Metabolites AM1 Research Articles

Functional evaluation of cyclosporine metabolism by CYP3A4 variants and potential drug interactions.

The aim of this study is to investigate the effects of CYP3A4 genetic polymorphisms on the metabolism of cyclosporine (CsA) in vitro and identify drugs that interact with CsA. An enzymatic incubation system was developed to evaluate the kinetic parameters of CYP3A4 on CsA catalysis. A total of 132 drugs were screened to identify potential drug-drug interactions. Sprague-Dawley rats were used to determine the interaction between CsA and nimodipine and nisoldipine. The metabolite AM1 was measured by ultra-performance liquid chromatography-tandem mass spectrometry. The results demonstrate that 16 CYP3A4 variants (CYP3A4.7, 8, 9, 12, 13, 14, 16, 18, 19, 23, 24, 28, 31, 32, 33, and 34) have a lower metabolic capacity for CsA, ranging from 7.19% to 72.10%, than CYP3A4.1. In contrast, the relative clearance rate of CYP3A4.5 is significantly higher than that of CYP3A4.1. Moreover, CYP3A4.20 loses its catalytic ability, and five other variants have no significant difference. A total of 12 drugs, especially calcium channel blockers, were found to remarkably inhibit the metabolism of CsA with an inhibitory rate of over 80%. Nimodipine inhibits the activity of CsA in rat liver microsomes with an IC50 of 20.54 ± 0.93 μM, while nisoldipine has an IC50 of 16.16 ± 0.78μM. In in vivo, three groups of Sprague-Dawley rats were administered CsA with or without nimodipine or nisoldipine; the AUC(0-t) and AUC(0-∞) of CsA were significantly increased in the nimodipine group but not obviously in the nisoldipine group. Mechanistically, the inhibition mode of nimodipine on cyclosporine metabolism is a mixed inhibition. Our data show that gene polymorphisms of CYP3A4 and nimodipine remarkably affect the metabolism of CsA, thus providing a reference for the precise administration of CsA.

Cyclosporine Inhibition of Hepatic and Intestinal CYP3A4, Uptake and Efflux Transporters: Application of PBPK Modeling in the Assessment of Drug-Drug Interaction Potential

To apply physiologically-based pharmacokinetic (PBPK) modeling to investigate the consequences of reduction in activity of hepatic and intestinal uptake and efflux transporters by cyclosporine and its metabolite AM1. Inhibitory potencies of cyclosporine and AM1 against OATP1B1, OATP1B3 and OATP2B1 were investigated in HEK293 cells +/- pre-incubation. Cyclosporine PBPK model implemented in Matlab was used to assess interaction potential (+/- metabolite) against different processes (uptake, efflux and metabolism) in liver and intestine and to predict quantitatively drug-drug interaction with repaglinide. Cyclosporine and AM1 were potent inhibitors of OATP1B1 and OATP1B3, IC(50) ranging from 0.019-0.093μM following pre-incubation. Cyclosporine PBPK model predicted the highest interaction potential against liver uptake transporters, with a maximal reduction of >70% in OATP1B1 activity; the effect on hepatic efflux and metabolism was minimal. In contrast, 80-97% of intestinal P-gp and CYP3A4 activity was reduced due to the 50-fold higher cyclosporine enterocytic concentrations relative to unbound hepatic inlet. The inclusion of AM1 resulted in a minor increase in the predicted maximal reduction of OATP1B1/1B3 activity. Good predictability of cyclosporine-repaglinide DDI and the impact of dose staggering are illustrated. This study highlights the application of PBPK modeling for quantitative prediction of transporter-mediated DDIs with concomitant consideration of P450 inhibition.

Gender- and dose-related effects of cyclosporin A on hepatic and bone metabolism

Previous data have shown gender-related differences in the skeletal effects of the immunosuppressive drug cyclosporin A (CsA) in rats. To test the hypothesis that the gender-related skeletal effects of CsA are caused by gender-specific metabolism of this drug, we treated aged male and female sham-operated, gonadectomized (GX) as well as sex hormone-supplemented GX rats with 5 mg/kg CsA three times per week for 2 months, and analyzed the bone phenotype as well as the concentrations of CsA and its major metabolites AM1, AM1c, AM9, and AM4N in blood, urine, and liver tissue. CsA treatment induced high turnover osteopenia in males, but not females. Male rats showed several-fold higher CsA and CsA metabolite blood levels compared with females. Renal clearance data revealed that CsA undergoes selective tubular reabsorption in male, but not female rats. However, a mathematical modeling approach demonstrated that the higher CsA blood levels in males were almost exclusively caused by a 6-fold lower hepatic clearance rate compared with females. In addition, we subcutaneously treated female rats with up to 6-fold higher doses of CsA. Similar to males, high dose CsA induced high turnover osteopenia in female rats. Our data show that the gender-related differences in the skeletal effects of CsA are caused by a higher hepatic clearance rate for CsA in female compared to male rats, and not by a differential skeletal response to CsA. Moreover, our study indicates that CsA blood levels of ≤200 ng/ml measured by HPLC do not induce high turnover osteopenia in aged rats.

Liquid chromatography–tandem mass spectrometry method for simultaneous determination of cyclosporine A and its three metabolites AM1, AM9 and AM4N in whole blood and isolated lymphocytes in renal transplant patients

A LC-MS/MS method was developed and validated for the determination of cyclosporine A (CsA) and its three phase 1 metabolites AM1, AM9, and AM4N in whole blood and lymphocytes isolated on the Histopaque gradient. 200 microL of whole blood was precipitated with 10 mol/L zinc sulfate in acetonitrile/methanol (40:60, v/v) and lymphocytes isolated from 1.5 mL blood were extracted with acetonitrile/methanol (40:60, v/v). The analytes and internal standard cyclosporine D were separated on RP column BEH C18, 2.1 x 50 mm, 1.7 microm using gradient LC-MS/MS analysis in positive electrospray mode. Time of analysis was 5 min. Linearity in blood was 5-2000 microg/L for CsA, AM1, and AM9; 2-500 microg/L for AM4N; and 2-500 microg/L for all substances in lymphocytes. Coefficient of variations was 1.8-9.8% and recovery was 92.0-110.0%. The method was used in early and chronic renal transplant patients for therapeutic drug monitoring of CsA to compare either its share in lymphocytes as target organ or binding to one lymphocyte. The same parameters were calculated for all metabolites tested.

Development of an Abbott ARCHITECT cyclosporine immunoassay without metabolite cross-reactivity

Objective : We investigated the mechanism by which the ARCHITECT cyclosporine (CsA) chemiluminescent microparticle immunoassay (CMIA) eliminates cross-reactivity to CsA metabolites AM1 and AM9, despite its use of a monoclonal antibody which shows cross-reactivity in fluorescence polarization immunoassays. Design and methods : The CMIA was accomplished by incubating an extracted blood sample with magnetic microparticles coated with a very low amount of anti-CsA antibody. After a wash step the microparticles were incubated with a chemiluminescent CsA tracer, followed by a second wash step and measurement of chemiluminescence. The reagent concentrations of salt and detergent were optimized to maximize CsA binding and minimize metabolite interference. Results : Elimination of CsA metabolite cross-reactivity was shown using purified metabolites and blood samples containing native CsA metabolites. The CMIA demonstrated precision and sensitivity acceptable for use in a clinical setting. Conclusion : We conclude that it is possible to eliminate CsA metabolite immuno-cross-reactivity by careful assay design.

Multisite Analytical Evaluation of the Abbott ARCHITECT Cyclosporine Assay

The objective of this study was to evaluate the analytical performance of the Abbott ARCHITECT Cyclosporine (CsA) immunoassay in 7 clinical laboratories in comparison to liquid chromatography/tandem mass spectrometry (LC/MS/MS), Abbott TDx, Cobas Integra 800, and the Dade Dimension Xpand immunoassay. The ARCHITECT assay uses a whole blood specimen, a pretreatment step with organic reagents to precipitate proteins and extract the drug, followed by a 2-step automated immunoassay with magnetic microparticles coated with anti-CsA antibody and an acridinium-CsA tracer. Imprecision testing at the 7 evaluation sites gave a range of total % coefficient of variations of 7.5%-12.2% at 87.5 ng/mL, 6.6%-14.3% at 411 ng/mL, and 5.2%-10.7% at 916 ng/mL. The lower limit of quantification ranged from 12 to 20 ng/mL. Purified CsA metabolites AM1, AM1c, AM4N, AM9, and AM19 were tested in whole blood by the ARCHITECT assay and showed minimal cross-reactivity at all 7 sites. In particular, AM1 and AM9 cross-reactivity in the ARCHITECT assay, ranged from -2.5% to 0.2% and -0.8% to 2.2%, respectively, and was significantly lower than for the TDx assay, in which the values were 3.2% and 16.1%, respectively. Comparable testing of metabolites in the Dade Dimension Xpand assay at 2 evaluation sites showed cross-reactivity to AM4N (6.4% and 6.8%) and AM9 (2.6% and 3.6%) and testing on the Roche Integra 800 showed cross-reactivity to AM1c (2.4%), AM9 (10.7%), and AM19 (2.8%). Cyclosporine International Proficiency Testing Scheme samples, consisting of both pooled specimens from patients receiving CsA therapy as well as whole-blood specimens supplemented with CsA, were tested by the ARCHITECT assay at 6 sites and showed an average bias of -24 to -58 ng/mL versus LC/MSMS CsA and -2 to -37 ng/mL versus AxSYM CsA. Studies were performed with the ARCHITECT CsA assay on patient specimens with the following results: ARCHITECT CsA assay versus LC/MSMS, average bias of 31 ng/mL; ARCHITECT versus the Dade Dimension assay (4 sites), average biases of -7 to -228 ng/mL; ARCHITECT versus AxSYM and TDx, average biases of -4 and -53 ng/mL, respectively. Spearman correlation coefficients were >or=0.89. The ARCHITECT CsA assay has significantly reduced CsA metabolite interference relative to other immunoassays and is a convenient and sensitive semiautomated method to measure CsA in whole blood.

Significant discrepancy in cyclosporin A post-dose concentrations when analyzed with specific RIA and HPLC method

C(2) or AUC sparse sampling methods are widely recommended for therapeutic monitoring of cyclosporin A (CsA). One additional reason for promoting the C(2) sampling time in place of commonly used C(0) is that the C(2) level may actually provide more accurate measurement of parent drug concentration by immunoassays, as lower portion of metabolites has been formed 2 hours post-dose than at the steady-state trough time point. HPLC and RIA whole blood levels of CsA and its main metabolites AM1, AM9 and AM4N were compared during 12 hours profile after chronic administration. 40 stable renal transplant male patients (age 49 +/- 6 years, body weight 76 +/- 7 kg) were treated with CsA (Sandimmun Neoral, Novartis s.r.o, Prague, Czech Republic) in doses 198 +/- 56 mg twice daily. Samples were collected in steady state (after 2 weeks of regular treatment regimen) as follows: pre-dose, 0.5, 1, 1.5, 2, 3, 5, 8 and 12 hours after dose. CsA concentrations were determined both specific RIA assay (Cyclo-Trac SP Whole, Dia Sorin) and HPLC method, where concentrations of metabolites AM1, AM9 and AM4N were simultaneously analyzed. The AUC(0-12) was calculated by the linear trapezoidal rule. The percentage prediction error defined as [(RIA value-HPLC value)/HPLC value] x 100 was used for estimation of differences. C(max), t(max), and C(avg) were compared using Student's t-test. RIA produced significantly higher CsA levels than HPLC method in the period of 0.5 - 5 hours after application. The greatest differences (43 - 56%) occurred between 1 and 3 hours after dose. AUC(0-12), C(max) a C(avg) calculated from RIA results were consequently significantly higher. Only t(max) remained unchanged. The ratio of metabolites/parent drug after CsA intake is decreasing but their absolute concentrations are significantly increasing. Mean levels at C(0)/C(2) were CsA-RIA 82/612, CsA-HPLC 89/425, AM1 121/179, AM9 4.1/81.4, AM4N 9.5/21.0 ng/ml. TDM using C(2) and AUC sparse sampling may cause misleading interpretation using both methods alternately for the same patient.

A Prospective Cross-Over Study Comparing the Pharmacokinetics of Cyclosporine A and Its Metabolites After Oral Versus Short-Time Intravenous Cyclosporine A Administration in Pre–Heart Transplant Patients

Sometimes intravenous administration of cyclosporine (CsA) is essential before oral administration is possible. There are only a few reports available on the interindividual variability of CsA metabolism and different metabolite pattern depending on intravenous versus oral administration of CsA in heart transplant (HTx) patients. For effective inhibition of calcineurin we used a short infusion reaching peak concentrations after 2 hours. In a prospective cross-over study we compared the pharmacokinetics of CsA and its metabolites after oral (2.0 mg/kg body weight) versus intravenous (0.7 mg/kg body weight; 2-hour infusion) CsA administration (single test dose) in 7 pre-HTx patients. The pharmacokinetic parameters of CsA and its metabolites were analyzed using high-pressure liquid chromatography. The pharmacokinetic parameter area under the concentration time curve (AUC (0-∞)) of CsA after intravenous administration was significantly lower (2903 ng*h*mL −1) than that after oral administration (4344 ng*h*mL −1; P = .01). Peak concentrations, time to peak concentration, and terminal elimination half life were not significantly different. Short-time infusion of CsA resulted in a significant decrease in the AUC of the metabolites AM1 (3-fold), AM9 (10-fold), and AM1c (3-fold). A 2-hour infusion of CsA is just as effective as oral administration and the reduced amount of metabolites is advantageous for the patient.

Determination of ciclosporin A and its six main metabolites in isolated T-lymphocytes and whole blood using liquid chromatography–tandem mass spectrometry

A specific and sensitive method for determination of intracellular ciclosporin A (CsA) and its six main metabolites AM1, AM9, AM1c, AM1c9, AM19 and AM4N, in isolated T-lymphocytes and whole blood is described. T-lymphocytes were separated from whole blood using Prepacyte ®. The analytes were extracted and purified from isolated lymphocytes and whole blood by protein precipitation followed by solid-phase extraction (SPE). The analytes and the internal standard, ciclosporin C (CsC), were separated on a reversed phase C8 column (30 mm × 2.1 mm, 3 μm) with a 10 mm × 2 mm, 5 μm Drop-In Guard Cartridge, using gradient elution chromatography and tandem ion trap mass spectrometry detection. The method has been validated in accordance with FDA guidelines and showed linear range from 0.25 to 500 ng/mL for CsA, 0.5 to 500 ng/mL for AM1, AM9 and AM19, 1 to 500 ng/mL for AM4N, AM1c and AM1c9 in intracellular matrix, and 2.5 to 3000 ng/mL for all analytes in whole blood. The applicability of the method is shown on patient samples.

Biotransformation of cyclosporin in primary rat, porcine and human liver cell co-cultures

The purpose of this study was to investigate the species-specific cyclosporin biotransformation in primary rat, human, and porcine liver cell cultures, and to investigate the suitability of a modified sandwich culture technique with non-purified liver cell co-cultures for drug metabolism studies. A sandwich culture was found to enhance hepatocellular metabolic activity and improve cellular morphology and ultrastructure. The cyclosporin metabolites AM9 and AM1 were formed in porcine and human liver cell sandwich co-cultures at levels corresponding to the respective in vivo situations. In contrast, metabolite profiles in rat hepatocytes were at variance with the in vivo situation. However, for all cell types, the overall metabolic activity was positively influenced by sandwich co-culture. The initial levels of albumin synthesis were higher in sandwich cultures than in those without matrix overlay. It is hypothesized that the sandwich culture system provides an improved microenvironment and is, therefore, an advantageous tool for in vitro studies of drug metabolism.

Evaluation of Microsomal Incubation Conditions on CYP3A4-Mediated Metabolism of Cyclosporine A by a Statistical Experimental Design

The effect of changes in microsomal incubation conditions (NADPH, Mg(2+), Cl(-), NADPH-regenerating system and pH) on the formation of the CYP3A4 metabolites AM1 and AM9 from CsA were studied by application of a fractional factorial design. Metabolism was studied in microsomes of transfected human liver epithelial (THLE) cells specifically expressing CYP3A4. Within the conditions tested, a 3-4-fold difference in formation of CsA metabolites was observed. Formation of both AM1 and AM9 was favoured by a low Mg(2+) concentration (0.5 mM) and no addition of Cl(-) to the incubation matrix. However, while a high NADPH concentration (1.75 mM) was the single most important factor for the formation of AM1, changes in NADPH concentration between 0.25 and 1.75 mM had no influence on AM9 formation. Formation of the two metabolites also differed in their influence by pH changes, as a change in pH from 7.2 to 7.5 significantly increased the formation of AM9, while formation of AM1 was unaffected by this change. The present study showed that relatively small changes in the incubation matrix had a significant influence on the microsomal CYP3A4-mediated metabolism of CsA. Systematic studies on microsomal incubation conditions could be a key to improve metabolic in vitro-in vivo extrapolations in drug development.

Determination of Cyclosporine and Its Metabolites in Blood via HPLC-MS and Correlation to Clinically Important Parameters

The narrow therapeutic window of the immunosuppressive drug cyclosporine (CsA), the interindividual variability of its metabolism, and the immunosuppressive activity/toxicity of some metabolites require investigation to correlate the parent substance and its metabolites and observed clinical parameters. Improved knowledge about these correlations may improve postoperative treatment of transplant patients. To observe such correlation therapeutic drug monitoring was performed by high-performance liquid chromatography-mass spectrometry (HPLC-MS) on 202 blood samples of kidney transplant patients. As CsA and its metabolites are preferably bound to lipoproteins in vivo, sample preparation included protein precipitation, solid phase extraction, and separation on a reversed phase column. Mass-spectrometric detection by an electrospray ionization chamber made the detection and quantification of the sodium adducts of CsA and its metabolites AM1, AM1c, DihydroAM1, AM19, and AM4N possible. With the presented HPLC-MS method, rapid information was achieved about the specific metabolization in a patient. Statistical computations related CsA and its metabolite concentrations to clinically important blood parameters. Significant correlation to the blood level of bilirubin and liver enzymes confirmed the presumed hepatotoxic potential of CsA and some metabolites. Furthermore, a strong correlation of AM19 to CRP and IL6 was observerd. These parameters may influence the prognosis for atherosclerosis, inflammation, and chronic allograft nephropathy.

Influence of Cyclosporine on the Serum Concentration and Biliary Excretion of Mycophenolic Acid and 7-O-Mycophenolic Acid Glucuronide

The authors have investigated whether cyclosporine decreases the serum concentration of mycophenolic acid, the active principle of the immunosuppressant mycophenolate mofetil, and increases that of the inactive metabolite 7-O-mycophenolic acid glucuronide by reducing their enterohepatic recirculation. Rats were treated daily with methylcellulose (1.66 mL/kg PO) plus 0.9% NaCl (6 mL/kg IP), mycophenolate mofetil (20 mg/kg PO) plus 0.9% NaCl (6 mL/kg IP), methylcellulose (1.66 mL/kg PO) plus cyclosporine (5 mg/kg IP), and mycophenolate mofetil (20 mg/kg PO) plus cyclosporine (5 mg/kg IP). After 14 days a bile fistula was installed to measure the biliary excretion of the immunosuppressants and their metabolites. After 90 minutes blood was taken to determine their concentrations in blood or serum by HPLC. Cyclosporine significantly decreased the serum concentration of mycophenolic acid by 39% and increased, not significantly, that of 7-O-mycophenolic acid glucuronide by 53%. The biliary excretion of 7-O-mycophenolic acid glucuronide was significantly reduced by cyclosporine by 57%, whereas that of mycophenolic acid was not affected. Mycophenolate mofetil did not show a significant effect on either the blood concentration or the biliary excretion of cyclosporine and its metabolites AM1, AM9, AM1c, and AM4N. Cyclosporine significantly decreased the serum concentration of active mycophenolate acid and increased, not significantly, the serum concentration of inactive 7-O-mycophenolic acid glucuronide, presumably by reducing the biliary excretion of this inactive metabolite.

Role of metabolites and calcineurin inhibition on C2 monitoring in renal transplant patients

Many transplantation centres have switched to C2 monitoring of cyclosporin-treated renal transplant patients. The rationale is that the C2 correlates best with AUC0-4 (area under the concentration-time curve), which again correlates with rejection and nephrotoxicity. It has also been demonstrated that calcineurin phosphatase is inhibited maximally 1-2 h after intake of cyclosporin in patients receiving their first dose. Cyclosporin is metabolized to many compounds, which may influence the results of immunoassays. Some metabolites may have immunosuppressive activity. Cyclosporin metabolites were added to whole blood from healthy volunteers and the calcineurin phosphatase activity (CaN) was determined. Twenty renal transplant patients at varying times after transplantation had blood samples drawn in the morning before and 1, 2, 3 and 4 h after intake of their usual dose of cyclosporin microemulsion. Whole blood samples were analysed by liquid chromatography/tandem mass spectrometry for cyclosporin blood concentration and for the cyclosporin metabolites AM1, AM9, AM1c and AM4n. All samples were analysed for CaN utilizing a 32P-labelled 19 amino-acid peptide. The concentrations of AM1c and AM4n were very low and cannot contribute to CaN inhibition. The ratio of AM1 and AM9 to cyclosporin was high before intake of the drug, but it was much lower during the following 4 h. The 2-h values of cyclosporin were the best predictors of AUC0-4. Calcineurin phosphatase was most inhibited in the 2-h samples and the 2-h value of CaN was the best predictor of CaN AUC0-4. The correlation with calcineurin inhibition seemed better for cyclosporin plus metabolites than for cyclosporin. Samples collected at 2 h are the best predictors of AUC0-4 for both cyclosporin and calcineurin inhibition. The impact of metabolites appears to be small; however, the temporal profile of calcineurin inhibition seemed to follow cyclosporin plus metabolites better than cyclosporin alone.

High-performance liquid chromatographic method for the simultaneous determination of cyclosporine A and its four major metabolites in whole blood

This study aimed to develop a simple and efficient optimized high-performance liquid chromatograph (HPLC) method for simultaneous determination of cyclosporine A (CyA) and its major, partly active metabolites AM1, AM9, AM4N, and AM19 in whole blood from transplant patients using cyclosporine D (CyD) as internal standard. The method used a CN analytical column maintained at 60 °C with hexan-isopropanol (93:7, v/v) as mobile phase; detection was at 212 nm. Linearity for all five compounds was tested in the range of 31–1500 ng ml −1 for CyA and of 31–1000 ng ml −1 for metabolites. The limit of detection was found to be 15 ng ml −1 for all compounds. This modified, inexpensive method is also suitable for measuring cyclosporine A and metabolite concentrations in routine monitoring of patients undergoing treatment with CyA.

Monitoring Cyclosporine of Pre-dose and Post-dose Samples Using Nonextraction Homogeneous Immunoassay

A nonextraction homogeneous immunoassay (CEDIA Cyclosporine Plus Assay) has been developed for the measurement of cyclosporine in predose (trough) and post-dose (C2 to C8) whole-blood samples. The method includes a low-range assay that measures cyclosporine from 25 to 450 ng/mL in pre-dose samples and a high-range assay that detects cyclosporine from 450 to 2000 ng/mL in post-dose samples. The high-range assay allows a direct measurement of post-dose samples without a dilution step. Alternatively, post-dose samples can be correctly measured by the low-range assay following a twofold dilution. Using an NCCLS precision protocol, the assay exhibited less than 10% CV or error less than the functional sensitivity. Functional sensitivity of the low-range assay was demonstrated at 20 ng/mL cyclosporine. Cross-reactivity was measured in the presence of cyclosporine and was found to be 4.4%, 19.8%, 16.4%, 0.9%, 1.0%, and 1.6% for metabolites AM1, AM9, AM4n, AM19, AM4n9, and AM1c, respectively. When 53 samples were evaluated using an HPLC method, the three most significant cross-reactive metabolites, AM1, AM4n, and AM9, exhibited an average concentration profile of 123%, 19%, and 0.06% of the parent cyclosporine, respectively. The average total contribution to cyclosporine quantification from these metabolites was estimated at 7.2% based on the percentage cross-reactivity of each metabolite in the CEDIA assay and the concentration of each metabolite as determined by HPLC. The method comparison study revealed a linear regression correlation of CEDIA = 1.095 x HPLC + 6.6, r = 0.972, for the low-range assay, and CEDIA = 1.018 x HPLC - 36.4, r = 0.968, for the high-range assay. In conclusion, the CEDIA Cyclosporine Plus Assay is a precise and accurate method for quantification of cyclosporine in pre-dose and post-dose samples.

Preliminary report on the effect of xenoperfusion with human blood on cyclosporin A metabolism and cytochrome-P-4503A4-mRNA expression in a pig liver perfusion model

Objectives: Little is known about the effect of ischemia/reperfusion with xenogenic blood on function and gene expression of CYP3A4, the enzyme largely responsible for the metabolism of the immunosuppressants Cyclosporin A (CsA) and Tacrolimus. Design and methods: In a pig liver perfusion model, we have compared the effect of perfusion (3 h) after 20 h cold storage, with either pig or human blood on CsA metabolism and CYP3A4-mRNA expression. mCYP3A4-mRNA was quantified by RT-PCR, CsA and its major metabolites AM1, AM9, AM4N by RP-HPLC. IL-6 served as inflammation marker, GLDH and ALT to estimate tissue damage. Results: Inflammatory response and tissue damage were more extensive during xenoperfusion. CYP3A4 expression decreased similarly during xenogenic and allogenic perfusion. CsA conversion to its metabolites was also comparable during xeno- and alloperfusion. Conclusion: There is no evidence that during the early reperfusion period pig liver CYP3A4 is severely affected if the organ is xenoperfused with human blood in comparison with alloperfusion.

Evaluation of the New AxSYM Cyclosporine Assay: Comparison with TDx Monoclonal Whole Blood and Emit Cyclosporine Assays

Cyclosporine (CsA) is a cyclic undecapeptide that has potent immunosuppressive activity, but its narrow therapeutic range and variable pharmacokinetics in humans make monitoring of CsA mandatory (1). The generally accepted whole-blood therapeutic ranges of CsA (with specific assay) are 100–200 μg/L for renal transplant patients and 150–250 μg/L for cardiac, hepatic, and pancreatic transplant patients during the maintenance phase (2)(3), whereas slightly higher ranges are recommended during the induction phase. The CsA metabolites arising from oxidative pathways (4) cross-react in immunoassays (5)(6), leading to results higher than those by HPLC. Trough concentrations of metabolite AM1, the major metabolite in human blood (1)(4), can exceed those of the parent drug (1). For metabolites AM1, AM9, and AM4N, immunosuppressive activity is <10% of that observed for CsA (7)(8), and at least in rats, various CsA metabolites are not nephrotoxic (9). Thus CsA itself, rather than its metabolites, is the major pharmacologically active substance (10), and specific analytical methods are recommended (3)(11)(12). HPLC is specific but time-consuming and labor-intensive; it also displays the highest between-center CV (1). Three major immunoassays based on different CsA monoclonal antibodies were developed by Abbott, IncStar, and Syva Laboratories: a fluorescence polarization immunoassay (FPIA; for the TDx analyzer), a Cyclo-Trac RIA, and an Emit, respectively (13)(14). All of these immunoassays yield slightly higher concentrations than does HPLC, with FPIA concentrations high by 20–30% (15). The results can be summarized as HPLC < Emit < RIA < TDx. Very recently, a new monoclonal CsA FPIA assay was introduced by Abbott Laboratories for the AxSYM analyzer; it was aimed at reducing metabolite cross-reactivity further and, therefore, at correlating better with HPLC. We first evaluated the analytical performances of this new assay by comparing it with the …

BIOEQUIVALENCE OF SANG-35 AND NEORAL FOR CYCLOSPORINE METABOLITES IN STABLE RENAL TRANSPLANT RECIPIENTS

16 Controversies exist over the role of cyclosporine (CyA) metabolites in terms of efficacy and toxicity, and the potential impact of formulation changes on the metabolite pharmacokinetic profile. To evaluate formulation effect on metabolite pharmacokinetic profiles between two CyA formulations(Sang-35 and Neoral), whole blood samples were analyzed by HPLC for concentrations of the parent compound, CyA, and metabolites, AM1, AM9, AM4N, and AM1c. Ten renal transplant recipients participated in a 2-treatment, 3-period, cross-over study to assess bioequivalence. Pharmacokinetic parameters were determined by analysis of concentrations at time 0, 1, 1.5, 2, 3, 4, 6, 8, 10, and 12 hours after a steady state morning dose. A paired t-test determined the variance between formulation Cmax, Tmax, AUC for CyA and its metabolites. Table There were no statistically significant differences between formulations for pharmacokinetic parameters measured by HPLC for CyA, AM1, or AM9. Only 5 patients had detectable concentrations of both formulations for AM4N, and two patients for AM1c. The concentrations observed were similar, and reflect expected low concentrations of these metabolites. Bioequivalence between CyA formulations Sang-35 and Neoral is established with regard to CyA and metabolites AM1, AM9, AM4N, and AM1c in stable renal transplant recipients. Study funded in part by SangStat Medical Corporation.

BIOEQUIVALENCE OF SANG-35 AND NEORAL FOR CYCLOSPORINE METABOLITES IN STABLE RENAL TRANSPLANT RECIPIENTS

16 Controversies exist over the role of cyclosporine (CyA) metabolites in terms of efficacy and toxicity, and the potential impact of formulation changes on the metabolite pharmacokinetic profile. To evaluate formulation effect on metabolite pharmacokinetic profiles between two CyA formulations(Sang-35 and Neoral), whole blood samples were analyzed by HPLC for concentrations of the parent compound, CyA, and metabolites, AM1, AM9, AM4N, and AM1c. Ten renal transplant recipients participated in a 2-treatment, 3-period, cross-over study to assess bioequivalence. Pharmacokinetic parameters were determined by analysis of concentrations at time 0, 1, 1.5, 2, 3, 4, 6, 8, 10, and 12 hours after a steady state morning dose. A paired t-test determined the variance between formulation Cmax, Tmax, AUC for CyA and its metabolites. TableThere were no statistically significant differences between formulations for pharmacokinetic parameters measured by HPLC for CyA, AM1, or AM9. Only 5 patients had detectable concentrations of both formulations for AM4N, and two patients for AM1c. The concentrations observed were similar, and reflect expected low concentrations of these metabolites. Bioequivalence between CyA formulations Sang-35 and Neoral is established with regard to CyA and metabolites AM1, AM9, AM4N, and AM1c in stable renal transplant recipients.