The Seraph®-100 Microbind Affinity Blood Filter Does Not Affect Vancomycin, Tacrolimus, and Mycophenolic Acid Plasma Concentrations.

Extracorporeal blood purification is considered an adjunct therapy in critically ill patients with life-threatening conditions such as sepsis and septic shock. It consists of cytokine removal, removal of endotoxins, a combination of both, or the removal of pathogens themselves. The latter technique was introduced for clinical application very recently. This case study describes a case of a 69-year-old female lung transplant recipient patient with a persistent VV-ECMO-related septic deep vein thrombosis with continuous renal replacement therapy-dependent acute kidney injury initiated on the Seraph®-100 Microbind Affinity Filter in order to control the persistent bacteraemia with coagulase-negative staphylococci. Drug plasma concentrations (vancomycin, tacrolimus, and mycophenolic acid) were measured before and after the device to calculate absorber-related drug clearance.

A new efficient method of generating photoaffinity beads for drug target identification.

Affinity purification is one of the most prevalent methods for the target identification of small molecules. Preparation of an appropriate chemical for immobilization, however, is a tedious and time-consuming process. A decade ago, a photoreaction method for generating affinity beads was reported, where compounds are mixed with agarose beads carrying a photoreactive group (aryldiazirine) and then irradiated with ultraviolet light under dry conditions to form covalent attachment. Although the method has proven useful for identifying drug targets, the beads suffer from inefficient ligand incorporation and tend to shrink and aggregate, which can cause nonspecific binding and low reproducibility. We therefore decided to craft affinity beads free from these shortcomings without compromising the ease of preparation. We herein report a modified method; first, a compound of interest is mixed with a crosslinker having an activated ester and a photoreactive moiety on each end. This mixture is then dried in a glass tube and irradiated with ultraviolet light. Finally, the conjugates are dissolved and reacted with agarose beads with a primary amine. This protocol enabled us to immobilize compounds more efficiently (approximately 500-fold per bead compared to the original method) and generated beads without physical deterioration. We herein demonstrated that the new FK506-immobilized beads specifically isolated more FKBP12 than the original beads, thereby proving our method to be applicable to target identification experiments.

Trends in the biosynthesis and production of the immunosuppressant tacrolimus (FK506).

The current off-patent state of tacrolimus (FK506) has opened the hunting season for new generic pharmaceutical formulations of this immunosuppressant. This fact has boosted the scientific and industrial research on tacrolimus for the last 5 years in order to improve its production. The fast discovery of tacrolimus producer strains has generated a huge number of producers, which presents the biosynthetic cluster of FK506 as a high promiscuous genetic region. For the first time, the current state-of-the-art on the tacrolimus biosynthesis, production improvements and drug purification is reviewed. On one hand, all the genes involved in the tacrolimus biosynthesis, in addition to the traditional PKS/NRPS, as well as their regulation are analysed. On the other hand, tacrolimus direct and indirect precursors are reviewed as a straight manner to improve the final yield, which is a current trend in the field. Twenty years of industrial and scientific improvements on tacrolimus production are summarised, whereas future trends are also drafted.

Identification of Ascomycin against Zika virus infection through screening of natural product library.

Zika virus (ZIKV) infection could lead to Guillain-Barré syndrome in adults and microcephaly in the newborns from infected pregnant women. To date, there is no specific drug for the treatment of ZIKV infection. In this study, we sought to screen inhibitors against ZIKV infection from a natural product library. A ZIKV replicon was used to screen a library containing 1680 natural compounds. We explored the antiviral mechanism of the compound candidate in vitro and in vivo infection models. Ascomycin, a macrolide from Streptomyces hygroscopicus, was identified with inhibitory effect against ZIKV in Vero cells (IC50 = 0.11 µM), hepatoma cell Huh7 (IC50 = 0.38 µM), and glioblastoma cell SNB-19 (IC50 = 0.06 µM), far below the cytotoxic concentrations. Mechanistic study revealed that Ascomycin suppressed ZIKV RNA replication step during the life cycle and the regulation of calcineurin-NFAT pathway maybe involved in this inhibitory effect, independent of innate immunity activation. Moreover, we found that Ascomycin also inhibited the infection of other Flaviviridae members, such as hepatitis C virus and dengue virus. Ascomycin reduced ZIKV load in blood by up to 3500-fold in A129 mice. Meanwhile, the infection in the mice brain was undetectable by immunohistochemistry staining. Together, these findings reveal a critical role of Ascomycin in the inhibition of ZIKV and related viruses, facilitating the development of novel antiviral agents.

Trough tacrolimus concentrations in the first week after kidney transplantation are related to acute rejection.

There is evidence showing the importance of reaching immunosuppressant target concentrations as soon as possible. The aim of this study was to evaluate the relationship between tacrolimus trough concentrations within the first week after transplantation and the rate of acute rejection. In this descriptive-analytic study, we included 57 renal transplant patients receiving tacrolimus as the primary immunosuppressive drug. After univariate analysis, donor age, duration of hospital stay, and creatinine clearance (third month) showed significant differences between rejecters and nonrejecters. In addition, mean tacrolimus trough concentrations on day 5, day 7, mean of days 1-7, and mean of days 5-7 were found to be significantly lower in rejecters (P = 0.009, P = 0.012, P = 0.006, and P = 0.035, respectively). Receiver operating characteristic curve analysis with tacrolimus trough concentrations measured on days 5 and 7 was able to discriminate between patients with and without acute rejection (P = 0.028 and P = 0.048 after Bonferroni correction). The tacrolimus trough concentration with the best sensitivity-specificity balance was 9.3 ng/mL on day 5 and 8.7 ng/mL on day 7. In the Kaplan-Meier analysis, patients with tacrolimus trough concentrations below 9.3 mg/mL on day 5 showed a lower survival time without acute rejection (P = 0.048 after correction) in comparison with patients with tacrolimus trough concentrations above this concentration. After logistic regression, we obtained a model relating rejection with sex, donor age, and tacrolimus trough concentrations on day 5 (P = 0.004). No significant relationship between tacrolimus trough concentrations and delta creatinine clearance from week 1 to month 3 was obtained. These results confirm that tacrolimus trough concentrations during the first week are an important predictor of acute rejection. Therefore, it is critical to reach target blood concentrations of tacrolimus as soon as possible to improve allograft survival.

Procedure for chromatography involving sample solvent with higher elution strength than the mobile phase.

In preparative chromatography, often the solubility of the sample in the mobile phase is limited, making the mobile phase unsuitable as a solvent for preparation of load. Generally, solvents that have high solubility for the sample also have higher elution strengths than the mobile phase. Additionally, at high loading volumes, these strong sample solvents are known to adversely affect the band profiles leading to poor chromatographic performance. Here, we show that controlling the mobile phase strength during loading and post-load elution resulted in improved band profiles when the sample solvent was stronger than the mobile phase. Such an approach improves performance in preparative chromatography by allowing either higher sample loading or higher organic content in mobile phase (without loss of yield). Alternately, the approach can be used for improvement in performance by increase in yield or product purity.

A high performance liquid chromatography tandem mass spectrometry for the quantification of tacrolimus in human bile in liver transplant recipients.

Tacrolimus whole-blood concentrations imperfectly reflect concentrations at the effect site. Tacrolimus concentrations in the transplanted organ could be more relevant to predict rejection events. Because liver biopsy cannot be repeatedly performed after liver transplantation, we suggested measuring tacrolimus in the bile to have a cost-effective and clinically implementable surrogate marker of intra-hepatic tacrolimus concentration. We developed and fully validated a liquid chromatography-tandem mass spectrometry method for the determination of tacrolimus in human bile. Sample purification was achieved using protein precipitation and liquid-liquid extraction with ethyl-acetate. Gradient elution was performed using a C18 analytical column with a 5min run-time. The method was linear from 0.5ng/mL to 20ng/mL. In this concentration range, within-day and between-day precisions as well as overall bias were within ±15%. Matrix effect was fully corrected by the internal standard (ascomycin). The assay was optimized to achieve good selectivity in this complex biological matrix. Tacrolimus was found to be stable in bile stored 6 months at -80°C, after 3 freeze and thaw cycles, 20h at room temperature and 24h in extracts kept at 15°C in the auto-sampler. The method was applied to quantify tacrolimus in bile from liver transplant recipients. It allowed getting preliminary data about tacrolimus excretion profile in bile and showed the lack of correlation between tacrolimus whole blood concentration and tacrolimus liver exposition. This alternative and innovative analytical approach of tacrolimus bio-analysis appears suitable for further studies evaluating relevance of biliary tacrolimus concentration as a new pharmacological marker of immunosuppressive activity.

Improvement of FK506 Production in the High-Yielding Strain Streptomyces sp. RM7011 by Engineering the Supply of Allylmalonyl-CoA Through a Combination of Genetic and Chemical Approach.

FK506, a widely used immunosuppressant, is a 23-membered polyketide macrolide that is produced by several Streptomyces species. FK506 high-yielding strain Streptomyces sp. RM7011 was developed from the discovered Streptomyces sp. KCCM 11116P by random mutagenesis in our previous study. The results of transcript expression analysis showed that the transcription levels of tcsA, B, C, and D were increased in Streptomyces sp. RM7011 by 2.1-, 3.1-, 3.3-, and 4.1- fold, respectively, compared with Streptomyces sp. KCCM 11116P. The overexpression of tcsABCD genes in Streptomyces sp. RM7011 gave rise to approximately 2.5-fold (238.1 µg/ml) increase in the level of FK506 production compared with that of Streptomyces sp. RM7011. When vinyl pentanoate was added into the culture broth of Streptomyces sp. RM7011, the level of FK506 production was approximately 2.2-fold (207.7 µg/ml) higher than that of the unsupplemented fermentation. Furthermore, supplementing the culture broth of Streptomyces sp. RM7011 expressing tcsABCD genes with vinyl pentanoate resulted in an additional 1.7-fold improvement in the FK506 titer (498.1 µg/ml) compared with that observed under nonsupplemented condition. Overall, the level of FK506 production was increased approximately 5.2-fold by engineering the supply of allylmalonyl-CoA in the high-yielding strain Streptomyces sp. RM7011, using a combination of overexpressing tcsABCD genes and adding vinyl pentanoate, as compared with Streptomyces sp. RM7011 (95.3 µg/ml). Moreover, among the three precursors analyzed, pentanoate was the most effective precursor, supporting the highest titer of FK506 in the FK506 high-yielding strain Streptomyces sp. RM7011.

Structure elucidation of new ascomycins produced by genetic engineering.

Three new ascomycins produced by genetic engineering of Streptomyces hygroscopicus ATCC 14891 have been purified and characterized. Replacement of the 13-methoxyl group of ascomycin was accomplished by substitution of the corresponding acyltransferase domain of the polyketide synthase with a domain specific for either malonyl-CoA or methylmalonyl-CoA. The strain containing the methylmalonyl-specific acyltransferase domain produced a compound with properties consistent with those expected for 13-demethoxy-13-methylascomycin. NMR analysis revealed this material to be predominantly the cis amide rotamer, similar to ascomycin. The strain containing the malonyl-specific acyltransferase domain produced a mixture of two compounds, 13-demethoxyascomycin and the 9,14-hemiacetal isomer of 13-demethoxyascomycin, in nearly equal amounts. NMR analysis revealed both compounds to be predominantly the trans amide rotamers.

The calcium release channel of sarcoplasmic reticulum is modulated by FK-506 binding protein: effect of FKBP-12 on single channel activity of the skeletal muscle ryanodine receptor.

The calcium release channel/ryanodine receptor of rabbit skeletal muscle sarcoplasmic reticulum is tightly associated with the immunophilin FK-506 binding protein (FKBP-12). The immunosuppressant drug FK-506 effectively dissociates FKBP-12 from the calcium release channel of terminal cisternae (TC) vesicles. Furthermore, calcium flux measurements of TC indicate that FKBP-12 stabilizes the closed conformation of the calcium release channel of TC [Timerman AP, Ogunbunmi E, Freund EA, Wiederrecht G, Marks AM, Fleischer S. (1993) J. Biol. Chem., 268, 22992-22999]. In this report, the effect of FKBP on single channel recordings of the calcium release channel/ryanodine receptor of TC is measured directly. Single channel recordings of the ryanodine receptor were obtained by fusion of TC vesicles into planar bilayers. The channel devoid of FKBP, retains key diagnostic features. That is, activation by Ca2+ and ryanodine, inhibition by Mg2+ (mM) and ruthenium red (microM), and its unitary conductance remain the same. Recordings of the calcium release channel obtained from the FKBP-deficient TC vesicles, as compared with control TC, have greater open probability and longer mean open times in a free calcium concentration range of 70 nM to 1.2 microM. The sensitivity of the channel to caffeine is also enhanced by the removal of FKBP. The enhanced channel activation of FKBP-deficient TC is reversed by rebinding recombinant FKBP-12 in a cyclical fashion. We conclude that FKBP modifies the channel behavior of the calcium release channel of skeletal muscle sarcoplasmic reticulum.

Identification of a 14 kDa FK-506/rapamycin binding immunophilin from calf thymus.

Specific binding proteins (immunophilins, 12-17 kDa) have been described in the cytosol for the immunosuppressant drugs cyclosporine, FK-506, and rapamycin. We describe the identification of a low abundant minor immunophilin (14 kDa) from calf thymus. Saturation experiments using dihydro[3H]-FK-506 gave a Kd of 2 nM and the Bmax of 72 nmol/mg protein. At saturation, 71.3 nmol of FK-506 is bound per mg of the 14 kDa protein (71 nmol) giving a drug/protein ratio of 1.0. Competition experiments using 3H-dihydro FK-506 and rapamycin showed displacement of rapamycin, with Kd in the range of 40 nM. The 14 kDa immunophilin does not have peptidyl-prolyl cis-trans isomerase activity with any of the four substrates investigated. Initial amino acid analysis and protein sequencing data indicate that the immunophilin is different from both the 12 kDa FK-506 binding protein and any other known protein.

Simultaneous simple and fast quantification of three major immunosuppressants by liquid chromatography--tandem mass-spectrometry.

OBJECTIVES: The aim of the current study was to develop a simple, fast and universal method for quantification of any combination of the three major immunosuppressants sirolimus, tacrolimus and cyclosporin in whole blood, using a LC-tandem mass spectrometer (API-2000, SCIEX, Toronto, Canada). METHODS: 250 microL whole blood was spiked with internal standard (ritonavir), and protein precipitated with 350 microL acetonitrile. The sample was centrifuged and 30 microL aliquot was injected onto the HPLC column, where it underwent an online extraction with ammonium acetate. After that the automatic switching valve was activated, changing the mobile phase to methanol and thereby eluting the analytes into the tandem mass spectrometer. The high selectivity of a tandem mass analyzer allows determination of any combination of the three drugs within a 5 min run. RESULTS: Between-day precision was between 2.4% and 9.7% for all analytes at the concentrations tested. Accuracy ranged between 98.8% and 103.2% (n = 20). The method was linear over the measuring ranges of all analytes. Within-run precision was below %CV = 6% for all analytes. Good correlation with other analytical methods was observed. CONCLUSIONS: The simplicity, universality and high throughput of the method make it suitable for application in a clinical laboratory. The method has been implemented in our laboratory for a routine use.

Partial characterization of a 52 kDa CsA/FK506/rapamycin binding protein.

Identification and characterization of the cellular proteins that specifically bind to the immunosuppressive drugs, cyclosporine (CsA), FK506, and rapamycin is necessary to understand their mechanism of action. We have isolated and partially characterized a 52 kDa binding protein (BP) from calf thymus. Using 12 peptide substrates we observed very low or no cis-trans peptidyl prolyl isomerase activity. We further tested the protein for catalytic activity including kinase activity, phosphatase activity, protein kinase C regulation, and LCK tyrosine kinase regulation. The 52 kDa BP was capable of blocking the cyclic AMP dependent, protein kinase mediated, phosphorylation of histones and casein. The protein did not demonstrate kinase activity, nor did it affect the activity of protein kinase C or LCK tyrosine kinase. Microsequencing of the 52 kDa BP was performed. A comparison of known sequences indicated that the protein is unique and has not been previously characterized.

Application of centrifugal counter-current chromatography to the separation of macrolide antibiotic analogues. I. Selection of solvent systems based on solubility and partition coefficient investigations.

As the first part of our studies on counter-current chromatography, the methodology for selecting suitable solvent systems was established based on detailed investigations of solubility and partition coefficients (log K) of macrolide antibiotic analogues. The solubility of two important macrolides, ascomycin and FK-506, was measured in a series of common solvents, where their polarities were ranked with dielectric constants. The partition coefficients of the two macrolides were compared in various binary, ternary, quaternary solvent systems. Hexane-tert.-butyl methyl ether-methanol-water system was selected based on suitable log K of solutes and hydrogen-bonding properties of solvents. In the further optimisation of composition proportions in the multicomponent solvent system, hexane-tert.-butyl methyl ether-methanol-water (1:3:6:5) showed the best solvent selectivity by giving the most prominent difference of partition coefficient (delta log K) between ascomycin and FK-506. With this solvent system, a baseline preparative separation of these two very closely related 23-membered macrolide antibiotics was successfully achieved by employing the newly introduced Quattro counter-current chromatograph.

Purification of an ascomycin derivative with simulated moving bed chromatography. A case study.

A purification process was developed for the separation of a semi-synthetic ascomycin derivative from its by-products. The process consists of a silica gel filtration and crystallization step prior to two simulated moving bed (SMB) separations, where in the first part the polar by-products and in the second part the apolar by-products were removed. The desired purity was achieved in a final crystallization step. Key elements of the whole process were the design of the first crystallization to obtain a product feasible for SMB chromatography and the specification of operating parameters for the two corresponding SMB steps. Starting from a crude product with an assay of only 44.9% an overall yield for the whole process of 81.0% was achieved with a final purity of >98%.

Tautomeric phenomenon of a novel potent immunosuppressant (FK506) in solution. I. Isolation and structure determination of tautomeric compounds.

Tautomeric phenomenon was observed in the HPLC chromatogram obtained from a novel potent immunosuppressant, FK506, in ethanol solution. Two tautomeric compounds derived from FK506 were isolated and purified by HPLC. Their structures were elucidated by spectral analyses. The mechanisms for this tautomerization were also established based on the results of structure analysis.

Isolation and characterization of the major equilibrium product of FK-520.

The existence of an isomeric form in equilibrium with the major component of FK-520 in polar solutions has been demonstrated. This minor component has been isolated in high yield and purity by a novel crystallization strategy and preparative HPLC. The equilibrium product was characterized by NMR and MS.

[Discovery and development of a novel immunosuppressant, tacrolimus hydrate].

Tacrolimus hydrate (FK506), a novel 23-membered macrolide, is an immunosuppressant isolated from Streptomyces tsukubaensis using extensive screening of fermentation products to identify a compound inhibiting the mixed lymphocyte reaction (MLR). The in vitro and in vivo immunosuppressive activities of FK506 were found to be more potent than those of cyclosporine (CyA). The superior immunosuppression with FK506 treatment was also confirmed in the skin allograft model in rats and liver transplantation in dogs. Clinical studies were initiated by Prof. Starzl at the University of Pittsburgh in 1989, and he demonstrated that FK506 surpassed CyA in the incidence of graft survival and the frequency of graft rejection. Multicenter randomized clinical studies, comparing FK506 to CyA corroborated the efficacy of FK506 on the survival of patients and of grafts, and especially on the appearance of severe refractory graft rejection. FK506 was marketed in 1993 in Japan, and was followed in 1994 in the U.S.A., U.K. and Germany. The mechanism of action of this compound was clarified by the endeavors of Prof. Schreiber, who demonstrated the existence of a binding protein for FK506 called FKBP, similar to cyclophilin for CyA. The FK506/FKBP complex binds with calcineurin, a serine/threonine phosphatase to inhibit the translocation of NFAT into the nucleus, resulting in inhibition of transcription of IL-2 mRNA. FK506 displays potent immunosuppressant activity, and contributes not only to the progress of transplantation therapy for clinical studies, but also to the clarification of signal transduction in T cell activation for basic science.

Designed biosynthesis of 36-methyl-FK506 by polyketide precursor pathway engineering.

The polyketide synthase (PKS) biosynthetic code has recently expanded to include a newly recognized group of extender unit substrates derived from α,ß-unsaturated acyl-CoA molecules that deliver diverse side chain chemistry to polyketide backbones. Herein we report the identification of a three-gene operon responsible for the biosynthesis of the PKS building block isobutyrylmalonyl-CoA associated with the macrolide ansalactam A from the marine bacterium Streptomyces sp. CNH189. Using a synthetic biology approach, we engineered the production of unnatural 36-methyl-FK506 in Streptomyces sp. KCTC 11604BP by incorporating the branched extender unit into FK506 biosynthesis in place of its natural C-21 allyl side chain, which has been shown to be critical for FK506's potent immunosuppressant and neurite outgrowth activities.

Evaluation, synthesis and characterization of tacrolimus impurities.

Tacrolimus is an immunosuppressant macrolactam of fermentative origin. By means of HPLC, LC-MS and NMR analyses, coupled with the reference standard synthesis, the main impurities of tacrolimus bulk drug samples were identified and their chemical-physical properties reported. Known ascomycin and tautomers I and II were detected. The correct relative retention time HPLC value of 39,40-dihydro tacrolimus was established. The not described 23,24-anhydro derivative was detected and completely characterized. A full characterization of ascomycin and 39,40-dihydro tacrolimus was also reported.