Cyclosporine Metabolites Research Articles

Biological effects of cyclosporin A: a new antilymphocytic agent. 1976.

The fungus metabolite cyclosporin A is a small peptide acting as a novel antilymphocytic agent. It strongly depressed appearance of both direct and indirect plaque-forming cells and produced a clear dose-dependent inhibition of haemagglutinin formation in mice upon oral administration. Skin graft rejection in mice and graft-versus-host disease in mice and rats were considerably delayed by cyclosporin A which also prevented the occurrence of paralysis in rats with experimental allergic encephalomyelitis. This compound was not only highly effective in preventing development of Freund's adjuvant arthritis, but in addition improved the symptoms in rats with established arthritis, although it is inactive in acute inflammation. This new agent contrasts with other immunosuppressives and cytostatic drugs in its weak myelotoxicity. Experimental evidence suggests that cyclosporin A, rather than being cytostatic or lympholytic, affects an early stage of mitogenic triggering of the immunocompetent lymphoid cell.

Immunoaffinity capillary electrophoretic analysis of cyclosporin in tears.

An immunoaffinity capillary electrophoresis technique has been developed for the analysis of cyclosporin A in human tear fluid following topical application of the drug. The technique combines the selectivity of immunoaffinity separation with the high-resolution of capillary electrophoresis by immobilizing monoclonal antibody fragments directly onto the internal surface of the capillary. This technique was used to measure cyclosporin levels in tears obtained from corneal transplant patients during normal and drug toxicity episodes in the course of their treatment. Comparison of this technique with HPLC detection of cyclosporin in tears showed a good correlation, with the immunoaffinity CE technique having the advantage of being able to simultaneously detect toxic metabolites of cyclosporin in the same sample.

Acute-phase response, interleukin-6, and alteration of cyclosporine pharmacokinetics

Administration of interleukin-6 partially reproduces the inhibitory effects of the acute-phase response on cytochrome P450-dependent drug metabolism. The aim of the study was to determine whether endogenous cytokine has such an effect in patients treated by cyclosporine, which is metabolized by the cytochrome P4503A subfamily. Blood cyclosporine and serum interleukin-6 levels were determined in six patients undergoing bone marrow transplantation, as long as they received cyclosporine by continuous infusion. Two serum acute-phase proteins, C-reactive protein and alpha 1-acid glycoprotein, and two cyclosporine metabolites, AM1 and AM9, were also determined. At the time of marrow infusion, levels of specific markers of inflammation were low. A peak in interleukin-6 level was then observed a mean of 10.8 days after transplantation, closely associated with variations in C-reactive protein levels. A parallel twofold increase in AM1 concentrations was observed, followed by a three-fold increase in cyclosporine levels, which peaked 4.8 days after interleukin-6. The times of peak cyclosporine and AM1 levels correlated with the time of peak interleukin-6 levels. AM9 was detectable in three patients but concentrations fell when interleukin 6 became detectable. An inflammatory reaction could be an important source of intraindividual variability in cyclosporine pharmacokinetics, possibly through an inhibition of cytochrome P4503A-dependent enzyme activities by endogenous interleukin-6. Blood AM1 accumulation might be explained by a secondary metabolic step that is highly sensitive to the inhibitory effect of interleukin-6.

Variable effect of ursodeoxycholic acid on cyclosporin absorption after orthotopic liver transplantation

Abstract The acute effects of administering a single dose of ursodeoxycholic acid (UDCA) on cyclosporin pharmacokinetics were recorded during paired studies in twelve liver transplant recipients, six of whom were cholestatic. Cyclosporin was measured using monoclonal selective antibodies (for parent drug) and non-selective antibodies (for cyclosporin plus metabolites). UDCA resulted in more rapid absorption of cyclosporin in 8 of 12 cases (67 %) but had no effect in two patients with and two patients without cholestasis. Median tmax did not change significantly after UDCA (3.0 vs 4.0 h without UDCA) and only 7 of 12 patients (58 %) showed a rise in the amount of cyclosporin absorbed over 24 h with the AUC not having changed significantly (median 4527 vs 4979 μg. h. 1-1 without UDCA). Median Cmax and C24 also increased only marginally following UDCA administration (616 vs 587 μg. 1-l and 87 vs 58 μg 1-1 without UDCA, respectively). In the cholestatic patients, median AUC was 50 % smaller (3223 vs 6439 μg. h. 1-1) and median t 1/2 a was 100% longer (1.58vs0.8 h) than in patients without cholestasis. There was no consistent improvement in cyclosporin pharmacokinetics in the cholestatic patients following UDCA, although the significantly elevated ratio of non-selective: selective AUC measurements (median 4.8 vs 2.7) fell markedly in the two most severely affected, possibly as a result of an increased clearance of cyclosporin metabolites by choleresis.

Variable effect of ursodeoxycholic acid on cyclosporin absorption after orthotopic liver transplantation.

The acute effects of administering a single dose of ursodeoxycholic acid (UDCA) on cyclosporin pharmacokinetics were recorded during paired studies in twelve liver transplant recipients, six of whom were cholestatic. Cyclosporin was measured using monoclonal selective antibodies (for parent drug) and non-selective antibodies (for cyclosporin plus metabolites). UDCA resulted in more rapid absorption of cyclosporin in 8 of 12 cases (67%) but had no effect in two patients with and two patients without cholestasis. Median tmax did not change significantly after UDCA (3.0 vs 4.0 h without UDCA) and only 7 of 12 patients (58%) showed a rise in the amount of cyclosporin absorbed over 24 h with the AUC not having changed significantly (median 4527 vs 4979 micrograms.h.l-1 without UDCA). Median Cmax and C24 also increased only marginally following UDCA administration (616 vs 587 micrograms.l-1 and 87 vs 58 micrograms.l-1 without UDCA, respectively). In the cholestatic patients, median AUC was 50% smaller (3223 vs 6439 micrograms.h.l-1) and median t1/2 a was 100% longer (1.58 vs 0.8 h) than in patients without cholestasis. There was no consistent improvement in cyclosporin pharmacokinetics in the cholestatic patients following UDCA, although the significantly elevated ratio of non-selective:selective AUC measurements (median 4.8 vs 2.7) fell markedly in the two most severely affected, possibly as a result of an increased clearance of cyclosporin metabolites by choleresis.

Effects of cyclosporine and its metabolites in the isolated perfused rat kidney.

The isolated perfused rat kidney (IPK) was used to study the acute effects of cyclosporin A (CsA) and its metabolites (M1, M17, M18, M21 and M-COOH). GFR, renal vascular resistance, and sodium, potassium and water reabsorption were measured before and after the addition of CsA/metabolites/vehicles. There was no difference in CsA effect (mild decrease in GFR and increase in renal vascular resistance with the inclusion of plasma (10 mL) or whole blood (20 mL) in the albumin perfusate (120 mL). Intralipid was used as the vehicle for CsA and the metabolites because methanol, ethanol, and Cremophor had significant effects on GFR. Intralipid enhanced the effect of CsA 25-fold, giving CsA dose responses comparable to those of human kidneys. This enhanced effect with intralipid was due to vasoconstriction, not vascular obstruction, and was apparently specific to CsA (no enhancement of norepinephrine with Intralipid). The primary metabolites (M1, M17, and M21) caused decreases in GFR comparable to or slightly less than those caused by CsA. The secondary metabolites (M18 and M-COOH) caused more modest declines in GFR. Cyclosporine metabolite levels in patient blood often greatly exceed levels of the parent drug; these studies suggest that the metabolites may contribute significantly to CsA nephrotoxicity in patients.

When to Measure Cyclosporin Metabolites in Liver Graft Recipients?

When to Measure Cyclosporin Metabolites in Liver Graft Recipients?

Binding of cyclosporine by erythrocytes: influence on cell shape and deformability

Cyclosporine is a widely used immunosuppressive drug with a high affinity for erythrocytes. It was hypothesized that the hydrophobic agent would interact with the erythrocyte membrane, which could cause a shape change and alter cell deformability. Administration of 300 mg cyclosporine in vivo and incubation of erythrocytes with concentrations up to 100 mg l-1 in vitro at room temperature showed that cyclosporine is found in the cytoplasm, but not in the membrane of erythrocytes and that cell shape or deformability were not affected. Incubation of erythrocytes with the highest cyclosporine concentration (100 mg l-1) at 37 degrees C lead to a time-dependent, slight stomatocytic shape transformation, indicating that the drug is intercalated preferentially into the inner hemileaflet of the membrane under these conditions. Cyclosporine metabolites had no effect on the cell shape. The shape of erythrocyte ghosts was neither affected by cyclosporine nor by its metabolites. It is concluded that cyclosporine is bound in the erythrocyte cytoplasm and does not affect the cell membrane and cell deformability at therapeutic concentrations. These results may contribute to a better understanding of the interactions of cyclosporine with cells.

Preparative chromatographic purification of cyclosporine metabolites

Polar and primary metabolites of cyclosporin A (CsA) have successfully been isolated by a novel separation protocol. An efficient, easy-to-scale-up chromatographic adsorption/desorption operation recovers polar and primary CsA metabolite pools from large volumes of urine; purified CsA metabolites are subsequently obtained by high-resolution preparative elution chromatography of the semipurified metabolite pools. Separations performed on a semipreparative scale [with a 250 x 9.4 mm (i.d.) reversed-phase HPLC column] yielded microgram quantities of CsA metabolites at > 97% purity, as determined by fast atom bombardment mass spectrometry. These separations also yielded two previously unreported CsA metabolites, similar to AM1A but with an additional hydroxylation. The yield of metabolites was increased to several milligrams by performing the separations with a preparative-scale [250 x 21.2 mm (i.d.)] reversed-phase column. The production rate of purified primary CsA metabolites was greatly increased by performing the separation with the preparative-scale column under conditions of severe mass overloading. In a single chromatographic run, we successfully isolated 11.0 and 5.0 mg of AM1 and AM1c, respectively, at a purity of > 97%. As expected, this increase in the yield of purified metabolites was accompanied by a decrease in the overall recovery. This separation scheme enables the rapid processing of large volumes of urine for isolation of the milligram quantities of CsA metabolites necessary to assess their biological activity. The procedure is applicable to small- or large-scale metabolite isolation and provides a ready source of purified metabolites for in vitro and whole-animal studies.

Cyclosporin metabolism in transplant patients

The immunosuppressant cyclosporin, a cyclic undecapeptide, is metabolized to more than 30 metabolites. Cytochrome P45IIIA enzymes located in liver and small are responsible for the biotransformation of cyclosporin and its metabolites and are the site of several drug interactions. It is still under discussion, whether the cyclosporin metabolites are involved in the immunosuppressive and/or toxic activities of cyclosporin. While isolated metabolites show not more than 10–20% of the activity of the mother compound in vitro, metabolite combinations have additive and synergistic effects. Isolated metabolites show no toxic effects in rat models while there is an association between metabolite blood concentrations and cyclosporin toxicity in several clinical studies. Possible mechanisms for the toxic effect of cyclosporin metabolites are covalent binding to macromolecules in liver and kidney, alteration of the cytochrome P450 pattern in liver and kidney, increased endothelin production in the kidney and synergistic effects of cyclosporin combinations on mesangial cells. Liver dysfunction leads to an alteration of the metabolite patterns and to increased concentrations of cyclosporin metabolites in blood. In conclusion there is evidence that cyclosporin metabolites may contribute to cyclosporin toxicity and high metabolite blood concentrations in patients should not be tolerated.

Cyclosporin G and metabolite binding to cyclophilin and a 50-kDa binding protein related to in vitro immunosuppression

Seven purified metabolites of cyclosporin G (CsG) were studied for binding to cyclophilin and a 50-kDa binding protein (50-kDa BP). The ratios of the metabolite dissociation constants with respect to CsG were compared with in vitro immunosuppression by using the primary mixed lymphocyte suppression assay. The immunosuppressive potency ratio of the parent compounds, both cyclosporin A (CsA) and CsG, compared favorably with the drug dissociation constants for cyclophilin and the 50-kDa BP. Three of the seven metabolites had comparable binding and potency ratios for the 50-kDa BP. In contrast, none of the seven metabolites appreciably bound to cyclophilin in the concentration range tested.

Flow cytometric analysis of altered mononuclear cell transmembrane potential induced by cyclosporin

Immunosuppression by the fungal metabolite cyclosporin A (CsA) is characterized by functional inhibition, rather than destruction of cells. Because activation of immune cells involves intracellular signalling events associated with modulations of cell transmembrane potential (TMP), we tested the ability of cyclosporin A (CsA) to modulate immune mononuclear cell TMP in vitro using a TMP sensitive cationic dye, dihexyloxacarbocyanine (DIOC6(3)). All analyses were performed by flow cytometry. CsA increased TMP in monocytes and lymphocytes isolated from the blood of healthy human volunteers. CsA-induced hyperpolarization was time and concentration dependent in monocytes while the lymphocyte hyperpolarization, although time dependent, was evident over the entire range of CsA concentrations tested. CsA-induced hyperpolarization of lymphocytes was dependent on potassium ion (K+) efflux as indicated by the absence of hyperpolarization in 154 mM KCl or with pretreatment with 100 microM quinine (an inhibitor of K+ channels). Monocyte hyperpolarization by CsA was not inhibited in either system. Dihydrocyclosporin C (DH-CsC), an immunosuppressive analog of CsA, also hyperpolarized mononuclear cells. The anionic TMP sensitive dye bis oxonal (diBA-C4) indicated that CsA treatment depolarized mononuclear cell plasma membranes. The mitochondrial poison carbonyl cyanide p-trifluoromethoxy-phenyl-hydrazine (FCCP) eliminated CsA induced hyperpolarization and also indicated that CsA caused plasma membrane depolarization. We conclude that brief in vitro exposure to cyclosporin alters the transmembrane electrical potential of human lymphocytes and monocytes.

A review of assay methods for cyclosporin. Clinical implications.

Cyclosporin is a unique immunosuppressive agent with a narrow therapeutic range. The pharmacokinetics of the drug present substantial within- and between-patient variability and drug interactions can significantly alter blood cyclosporin concentrations. Monitoring of cyclosporin concentrations in blood is an invaluable and essential aid in adjusting dosage to ensure adequate immunosuppression while minimising toxicity. The principal rationale behind therapeutic monitoring of cyclosporin is the fact that the incidence of rejection is higher at low cyclosporin concentrations and toxicity occurs more often at high concentrations. In renal transplant recipients, cyclosporin concentrations help to discriminate between insufficient immunosuppression and cyclosporin-induced nephrotoxicity. There are several methods available, both specific and nonspecific, for the routine measurement of cyclosporin. Radioimmunoassay and fluorescence polarisation immunoassay are most widely employed, while high performance liquid chromatography remains the reference procedure. The allegedly specific immunoassays tend to slightly overestimate the actual blood cyclosporin concentrations. There is a need for assay systems capable of measuring the biological activity of cyclosporin. Cyclosporin concentrations should be determined by a specific method, using whole blood as the sample matrix. The routine monitoring of individual cyclosporin metabolites is not warranted, but characterising the metabolite pattern of cyclosporin by concomitant use of a nonspecific and a specific assay can be clinically useful in patients with cyclosporin-associated toxicity or impaired liver function. In organ transplantation, measurement of blood cyclosporin concentration should be continued periodically as long as the therapy continues, whereas monitoring is only indicated in special circumstances in patients with autoimmune and other nontransplant diseases. The assessment of a 'therapeutic window' for cyclosporin is complicated for several reasons and definite target ranges cannot be given. Cyclosporin concentrations should always be interpreted in conjunction with the recent blood concentration history and other relevant clinical and laboratory data.

Structure of Cyclosporine and Its Metabolites: Total Synthesis of Cyclosporine Metabolites Formed by Oxidation at Positions 4 and 9 of Cyclosporine. Preparation of Leucine-4-cyclosporine, (γ-Hydroxy)-N-methyl-leucine-9-cyclosporine and Leucine-4-(γ-hydroxy)-N-methyl-leucine-9-cyclosporine

The syntheses of the cyclosporine metabolites Leu-4-cyclosporine (AM4N), (γ-OH)MeLeu-9-cyclosporine (AM9) and Leu-4-(γ-OH)MeLeu-cyclosporine (AM4N9) were completed. Aspartic acid was used as starting material to prepare peptides containing (γ-OH)MeLeu residues. The structure of metabolite M13 was definitively established as being Leu-4-(γ-OH)MeLeu-9-cyclosporine. The immunosuppressive activity in vitro of these synthesized metabolites was compared with that of cyclosporine and metabolite AM1.

The effect of cyclosporine administration on the cellular distribution and content of cyclophilin.

Cyclophilin (CYP), an intracellular protein sharing amino acid sequence identity with the enzyme peptidyl-prolyl cis-trans isomerase has become the leading candidate for the receptor responsible for cyclosporine biological effects. Avid binding of CYP to cyclosporine and immunosuppressive cyclosporine metabolites has been demonstrated, while nonimmunosuppressive cyclosporine metabolites have tended not to bind to cyclophilin. A previous immunohistochemical analysis documented that CYP localized principally to the cytoplasmic cellular compartment, but nuclear staining was observed among some cells. This study was undertaken to more precisely define the ultrastructural distribution of CYP, and to determine whether CYP cellular content was affected by CsA therapy. Untreated Wistar rats or those receiving 7 days of CsA (15 mg/kg/day, i.p.) were anesthetized, perfusion-fixed in situ, and sacrificed. Analyses of lymph node, spleen, thymus, kidney, liver, heart, brain, and ileum used an affinity purified, rabbit anticyclophilin IgG. Transmission electron microscopy was performed after staining with anti-CYP using a horseradish peroxidase/biotin/avidin technique. Quantitative immunofluorescence was measured by confocal microscopy using anti-CYP, with a biotin/avidin/phycoerythrin technique. Cyclophilin localized to the cytoplasmic compartment--however, association with mitochondria endoplasmic reticulum, Golgi, and with the nuclear membrane among lymphocytes, as well as cells from kidney, liver and ileum--was documented. Cyclophilin was not identified within the nucleus proper. Tissues obtained from animals receiving CsA exhibited a generalized increase in CYP content compared with tissues from untreated controls, suggesting the possibility that CsA may exert a regulatory influence upon CYP gene activation. Collectively, the data were consistent with the hypothesis that CYP exerts a central role in cellular metabolism, and that CsA-mediated biologic effects result from the CsA/CYP interaction.

Cholestasis and kidney dysfunction in liver transplant patients reduces cyclosporine metabolite excretion

Cyclosporin A (CsA) is metabolized principally by the hepatic cytochrome P 450-dependent microsomal enzyme system and eliminated virtually entirely as metabolites, mainly in the bile [1, 4, 6]. Only less than 1% of the oral dose is excreted unmetabolized in the urine or bile [5, 7]. Metabolites account for 50–70% of the total CsA in whole blood [3, 5, 8]. Some of the metabolites have been shown to possess an immunosuppressive and even toxic effect but the role of this effect remains uncertain [2, 9]. In order to evaluate the effect of liver and kidney failure on the metabolism of CsA, we studied twelve patients who had undergone liver transplantation. The samples were collected during the first 4 postoperative weeks. The aim of the study was threefold: to evaluate (1) whether an impairment of liver function, as measured by standard biochemical liver function tests, decreased the metabolism or excretion of CsA; (2) whether an induction of either the CsA metabolites or the parent compound took place in the first postoperative period; and (3) whether kidney failure, as measured by serum creatinine, correlated with blood levels of CsA or its metabolites.

Monitoring Cyclosporine in Liver-Transplant Recipients: Effects of Clinical Status on the Performance of Two Monoclonal Antibody-Based Methods

The characteristics of the Abbott selective fluorescence polarization immunoassay for cyclosporine in whole blood (TDXSP) were compared with the Incstar radioimmunoassay by using kit standards (INCSP) or TDXSP standards (INCTDX) with samples from adult (48) and pediatric (24) liver-transplant recipients and a further 32 control specimens. The TDXSP assay was faster, less labor intensive, and more precise at higher concentrations; it gave results a median of 5 micrograms/L lower than INCSP values, irrespective of biliary T-tube status, patient's age, or severity of cholestasis in nine patients, comparisons being made for serum bilirubin more than eight times normal or less than 50 mumol/L. However, both assays overestimated INCTDX values by approximately 10%. There was a progressive increase in the ratio of TDXSP to both INCSP and INCTDX results in 57 samples stratified in relation to serum bilirubin concentrations. Cross-reactivity of TDXSP with cyclosporine metabolites M1 (23%) or M17 (12%) was greater than for INCSP (15% and 5%, respectively) or INCTDX (22% and 4%, respectively), but the converse was true with M21 (2% for TDXSP vs 4% and 8%, respectively). Some overestimation of cyclosporine whole-blood concentrations (relative to INCTDX results) may be detected with either the TDXSP (because of cross-reactivity with metabolites) or the INCSP assays (probably because of matrix effects). The similar magnitude of these overestimations suggests that alternative factors such as speed, precision, and cost may influence which method one selects for use in routine monitoring.

Cholestasis and kidney dysfunction in liver transplant patients reduces cyclosporine metabolite excretion.

Cyclosporin A (CsA) is metabolized principally by the hepatic cytochrome P 450-dependent microsomal enzyme system and eliminated virtually entirely as metabolites, mainly in the bile. Only less than 1% of the oral dose is excreted unmetabolized in the urine or bile. Metabolites account for 50-70% of the total CsA in whole blood. Some of the metabolites have been shown to possess an immunosuppressive and even toxic effect but the role of this effect remains uncertain. In order to evaluate the effect of liver and kidney failure on the metabolism of CsA, we studied twelve patients who had undergone liver transplantation. The samples were collected during the first 4 postoperative weeks. The aim of the study was threefold: to evaluate (1) whether an impairment of liver function, as measured by standard biochemical liver function tests, decreased the metabolism or excretion of CsA; (2) whether an induction of either the CsA metabolites or the parent compound took place in the first postoperative period; and (3) whether kidney failure, as measured by serum creatinine, correlated with blood levels of CsA or its metabolites.

First-pass metabolism of cyclosporin by the gut

Cyclosporin is thought to be exclusively metabolised in the liver. We instilled cyclosporin into the small bowel of 2 patients during the anhepatic phase of liver transplantation; cyclosporin metabolites were readily detected in portal venous blood. Our findings indicate that the small intestine is a major site of cyclosporin breakdown: such intestinal metabolism might help to explain the poor oral bioavailability and drug interactions of cyclosporin.

Recent advances in cyclosporine pharmacokinetics.

Although cyclosporine has been in clinical use for more than a decade, many questions persist concerning its pharmacokinetics. Gastric emptying, bile acids, and gastrointestinal motility can influence drug absorption after oral administration, but the relative importance of each of these factors and their interactions as determinants of absorption are not clear. The fraction unbound in plasma has been determined in several studies and may be associated with clinical effects. Biochemical and molecular studies have identified the specific cytochrome P-450 enzymes that metabolize cyclosporine to its primary metabolites. The drug's basic pharmacokinetic profile has been defined and many factors have been identified that can influence clearance, such as age, hepatic function, hematocrit, and lipoprotein concentrations. With the development of sensitive and specific analytical methods, many new cyclosporine metabolites have been identified in humans. The major metabolites are present in relatively high concentrations in human blood and have some immunosuppressive activity in vitro, but their contribution to in vivo immunosuppression is not clear.