Analysis of a substrate specificity switch residue of cephalosporin acylase.

Residue Phe375 of cephalosporin acylase has been identified as one of the residues that is involved in substrate specificity. A complete mutational analysis was performed by substituting Phe375 with the 19 other amino acids and characterising all purified mutant enzymes. Several mutations cause a substrate specificity shift from the preferred substrate of the enzyme, glutaryl-7-ACA, towards the desired substrate, adipyl-7-ADCA. The catalytic efficiency ( [Formula: see text] (cat)/ [Formula: see text] (m)) of mutant SY-77(F375C) towards adipyl-7-ADCA was increased 6-fold with respect to the wild-type enzyme, due to a strong decrease of [Formula: see text] (m). The [Formula: see text] (cat) of mutant SY-77(F375H) towards adipyl-7-ADCA was increased 2.4-fold. The mutational effects point at two possible mechanisms by which residue 375 accommodates the long side chain of adipyl-7-ADCA, either by a widening of a hydrophobic ring-like structure that positions the aliphatic part of the side chain of the substrate, or by hydrogen bonding to the carboxylate head of the side chain.

Directed evolution of a glutaryl acylase into an adipyl acylase.

Semi-synthetic cephalosporin antibiotics belong to the top 10 of most sold drugs, and are produced from 7-aminodesacetoxycephalosporanic acid (7-ADCA). Recently new routes have been developed which allow for the production of adipyl-7-ADCA by a novel fermentation process. To complete the biosynthesis of 7-ADCA a highly active adipyl acylase is needed for deacylation of the adipyl derivative. Such an adipyl acylase can be generated from known glutaryl acylases. The glutaryl acylase of Pseudomonas SY-77 was mutated in a first round by exploration mutagenesis. For selection the mutants were grown on an adipyl substrate. The residues that are important to the adipyl acylase activity were identified, and in a second round saturation mutagenesis of this selected stretch of residues yielded variants with a threefold increased catalytic efficiency. The effect of the mutations could be rationalized on hindsight by the 3D structure of the acylase. In conclusion, the substrate specificity of a dicarboxylic acid acylase was shifted towards adipyl-7-ADCA by a two-step directed evolution strategy. Although derivatives of the substrate were used for selection, mutants retained activity on the beta-lactam substrate. The strategy herein described may be generally applicable to all beta-lactam acylases.

Altering the substrate specificity of cephalosporin acylase by directed evolution of the Beta -subunit.

Using directed evolution, we have selected an adipyl acylase enzyme that can be used for a one-step bioconversion of adipyl-7-aminodesacetoxycephalosporanic acid (adipyl-7-ADCA) to 7-ADCA, an important compound for the synthesis of semisynthetic cephalosporins. The starting point for the directed evolution was the glutaryl acylase from Pseudomonas SY-77. The gene fragment encoding the beta-subunit was divided into five overlapping parts that were mutagenized separately using error-prone PCR. Mutants were selected in a leucine-deficient host using adipyl-leucine as the sole leucine source. In total, 24 out of 41 plate-selected mutants were found to have a significantly improved ratio of adipyl-7-ADCA versus glutaryl-7-ACA hydrolysis. Several mutations around the substrate-binding site were isolated, especially in two hot spot positions: residues Phe-375 and Asn-266. Five mutants were further characterized by determination of their Michaelis-Menten parameters. Strikingly, mutant SY-77(N266H) shows a nearly 10-fold improved catalytic efficiency (k(cat)/K(m)) on adipyl-7-ADCA, resulting from a 50% increase in k(cat) and a 6-fold decrease in K(m), without decreasing the catalytic efficiency on glutaryl-7-ACA. In contrast, the improved adipyl/glutaryl activity ratio of mutant SY-77(F375L) mainly is a consequence of a decreased catalytic efficiency toward glutaryl-7-ACA. These results are discussed in the light of a structural model of SY-77 glutaryl acylase.