Cyclosporin A Metabolites Research Articles

Dextran sulfate sodium-induced colitis and ginseng intervention altered oral pharmacokinetics of cyclosporine A in rats

Ethnopharmacological relevanceApplication of cyclosporine A (CsA) as a rescue treatment in acute severe ulcerative colitis (UC) is limited by its narrow therapeutic window and great interpatient variability. As a substrate of cytochrome P450 3A enzyme (CYP3A) and P-glycoprotein (P-gp), the oral pharmacokinetics of CsA is susceptible to disease status and concomitant medications. Combined treatment with ginseng, a famous medicinal herb frequently prescribed for ameliorating abnormal immune response in many diseases including UC, showed immunologic safety in CsA-based immunosuppression. Aim of the studySince the therapeutic levels of CsA can be achieved within 24 h, this study first assessed the impact of acute colitis and ginseng intervention on the single oral dose pharmacokinetics of CsA and explored the underlying mechanisms in dextran sulfate sodium (DSS)-induced colitis rats and Caco-2 cells. Materials and methodsRats received drinking water (normal group), 5% DSS (UC group), or 5% DSS plus daily oral ginseng extract (GS+UC group). On day 7, GS+UC group only received an oral dose of CsA (5 mg/kg), while animals of normal or UC group received an oral, intravenous (1.25 mg/kg), or intraperitoneal dose of CsA (1.25 mg/kg), respectively. Blood, liver/intestine tissues and fecal samples were collected for determining CsA and main hydroxylated metabolite HO-CsA or measuring hepatic/intestinal CYP3A activity. Caco-2 cells were incubated with gut microbial culture supernatant (CS) of different groups or ginseng (decoction or polysaccharides), and then CYP3A, P-gp and tight junction (TJ) proteins were determined. ResultsOral CsA exhibited enhanced absorption, systemic exposure and tissue accumulation, and lower fecal excretion, while intravenous or intraperitoneal CsA showed lower systemic exposure and enhanced distribution, in colitis rats. Diminished intestinal and hepatic P-gp expression well explained the changes with DSS-induced colitis. Moreover, blood exposures of HO-CsA in both normal and colitis after oral dosing were significantly higher than intravenous/intraperitoneal dosing, supporting the dominant role of intestinal first-pass metabolism. Interestingly, colitis reduced CYP3A expression in intestine and liver but only potentiated intestinal CYP3A activity, causing higher oral systemic exposure of HO-CsA. Oral ginseng mitigated colitis-induced down-regulation of CYP3A and P-gp expression, facilitated HO-CsA production, biliary excretion and colonic sequestration of CsA, while not affected CsA oral systemic exposure. In Caco-2 cells, gut microbial CS from both colitis and GS+UC group diminished P-gp function, while ginseng polysaccharides directly affected ZO-1 distribution and suppressed TJ proteins expression, explaining unaltered oral CsA systemic exposure. ConclusionsDSS-induced colitis significantly altered oral CsA disposition through regulating intestinal and hepatic P-gp and CYP3A. One-week ginseng treatment enhanced colonic accumulation while not altered the systemic exposure of CsA after single oral dosing, indicating pharmacokinetic compatibility between the two medications.

Cardiovascular Disease in Kidney Transplantation and Its Association With Blood Concentrations of Cyclosporine and Cyclosporine Metabolites

Cyclosporine A (CsA) is the first calcineurin inhibitor used as immunosuppressive agent. Its administration is associated with multiple adverse effects including cardiovascular diseases (CVDs), but their mechanisms have not been fully elucidated. Cyclosporine metabolites are not well studied in this context. This study was aimed at analysis of the incidence of CVDs and their association with concentrations of cyclosporine and its metabolites.Sixty patients after kidney transplantation (KTX) taking an immunosuppressive regimen including CsA participated in the study. There were 22 women (36.67%) and 38 men (63.33%), mean age 51.73 years, mean 109.38 months after KTX.We observed a correlation between mean diastolic blood pressure and concentrations of metabolite to parent drug ratios of AM1-CsA/CsA (r = 0.35, P = .006), dihydroxy-CsA/CsA (r = 0.42, P = .001), trihydroxy-CsA/CsA (r = 0.42; P = .003) and desmethyl-carboxy-CsA/CsA (r = 0.65, P = .003). There were no significant associations of other CsA metabolites' parameters with CVDs (coronary disease, hypertension, stroke, arrhythmia, diabetes mellitus, obesity).Study results suggest that blood pressure increases associated with CsA therapy could be caused by CsA metabolites that influence mainly diastolic blood pressure levels. A lack of such differences in relation with other CVDs may suggest that more complex mechanisms are involved in the development of cardiovascular injury and disease after kidney transplantation.

CYP3A5 Gene Variation Influences Cyclosporine A Metabolite Formation and Renal Cyclosporine Disposition

Higher concentrations of AM19 and AM1c9, secondary metabolites of cyclosporine A (CsA), have been associated with nephrotoxicity in organ transplant patients. The risk of renal toxicity may depend on the accumulation of CsA and its metabolites in the renal tissue. We evaluated the hypothesis that CYP3A5 genotype, and inferred enzyme expression, affects systemic CsA metabolite exposure and intrarenal CsA accumulation. An oral dose of CsA was administered to 24 healthy volunteers who were selected based on their CYP3A5 genotype. CsA and its six main metabolites in whole blood and urine were measured by liquid chromatography-mass spectometry. In vitro incubations of CsA, AM1, AM9, and AM1c with recombinant CYP3A4 and CYP3A5 were performed to evaluate the formation pathways of AM19 and AM1c9. The mean CsA oral clearance was similar between CYP3A5 expressors and nonexpressors. However, compared with CYP3A5 nonexpressors, the average blood area under the concentration-time curve (AUC) for AM19 and AM1c9 was 47.4% and 51.3% higher in CYP3A5 expressors (P=0.040 and 0.011, respectively), corresponding to 30% higher AUCmetabolite/AUCCsA ratios for AM19 and AM1c9 in CYP3A5 expressors. The mean apparent urinary CsA clearance based on a 48-hr collection was 20.4% lower in CYP3A5 expressors compared with CYP3A5 nonexpressors (4.2±1.0 and 5.3±1.3 mL/min, respectively; P=0.037), which is suggestive of CYP3A5-dependent intrarenal CsA metabolism. At steady state, intrarenal accumulation of CsA and its secondary metabolites should depend on the CYP3A5 genotype of the liver and kidneys. This may contribute to interpatient variability in the risk of CsA-induced nephrotoxicity.

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.

Individual Differences in Cyclosporine A Pharmacokinetics and Its Association with Acute Renal Function Following Heart Transplantation

Background: The secondary metabolites of cyclosporine A (CsA), AM19, AM1c and AM1c9, have been indi- cated to be nephrotoxic. The aim of the present pilot study was to investigate the relationship between acute renal failure and CsA metabolite levels, including relevant pharmacokinetic genotypes, following heart transplantation. Methods: Whole-blood samples were drawn the first posttransplant week in 22 patients (median 54 years, range from 27 to 65). Whole blood concentrations of CsA and its six main metabolites were analyzed with a validated HPLC-MS/MS method, and relevant CYP3A5 and ABCB1 genotypes determined. Renal function was monitored daily during the first posttransplant month and also at months 3, 6 and 12. Results: One patient died early posttransplant. Six patients were in need of dialysis directly after transplantation. Nine pa- tients developed sustained impaired renal function, while six had stable renal function. Sustained renal impairment tended to be associated with high levels of toxic metabolites (P=0.08). Six of the patients with possible ABCB1 TTT-haplotype developed renal impairment (P=0.12). Conclusion: The present study indicates that toxic CsA metabolites seems to be associated with development of impaired renal function and together with ABCB1 genotyping they might be promising biomarkers for optimalization of immuno- suppressive drug treatment in future studies.

Cinacalcet's effect on the pharmacokinetics of tacrolimus, cyclosporine and mycophenolate in renal transplant recipients

The calcimimetic drug cinacalcet offers a novel therapeutic option to treat post-transplant hypercalcemia and hyperparathyroidism; however, the interaction with calcineurin inhibitors and mycophenolate has not been evaluated. In the present study the effects of cinacalcet on the pharmacokinetics of cyclosporine A (CsA), tacrolimus (Tac) and mycophenolate were investigated in 14 renal transplant recipients with stable renal function (mean creatinine 126.4 +/- 45.3 micromol/L). The patients were treated with either CsA (n = 8) or Tac (n = 6) in combination with mycophenolate/azathioprine and steroids. Twelve-hour pharmacokinetic investigations to measure CsA and its six main metabolites, Tac and mycophenolate concentrations were performed before and after 1-week treatment with 30 mg cinacalcet once daily. Cinacalcet treatment induced a significant 14.3 +/- 12.1% decrease in Tac AUC(0-12) (P = 0.039). Tac C(max), T(max) and T(1/2) also tended to decrease. The pharmacokinetics of CsA and mycophenolate were not significantly affected by concomitant treatment with cinacalcet. However, the secondary CsA metabolite, AM19, showed a significant increase of 9.0 +/- 9.5% during cinacalcet treatment (P = 0.040). Renal function decreased significantly from 78 +/- 11 to 72 +/- 12 mL/min (P = 0.019) and correlated with the increased levels of metabolite AM19 in the CsA group. Renal function was unchanged in the Tac group. Cinacalcet treatment showed a moderate effect on the Tac, but not CsA or mycophenolate, pharmacokinetics after 1-week concomitant treatment. This interaction appears to have minor clinical relevance. However, it is advisable to monitor renal function in CsA-treated patients due to the observed decrease in renal function.

A Review of the Immunosuppressive Activity of Cyclosporine Metabolites:New Insights into an Old Issue

Cyclosporine A (CsA) is metabolized into a vast spectrum of metabolites. The potential immunosuppressive action of CsA's metabolites has been studied extensively in the early 1990's, with conflicting and inconclusive results. Since then, the pharmacological and clinical consensus guidelines recommend the use of specific monoclonal assays for measurement of CsA's concentrations thus avoiding metabolite interference. Nevertheless, clinical benefit or superiority of these assays was never convincingly demonstrated. We provide a review of the performed in vivo, in vitro and animal studies and their conclusions. During the last years, many transplantation centres have employed the C(2) monitoring of CsA (2 hours post-dose concentration). The metabolites / parent drug ratio two hours post dose is completely different from trough levels (predose concentration). Cyclosporine exerts its immunosuppressive action by inhibiting the enzyme calcineurin phosphatase (CaN). Currently, our laboratory, among others, is working on determination of the enzyme's inhibition and its potential use as a pharmacodynamic biomarker. Utilizing this novel pharmacodynamic approach, we have performed in vitro and in vivo studies investigating the immunosuppressive impact of CsA's metabolites on C2 monitoring and on various monitoring assays (mono-/polyclonal). Interestingly, even though we have estimated in vivo that the potential immunosuppressive action of metabolites is less than 10% of the parent drug, we have found assays that take metabolites into consideration to correlate stronger with calcineurin phosphatase inhibition. We believe that the old controversial issue of metabolite induced immunosuppressive action examined under the light of newer pharmacodynamic approaches is still intriguing. Instead of a priori neglecting CsA's metabolites maybe we should investigate the potential of utilizing them as an additional tool towards better therapeutic drug monitoring of cyclosporine.

The measurement of whole blood pre-treatment cyclosporine A: metabolite ratios predicts the onset of renal dysfunction in recipients of allogeneic stem cell transplantation

In the context of post-transplant immunosuppression, cyclosporine A (CSA) is dose adjusted in accordance with whole blood drug monitoring. While currently available immunoassay systems primarily target the parent drug, cross-reactivity results in the detection of the major circulating CSA metabolites, though their contribution to both immunosuppression and toxicity remain unclear. This study examines the relationship of CSA metabolites to hepatic and renal dysfunction and the incidence of graft-versus-host disease (GvHD) through parallel assaying of parent drug and drug/metabolites expressed as a metabolite ratio (Cp:mR). Sequential pre-treatment (trough) whole blood samples (n=527) were collected from 31 allo-stem cell transplantation (SCT) recipients. Both parent drug and drug/metabolite levels were determined using the Abbott fluorescence polarization immunoassay. The average mean Cp:mR was significantly higher in patients with hepatic (P=0.004) and renal dysfunction (P=0.004) than in those without. Significantly higher Cp:mR were also found in patients with grades II-IV GvHD (P=0.001) than were observed in patients who did not experience significant GvHD. When measured prospectively, an increasing Cp:mR predated the rise in serum creatinine concentration by a median of two weeks. This study demonstrates a clinically useful CSA metabolite ratio that shows association with hepatic and renal dysfunction and with GvHD. The measure can be used to predict those patients on CSA therapy who are likely to develop renal dysfunction.

In vitro metabolism of cyclosporine A by human kidney CYP3A5

The objectives of this study were to characterize and compare the metabolic profile of cyclosporine A (CsA) catalyzed by CYP3A4, CYP3A5 and human kidney and liver microsomes, and to evaluate the impact of the CYP3A5 polymorphism on product formation from parent drug and its primary metabolites. Three primary CsA metabolites (AM1, AM9 and AM4N) were produced by heterologously expressed CYP3A4. In contrast, only AM9 was formed by CYP3A5. Substrate inhibition was observed for the formation of AM1 and AM9 by CYP3A4, and for the formation of AM9 by CYP3A5. Microsomes isolated from human kidney produced only AM9 and the rate of product formation (2 and 20 μM CsA) was positively associated with the detection of CYP3A5 protein and presence of the CYP3A5*1 allele in 4 of the 20 kidneys tested. A kinetic experiment with the most active CYP3A5*1-positive renal microsomal preparation yielded an apparent K m (15.5 μM) similar to that of CYP3A5 (11.3 μM). Ketoconazole (200 nM) inhibited renal AM9 formation by 22–55% over a CsA concentration range of 2–45 μM. Using liver microsomes paired with similar CYP3A4 content and different CYP3A5 genotypes, the formation of AM9 was two-fold higher in CYP3A5*1/*3 livers, compared to CYP3A5*3/*3 livers. AM19 and AM1c9, two of the major secondary metabolites of CsA, were produced by CsA, AM1 and AM1c when incubated with CYP3A4, CYP3A5, kidney microsomes from CYP3A5*1/*3 donors and all liver microsomes. Also, the formation of AM19 and AM1c9 was higher from incubations with liver and kidney microsomes with a CYP3A5*1/*3 genotype, compared to those with a CYP3A5*3/*3 genotype. Together, the data demonstrate that CYP3A5 may contribute to the formation of primary and secondary metabolites of CsA, particularly in kidneys carrying the wild-type CYP3A5*1 allele.

Lack of specificity of cyclosporine immunoassays. Results of a College of American Pathologists Study.

To evaluate the cross-reactivity of the 6 most abundant cyclosporine A (CsA) metabolites in commonly used assays for CsA. Whole blood samples containing either only 62 ng/mL CsA (A) or 62 ng/mL CsA and between 49 and 86 ng/mL of 1 of the 6 most abundant CsA metabolites (B) were lyophilized. One sample of A and 1 of B were mailed to each of the laboratories participating in the College of American Pathologists Proficiency Testing Program quarterly during a 3-year period (1999-2001). Method means and coefficients of variation were calculated for each mailing. The study showed significant cross-reactivity of metabolites in all the immunoassay systems studied. Overall degree of interference decreased from Abbott TDx polyclonal > Abbott TDx monoclonal > DiaSorin > Syva EMIT. High-performance liquid chromatography methods gave results close to those found using mass spectrometric techniques. Significant metabolite interference was found to occur with the immunoassay systems studied.

Lack of specificity of cyclosporine immunoassays - Results of a college of American Pathologists Study

Objective.-To evaluate the cross-reactivity of the 6 most abundant cyclosporine A (CsA) metabolites in commonly used assays for CsA. Design.-Whole blood samples containing either only 62 ng/mL CsA (A) or 62 ng/mL CsA and between 49 and 86 ng/ mL of 1 of the 6 most abundant CsA metabolites (B) were lyophilized. One sample of A and 1 of B were mailed to each of the laboratories participating in the College of American Pathologists Proficiency Testing Program quarterly during a 3-year period (1999-2001). Method means and coefficients of variation were calculated for each mailing. Results.-The study showed significant cross-reactivity of metabolites in all the immunoassay systems studied. Overall degree of interference decreased from AbbottTDx polyclonal > Abbott TDx monoclonal > DiaSorin > Syva EMIT. High-performance liquid chromatography methods gave results close to those found using mass spectrometric techniques. Conclusions.-Significant metabolite interference was found to occur with the immunoassay systems studied.

Evaluation of a No-Pretreatment Cyclosporin A Assay on the Dade Behring Dimension RxL Clinical Chemistry Analyzer

Monitoring whole-blood concentrations of cyclosporin A (CsA) is common practice in the management of solid organ and bone marrow transplant recipients. In a multicenter study we evaluated a new, direct (no pretreatment) CsA assay on the Dade Behring Dimension RxL system and compared results with those from the Abbott TDx CsA immunoassay and a HPLC method. Whole-blood samples from heart (n = 111; 35 patients), liver (n = 201; 44 patients), kidney (n = 279; 65 patients), and miscellaneous organ (n = 77; 12 lung, 12 bone marrow, 5 kidney/pancreas, and 1 pancreas patient) recipients were obtained from patient populations of the participating institutions. Routine clinical monitoring of CsA was performed using either the TDx method or HPLC. The minimum detectable concentration of CsA averaged 9.4 microg/L, and the lower limit of quantification was 30 microg/L. The method was linear from 30 to 500 microg/L. Cross-reactivity with seven different CsA metabolites ranged from 0.0% to 5.7% for the Dimension RxL assay compared with 0.4-15.9% for the TDx assay. Total imprecision (CV) averaged 6.2%, and within-run imprecision averaged 4.9%. Passing-Bablok linear regression analyses of all samples from two sites yielded the following: RxL = 0.81 x TDx - 16.8; and RxL = 1.12 x HPLC - 1.7. The Dade Behring CsA assay for the random-access Dimension platform offers adequate performance characteristics for routine clinical use, does not require a manual pretreatment step, and demonstrates less cross-reactivity with CsA metabolites than another commonly used immunoassay.

Evaluation and comparison of therapeutic monitoring of whole-blood levels of cyclosporin A and its metabolites in renal transplantation by HPLC and RIA methods

Background: The aim of the work was to evaluate the possibility to estimate the level of cyclosporin A (CyA) metabolites as the difference of radioimmunoassay (RIA) non-specific and RIA specific methods. Methods: Blood samples of renal transplant patients were analyzed by three different methods: RIA specific method (CYCLO-Trac, DiaSorin, USA) (RIA SP), RIA non-specific method (Immunotech, Czech Republic) (RIA NS), and high performance liquid chromatography (HPLC) method. Results: Although values obtained by RIA SP correlated well those obtained by HPLC (RIA SP=0.995·HPLC+9.68; r 2=0.962, n=448), the results of HPLC methods were lower by 8%. The values obtained by RIA NS were 2.57 times higher than the values obtained by RIA SP (RIA SP=0.356RIA NS; r 2=0.713, n=448). The ratio (CyA+CyA metabolites)/(CyA) calculated as the ratio RIA NS/RIA SP values for 42 renal transplant patients was relatively stable for each particular patient. The sum of selected CyA metabolites (M1+M17+M21) measured by HPLC correlated well with that estimated from the difference of RIA NS−RIA SP: HPLC metab=0.921·(RIA NS−RIA SP)+21.3; ( r 2=0.746, n=448). Conclusion: The combination of both the specific and non-specific methods for the determination of CyA presents an improved means for the TDM of CyA and CyA metabolites in renal transplant patients. Moreover, a combination of both methods can help to elucidate some unexpected events, such as the persistence of high cyclosporin blood levels.

The potential replacement of HPLC by 125I-RIA for the characterization of cyclosporin A: a bioavailability study after oral administration in healthy human subjects

A significant overestimation of cyclosporin A (CsA) by a radioimmunoassay using 125I-labeled monoclonal antibody ( 125I-RIA), compared to the reference HPLC method, has been reported for a limited number of samples from transplant patients. However, the extent of the discrepancy, with respect to bioavailability parameters, has not been examined for the case of the oral administration of a single dose CsA to healthy subjects where a number of factors which might be involved in this overestimation (e.g. under steady state condition and a significant accumulation of CsA metabolites) would be absent. Therefore, the objective of this study was to assess the effect of potential difference manifested by the two analytical procedures, 125I-RIA and HPLC, on the bioavailability analysis of CsA. An oral CsA formulation was administered to 22 healthy male subjects and the blood samples were analysed by both 125I-RIA and HPLC. Significant discrepancies in the estimated CsA concentrations by the two methods (paired t-test, P<0.001) were found. The difference (bias) increased with increasing concentrations of blood CsA ( P<0.001). However, despite the bias in CsA estimations, the AUC and C max, obtained by 125I-RIA and HPLC methods showed only small differences (i.e. 2% for AUC and 7% for C max,). Thus, our results suggest that the bias of the 125I-RIA vis-a-vis the HPLC method in the estimation of CsA blood levels may not, in practice, affect the bioavailability analysis (e.g. bioequivalence study) of CsA in a situation where a single dose CsA is orally administered to healthy subjects.

Performance and Specificity of Monoclonal Immunoassays for Cyclosporine Monitoring: How Specific Is Specific?

Immunoassays designed for the selective measurement of cyclosporin A (CsA) inadvertently show cross-reactivity for CsA metabolites. The extent and clinical significance of the resulting overestimation is controversial. A comprehensive assessment of old and new methods in clinical specimens is needed. In a comprehensive evaluation, CsA was analyzed in 145 samples with the new CEDIA assay and compared with the Emit assay with the old and new pretreatments, the TDx monoclonal and polyclonal assays, the AxSYM, and HPLC. All samples were from patients with liver and/or kidney transplants. The CEDIA offered the easiest handling, followed by the AxSYM, which showed the longest calibration stability. The TDx monoclonal assay provided the lowest detection limit and the lowest CVs. The mean differences compared with HPLC were as follows: Emit, 9-12%; CEDIA, 18%; AxSYM, 29%; and TDx monoclonal, 57%. The CycloTrac RIA paralleled the Emit results. In contrast to the mean differences, substantial (>200%) and variable overestimations of the CsA concentration were observed in individual patient samples. Metabolic ratios, estimates of the overall concentrations of several cross-reacting metabolites (nonspecific TDx polyclonal/specific reference method), correlated with the apparent biases of the various monoclonal assays. Metabolic ratios varied up to 10-fold, which translated into biases for individual samples between -7% and +174%. The higher the cross-reactivity of an assay was, the higher was the range of biases observed. The interindividual differences markedly exceeded other factors of influence (organ transplanted, hepatic function). Because assay bias cannot be predicted in individual samples, substantially erratic CsA dosing can result. The specificity of CsA assays for parent CsA remains a major concern.

Inhibition of P-glycoprotein by cyclosporin A analogues and metabolites

The interaction between P-glycoprotein (P-gp) from membranes isolated from multidrug-resistant Chinese hamster ovary cells and cyclosporin A (CsA) analogues and its metabolites was characterized. Screening of these latter as chemosensitizers was performed using three different assays: (i) vinblastine uptake, (ii) photoaffinity labeling by [125I]iodoaryl azidoprazosin, and (iii) P-gp ATPase activity. Oxidation of the hydroxyl group at position 1 of CsA (200-096), CsG (215-834), or CsD (PSC-833) increased their inhibition of P-gp. CsA analogues (208-032, 208-183) modified at position 11 retained their ability to inhibit P-gp while analogues modified at position 2 (CsC and CsD) lost their efficiency. The inhibitions induced by metabolites of CsA were also compared to those obtained with CsG metabolites. From all the molecules tested, PSC-833 and 280-446 peptolide were the strongest inhibitors. Our results indicate that modifications of CsA analogues at position 1 and 2 are critical for their interaction with P-gp and that CsA metabolites retain a portion of the inhibitory activity of the parent drug.Key words: P-glycoprotein, cyclosporin A, vinblastine uptake, photolabeling, ATPase activity.

Inhibition of P-glycoprotein by cyclosporin A analogues and metabolites

The interaction between P-glycoprotein (P-gp) from membranes isolated from multidrug-resistant Chinese hamster ovary cells and cyclosporin A (CsA) analogues and its metabolites was characterized. Screening of these latter as chemosensitizers was performed using three different assays: (i) vinblastine uptake, (ii) photoaffinity labeling by [125I]iodoaryl azidoprazosin, and (iii) P-gp ATPase activity. Oxidation of the hydroxyl group at position I of CsA (200-096), CsG (215-834), or CsD (PSC-833) increased their inhibition of P-gp. CsA analogues (208-032, 208-183) modified at position 11 retained their ability to inhibit P-gp while analogues modified at position 2 (CsC and CsD) lost their efficiency. The inhibitions induced by metabolites of CsA were also compared to those obtained with CsG metabolites. From all the molecules tested, PSC-833 and 280-446 peptolide were the strongest inhibitors. Our results indicate that modifications of CsA analogues at position 1 and 2 are critical for their interaction with P-gp and that CsA metabolites retain a portion of the inhibitory activity of the parent drug.

Competitive immunoassay for cyclosporine using capillary electrophoresis with laser induced fluorescence polarization detection

Frequent monitoring of immunosuppressive drug cyclosporine A (CsA) in blood samples of tissue transplant patients is required in clinical practice because of the narrow therapeutic range between the immunosuppressive effect and the toxic effect of this drug. We describe a competitive immunoassay capillary electrophoresis (CE) with laser induced fluorescence polarization detection method, which is rapid and sensitive for the determination of CsA. The method is based on the competitive immunochemical reaction between the analyte and fluorescent hapten (CsA *) with the antibody, CE separation of the antibody bound and free fluorescent CsA *, followed by the laser induced fluorescence polarization detection (LIFP) of the fluorescent species. The method detection limit is governed by the stability of the antibody–CsA * complex rather than by the detector noise. The use of post-column sheath flow cuvette LIFP detection resulted in excellent detection limit, typically 0.9 n M (or 9·10 −19 mol for 1 nl injection) of CsA. CsA in whole blood samples from organ transplant patients were measured and results agreed well with those obtained by using a standard fluorescence polarization immunoassay. Each determination took less than 3 min. The CsA metabolites AM9 and AM19 were also determined by using this technique, and their cross-reactivities with the antibody were 13% and 2%, respectively.

Drug resistance in multiple myeloma: cyclosporin A analogues and their metabolites as potential chemosensitizers

The malignant clone in myeloma is not eradicated by chemotherapy. Cyclosporins inhibit drug transport mechanisms, particularly the multidrug transporter p-glycoprotein 170, leading to their use as chemosensitizers. In myeloma, clonotypic blood B cells represent the major drug-resistant subset. This study compares the ability of cyclosporin A analogues and metabolites to inhibit cellular transporter(s) in myeloma and normal B cells in vitro, and evaluates their potential role in vivo. Cyclosporin A (CsA), CsG, PSC 833 or SDZ 280-446, and primary CsA and CsG metabolites, were tested for their ability to inhibit drug transport mechanisms of ex vivo malignant B cells from 81 patients with multiple myeloma as compared to B cells from normal donors, as measured by the export of the dye rhodamine 123 (Rh123) using multiparameter flow cytometry. The majority of myeloma B and normal B cells had efficient transporter function as measured by their CsA-sensitive export of Rh123. CsA and CsA analogues mediated efficient inhibition of this transport. Inhibition of dye transport by normal B cells required an approximately six-fold greater concentration of the synthetic peptolide SDZ 280-446 than was needed to optimally inhibit transport by myeloma B cells. PSC 833 and CsG were inhibitory at concentrations approximately five-fold lower than were required for CsA. Assessment of inhibitory potency in vivo indicated that the in vivo chemosensitizer levels of CsA and PSC 833 exceeded the transporter inhibitory concentration by four- and 20-fold respectively. In vivo, cyclosporins are rapidly and almost completely converted to metabolites. AM1 and AM4N, primary metabolites of CsA, mediated inhibition of transport, as did CsG metabolites GM1, GM4N and GM9. AM1 and GM9 are known to reach steady-state in vivo levels that exceed the inhibitory concentration identified here by 1.1- to 1.9-fold. Thus, cyclosporin metabolites, which accumulate in the blood during infusion of CsA and other cyclosporins, are shown here to be effective chemosensitizers for normally drug-resistant myeloma cells in vitro. Cyclosporin metabolites are considered to be less toxic than the parent drugs, suggesting that novel chemosensitization strategies designed to minimize concentrations of parent drug and maximize accumulation of primary metabolites in vivo may optimize cytotoxicity to the malignant clone in myeloma.

The determination of metabolite M17 and its meaning for immunosuppressive cyclosporin therapy.

Cyclosporin A (CyA) is intensively metabolized by the hepatic cytochrome p450 III monooxygenase A system in the human liver, the most important metabolites being M1, M17, and M21. Because CyA and its metabolites have nephrotoxic, hepatotoxic, and neurotoxic side effects, CyA dosage must be calculated to avoid the risk of organ rejection through underdosage and toxic organ damage through overdosage or accumulation of metabolites. In this study, we determined the whole-blood concentrations of cyclosporin and metabolite M17 by high-pressure liquid chromatography (HPLC) and by monoclonal specific and polyclonal nonspecific fluorescence polarization immunoassay (Abbott) in patients after immunosuppressive treatment. Patients with different resorption and metabolization rates showed high individual variations. CyA concentrations in patients with good liver function and low concentrations of CyA metabolites showed a good correlation between the HPLC and the FPIA (TDx-monoclonal assay) methods in ranges between 25 and 180 ng/mL. TDx-monoclonal was not always as precise as HPLC. In cases of metabolic disorders, we found false high CyA concentrations assayed with the immunologic method, caused by a crossreaction of the elevated metabolite concentration. We found that HPLC rendered more information about the extent of immunosuppressive activity and the metabolization rate and showed a good correlation with the concentration of metabolite M17 and total metabolites measured with the Abbott CyA polyclonal kit.