Surface plasmon resonance biosensor combined with lentiviral particle stabilization strategy for rapid and specific screening of P-Glycoprotein ligands.

A novel surface plasmon resonance-based P-gp ligand screening system (SPR-PLSS) combined with lentiviral particle (LVP) stabilization strategy was constructed to screen out potential P-gp inhibitors from natural products. Firstly, we constructed LVPs with high and low expression levels of P-gp. The LVPs can ensure the natural conformation of P-gp based on the principle that LVPs germinated from packaging cells will contain cell membrane fragments and P-gp they carry. Then the LVPs with high P-gp expression for active channel and LVPs with low P-gp expression for reference channel were immobilized on CM5 chip respectively. The affinity detection was thus carried out with the signal reduction on the two channels. The P-gp inhibitors, Valspodar (Val) and cyclosporin (CsA), as positive compounds, were detected to characterize the chip's activity, and the KD of Val and CsA were 14.09 µM and 16.41 µM, respectively. Forty compounds from natural product library were screened using the SPR CM5 chip, and magnolol (Mag), honokiol (Hon), and resveratrol (Res) were screened out as potential P-gp ligands, showing a significant response signal. This work presented a novel P-gp ligand screening system based on LVP-immobilized biosensor to rapidly screen out P-gp ligands from natural product library. Compared with traditional cell experiments which the screening time may take up to several days, our method only takes several hours. Furthermore, this study has also provided solid evidences to support that some complicated membrane proteins would apply to the lentivirus-based SPR screening system.

Differentiation of cyclosporin A from isocyclosporin A by liquid chromatography/electrospray ionization mass spectrometry with post-column addition of divalent metal salt.

RATIONALE: Cyclosporin A (CsA) rearranges to its isomer isocyclosporin A (isoCsA) upon acid hydrolysis and also during ionization in the ion source of the mass spectrometer. It has been reported that both compounds could not be differentiated by tandem mass spectrometry (MS/MS) using atmospheric pressure ionization (API) sources and ambiguously differentiated by using other sources. In order to analyze these compounds which are common fungal metabolites, it is relevant to develop a simple method for their differentiation. METHODS: CsA and isoCsA were analyzed by liquid chromatography/mass spectrometry (LC/MS) with post-column addition of metal ion solutions in a quadrupole time-of-flight instrument equipped with an electrospray ionization (ESI) source. RESULTS: Mass spectra of CsA obtained upon post-column addition of solutions of Ca(II), Cu(II) and Zn(II) showed complexes between cyclosporin and the metal, including [2CsA + Me](2+) and [CsA-H + Me](+). These complexes were not observed in the spectra of isoCsA. The same results were observed at different metal concentrations. CONCLUSIONS: Differentiation via metal complexation in positive ion mode LC/ESI-MS was performed to simultaneously distinguish CsA and its isomer isoCsA.

Liquid chromatography coupled to different atmospheric pressure ionization sources-quadrupole-time-of-flight mass spectrometry and post-column addition of metal salt solutions as a powerful tool for the metabolic profiling of Fusarium oxysporum.

Fusarium oxysporum L11 is a non-pathogenic soil-borne fungal strain that yielded an extract that showed antifungal activity against phytopathogens. In this study, reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to different atmospheric pressure ionization sources-quadrupole-time-of-flight mass spectrometry (API-QTOF-MS) was applied for the comprehensive profiling of the metabolites from the extract. The employed sources were electrospray (ESI), atmospheric pressure chemical ionization (APCI) and atmospheric pressure photoionization (APPI). Post-column addition of metal solutions of Ca, Cu and Zn(II) was also tested using ESI. A total of 137 compounds were identified or tentatively identified by matching their accurate mass signals, suggested molecular formulae and MS/MS analysis with previously reported data. Some compounds were isolated and identified by NMR. The extract was rich in cyclic peptides like cyclosporins, diketopiperazines and sansalvamides, most of which were new, and are reported here for the first time. The use of post-column addition of metals resulted in a useful strategy for the discrimination of compound classes since specific adducts were observed for the different compound families. This technique also allowed the screening for compounds with metal binding properties. Thus, the applied methodology is a useful choice for the metabolic profiling of extracts and also for the selection of metabolites with potential biological activities related to interactions with metal ions.

Comparison of specific radioimmunoassays for cyclosporine.

This study compares two recently introduced radioimmunoassay kits involving specific monoclonal antibodies to cyclosporine. One kit (Sandimmun) involved 3H-labeled CsA (3H-CsA) as tracer and the other (CY-CLO-Trac-SP) involved a 125I-labeled conjugated derivative of CsA. The kits were nearly equivalent in method performance characteristics. They produced superimposed standard curves and equivalent values to transplanted patient samples. Concentrations of CsA determined by either kit were apparently equivalent to values measured by high-performance liquid chromatography, suggesting that the specific monoclonal antibodies used with the kits detect in trough blood mainly native CsA. The 125I-labeled CsA, when compared with the 3H-CsA alternative, increased the sensitivity and precision, decreased the turnaround time, and provided a technically efficient and conveniently capable method of replacing HPLC for measuring native CsA.

Monitoring of cyclosporine therapy with in vitro biological assays.

We examined the correlation between cyclosporine (CsA) levels and in vitro assays of immune function and hematopoiesis. Mixed lymphocyte reaction (MLR), mitogen responses, suppressor cell (SC), cell-mediated lympholysis (CML), and erythroid colony (EC) assays were studied in dogs, and in vitro megakaryocytopoiesis was studied in mice. Serum CsA concentrations were measured by radioimmunoassay. After oral or intramuscular CsA dosing, lymphocyte proliferation, as measured by MLR, inversely correlated with in vivo serum CsA concentration. MLR responses decreased rapidly, and nearly complete inhibition coincided with peak CsA levels. While CsA concentration-related suppression of lymphocyte stimulation was also observed in mitogen-stimulated cultures, results were less predictable and similar to results with in vitro CsA addition, and higher serum CsA levels were required to achieve comparable suppression. In vivo serum or in vitro CsA levels greater than 300 ng/ml completely inhibited the development of cytotoxic effector cells but had no measureable effect on the expression of suppressor cells in the same cultures. Furthermore, CsA serum also caused concentration-related inhibition of EC growth. The addition of human embryonic kidney-conditioned medium, however, abrogated CsA-related inhibition of EC growth, which suggested that CsA indirectly inhibited EC growth, presumably by interfering with CsA-sensitive accessory cells. This was supported by studies in an in vitro model of murine megakaryocytopoiesis. In normal conditioned medium, megakaryocyte colonies were unaffected by the presence of CsA. However, when cells were cultured in conditioned medium prepared in the presence of CsA, profound inhibition of megakaryocyte growth was observed. These studies show that biologic assays can be used reliably to measure concentration-related changes in immunosuppressive activity of CsA. Further clinical studies are needed to evaluate the usefulness of pharmacodynamic monitoring of CsA therapy.

Pharmacokinetic of topical cyclosporin A in the rabbit eye.

3H labelled cyclosporin A (CS-A) eye drops (2% in castor oil) were applied in rabbits. When a single drop of 10 microliters was given CS-A reached a maximal concentration of 900-1400 ng/ml in the cornea 6 hr after application and then slowly decreased. A substantial level of 600-900 ng/ml was still present after 48 hr. When corneas were analysed for their cellular compartments, 67% of CS-A was found in the epithelium, 25% in the stroma (probably in keratocytes), and 8% in the endothelium. In corneas denervated by circular keratotomy, slightly reduced concentrations were found after surgery, the reduction was not therapeutically significant. Using the HPLC method a significant corneal metabolism of CS-A could be excluded. Scleral concentrations of CS-A as high as 180 ng/ml were found. Concentrations of CS-A in aqueous humor, lens, vitreous body and uvea/retina were below the therapeutic range.

Clinical pharmacokinetics of cyclosporin.

Cyclosporin (cyclosporin A) is a unique immunosuppressant used to prevent the rejection of transplanted organs and to treat diseases of autoimmune origin. Therapeutic drug monitoring of cyclosporin is essential for several reasons: wide variability in cyclosporin pharmacokinetics has been observed after the oral or intravenous administration of the drug. The variability in the kinetics of cyclosporin is related to a patient's disease state, the type of organ transplant, the age of the patient and therapy with other drugs that interact with cyclosporin; maintaining a blood concentration of cyclosporin required to prevent rejection of the transplanted organ; minimising drug toxicity by maintaining trough concentrations below that which toxicity is most likely to occur; and monitoring for compliance since patient non-compliance with drug regimens is a significant reason for graft loss after 60 days. Clinical monitoring and pharmacokinetic studies of cyclosporin can be performed using different biological fluids (plasma, serum or whole blood) and different analytical techniques (radioimmunoassay or high pressure liquid chromatography). The available analytical methods provide different results when using blood, plasma, or serum. Comparison of therapeutic ranges and pharmacokinetic parameters should be made with careful attention given to the method of cyclosporin analysis. Following oral administration, the absorption of cyclosporin is slow and incomplete. Peak concentrations in blood or plasma are reached in 1 to 8 hours after dosing. The bioavailability of cyclosporin ranges from less than 5% to 89% in transplant patients; poor absorption has frequently been observed in liver and kidney transplant patients and in bone marrow recipients. Factors that affect the oral absorption of cyclosporin include the elapsed time after surgery, the dose administered, gastrointestinal dysfunction, external bile drainage, liver disease, and food. Cyclosporin is widely distributed throughout the body. Following intravenous administration, the drug exhibits multicompartmental behaviour. The volume of distribution (whole blood; HPLC) ranges from 0.9 to 4.8 L/kg. Cyclosporin is highly bound to erythrocytes and plasma proteins and has a blood to plasma ratio of approximately 2. In plasma, approximately 80% of the drug is bound to lipoproteins. The distribution of cyclosporin in blood can be affected by a patient's haematocrit and lipoprotein profile. Cyclosporin is extensively metabolised, primarily by mono- and dihydroxylation as well as N-demethylation, and is considered a low-to-intermediate clearance drug.(ABSTRACT TRUNCATED AT 400 WORDS)

Complexation and medium effects on the conformation of cyclosporin A studied by NMR spectroscopy and molecular dynamics calculations.

The conformation of cyclosporin A was investigated in different environments. Whereas there was no distinct difference between the structure of CSA in CDCl3 and THF, the addition of LiCl caused a conformational change. Also, using polar solvents (DMSO, water) induced conformational changes in the backbone as well as the side chains.

Effect of cyclosporin A on daunorubicin accumulation in multidrug-resistant P388 leukemia cells measured by real-time flow cytometry.

We investigated the mode of action of cyclosporin A (Cy-A) as a modifier of multidrug resistance in P388 mouse leukemia cells. A fluorescence-activated flow cytometer (FCM) was modified with a flow-through cuvette to allow continuous on-line monitoring of daunorubicin uptake in vitro. The addition of Cy-A to multidrug-resistant P388/R cells at steady-state daunorubicin uptake, led to a dose-dependent increase in cellular daunorubicin accumulation, as measured by FCM and high-performance liquid chromatography (HPLC). A linear relationship was found between the daunorubicin concentration in the incubation medium and the Cy-A concentration required for optimal stimulation of cellular anthracycline accumulation. The results of a cytotoxicity assay indicated that Cy-A completely restored the chemosensitivity of the P388/R cells. Intracellular Cy-A measurements in P388/S and P388/R cells showed that P388/R cells accumulated significantly less Cy-A than P388/S cells. Relatively high daunorubicin concentrations could not restore that accumulation defect. These results suggest that Cy-A promotes cellular anthracycline accumulation by competing for an outward drug-transport system that operates in multidrug-resistant cells.

Evaluation of the TDx method for cyclosporine and a comparison to CYCLO-Trac RIA in renal transplant patients.

The recent introduction of a fluorescence polarization immunoassay (FPIA) for cyclosporine by Abbott for their TDx analyzer provides an alternative to radioimmunoassay (RIA) and high performance liquid chromatography procedures. This new method is easier and faster than the Incstar CYCLO-Trac RIA assay. The TDx method has good precision except at low levels of cyclosporine. RIA gives consistently higher values than FPIA for samples containing only cyclosporine and in those from renal transplant patients. The FPIA procedure may be modified to allow up to a 50-min pause during the protein precipitation step, allowing more efficient use of technologists' time.

Evaluation of cyclosporine kit utilizing an iodinated tracer and double antibody precipitation.

Due to the increasing number of requests for Cyclosporine quantification, less time-consuming methods are being sought. A commercially available radioimmunoassay which utilizes an 125I-Cyclosporine tracer and double antibody precipitation to separate free and bound fraction is described. The assay was evaluated with standards prepared in-house as well as those supplied by the manufacturer. With both types of standards, the assay exhibited between-run coefficients of variation of less than 14.3%, recoveries near 100% and good parallelism. When the Cyclosporine values obtained with the 125I-RIA calibrated with standards provided in the kit were compared with those obtained with the RIA supplied by Sandoz, a relatively large proportional and constant error was noted. When the comparison was repeated, with the former assay being calibrated with standards prepared in-house, the magnitude of these errors was significantly reduced. With both types of standards the 125I-RIA exhibited good correlation with Cyclosporine values obtained by HPLC. This RIA is suitable for the relatively rapid quantification of Cyclosporine.

Isolation of an immunosuppressive metabolite of FK506 generated by human microsome preparations.

Metabolism of FK506, a 23 member macrolide under clinical investigation as immunosuppressant after transplantation, was studied using human liver microsomes. Two fractions isolated by semi-preparative HPLC were identified by negative fast atom bombardment mass spectrometry as FK506 metabolites with mass peaks at m/z = 790 indicating demethylation of the mother compound. The immunosuppressive activity of one metabolite was evaluated in a ConA-stimulated peripheral rat lymphocyte assay. FK506 had an IC50 of 0.186 nmol/L and the metabolite tested of 1.89 nmol/L.

Synthesis of a fluorescent derivative of cyclosporin A for high-performance liquid chromatography analysis.

A direct assay method for use in studies of cyclosporin binding must be highly sensitive and selective since it must be capable of measuring the concentrations encountered in the protein-free matrix. The failure of current HPLC methods to achieve the sensitivity required for binding studies may be attributed to the use of UV detection, which relies on the relatively weak end-absorption of cyclosporin A. A method involving fluorescence derivatization was sought with the aim of increasing HPLC assay sensitivity. A method is described for producing a fluorescent derivative of cyclosporin A, a compound which has no functional groups which are easily derivatized. However, intramolecular rearrangement of cyclosporin A to form its structural isomer, isocyclosporin A, exposes a secondary amine which can be reacted with dansyl chloride to produce a fluorescent derivative. This two-step derivatization procedure was used as the basis of an HPLC fluorescence assay. Although this assay is not sufficiently sensitive to measure concentrations encountered in the protein-free matrix during plasma binding studies, the method does point to the possible development of a more sensitive assay using a derivatizing reagent other than dansyl chloride.

Excretion of cyclosporine and its metabolites in human bile.

Quantitative and qualitative studies of cyclosporine and its metabolites were performed on human bile from liver transplant and liver disease patients. The concentration of CsA in bile is higher in patients with normal liver function than in those with poor liver function but in neither case could account for more than 2% of an absorbed dose of CsA. Although concentrations of CsA plus metabolites in bile measured by RIA were 18 to 36 times higher than HPLC concentrations, they accounted for less than 50% of an absorbed CsA dose. By means of mass spectrometry and HPLC retention times of known metabolites, peaks equivalent to the previously isolated CsA M8, M13, M17, M1, M18, and M21 of Maurer et al were found in the ether extracts of bile. Future studies should not only concentrate on the pharmacologic and toxicologic effects of the metabolites but should also accurately quantitate these compounds in blood, plasma, urine, and bile.

Pharmacokinetics of cyclosporine in toxicological studies.

Pharmacokinetic studies performed in toxicological studies indicate that CsA is well absorbed in rats and dogs with absolute bioavailability in the range of 10% to 30% when administered by gavage (olive oil). Exceptions are the guinea pig, the rabbit and marmoset with low bioavailability (less than 5%). CsA is also absorbed in rats when given mixed in the feed. Vehicles may have marked effects on the absolute bioavailability. The correlation between areas under the plasma concentration time curves (AUC) and dose levels is linear in most species up to a dose of 50 mg/kg/d. CsA steady state plasma levels are generally 2 to 3 times higher than after single administration. Measures to reduce CsA nephrotoxicity must consider possible changes of oral bioavailability and immunosuppressive activity as shown for dmPGE2. Drug interactions may be predicted in animal experiments.

Micro method for liquid chromatographic determination of cyclosporin A in whole blood with use of a rapid extraction procedure.

In this precise, reversed-phase liquid chromatographic procedure, Cyclosporin A (Cy A) is extracted from 300 microL of whole blood with cyclosporin D as internal standard by liquid-liquid extraction. A 20-microL aliquot of the extract, injected onto a Nucleosil octyl analytical column heated at 72 degrees C, is eluted with a mixture of acetonitrile and 0.01 M phosphate buffer, pH 5.5 at a flow rate of 1 mL/min. Detection is set at 210 nm. The chromatography is complete within 10 min. The detection limit is 25 micrograms/L. Between-run CVs range from 2.3 to 6.2% and recovery is 92.1 +/- 6.3%. The major advantages of our extraction procedure are the rapid clean-up method and the small volume required. This procedure is especially suitable for cyclosporine determination in pediatric transplantations.

Routine monitoring of cyclosporine in whole blood and in kidney tissue using high performance liquid chromatography.

A simple isocratic reversed-phase high pressure liquid chromatographic procedure for the specific estimation of cyclosporine in 1 mL of whole blood using cyclosproin D as internal standard is described. The chromatographic conditions chosen afford a high degree of resolution and sensitivity, a prolonged analytical column life (four months), and complete analysis of ether extracts within 8 min. The limit of detection is 10 micrograms/L. Thirty-five patient samples can be analyzed in a single batch with a turnaround time of 7 hr. This method has been in use in the authors' therapeutic drug monitoring laboratory for nearly two years. It can be simply adapted for the analysis of cyclosporine in kidney samples as a possible aid in the diagnosis of therapeutic failure or toxicity in renal allograft recipients. Examples are given to illustrate the usefulness of frequent monitoring of cyclosporine in stabilizing graft function after organ transplantation.

[Cyclosporin levels in semen of renal transplant patients].

Cyclosporin (CsA) has powerful immunosuppressive properties, and its recent application to organ transplantation has resulted in markedly improved graft survival. However, CsA has certain adverse side effects, the most notable being nephrotoxicity and hepatotoxicity. Among other untoward effects, little is known about the effects of CsA on the reproductive organs. Since good graft survival is now expected with CsA, the long term effect of the drug on the gonads should be monitored carefully, particularly for young recipients. Prior to investigation of the effect of CsA on the testis, the level of CsA in semen of the adult renal transplant patients were measured. Twelve samples of semen from eight recipients, mean age 35 (SD 9), were collected by masturbation in the morning just before taking CsA after more than 5 day abstinence, and it was frozen at -80 degrees C. They had been taking immunosuppressants that were a combination of either CsA and prednisolone, or CsA, azathioprine and prednisolone for 5 to 63 months. The dose of CsA was 3.7 mg/kg to 5.0 mg/kg. The range of the level of serum creatinine was 1.1 mg/dl to 2.9 mg/dl. CsA in whole blood was measured by high performance liquid chromatography (HPLC), and CsA in semen was also measured by HPLC after extracting CsA with diethylether. The level of CsA in semen was 27 ng/ml to 165 ng/ml and that in whole blood was 51 ng/ml to 133 ng/ml. The relation between both levels was linear (r = 0.68, p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)