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Cyclosporine blood level monitoring
Molecular Immunology 39 (2003) 1059–1060
Short communication
Cyclosporine blood level monitoring Cross-reactivity of anti-Cyclosporine A monoclonal with its sulphate metabolite: an in vitro study Marwan Masri a,∗ , Sylvana Rizk a , Tomas Andrysek b , Vladimir Matha b b
a Rizk Hospital, P.O. Box 11 2388, Beirut, Lebanon IVAX-CR, Ostravska 29, 74770 Opava 9, Czech Republic
Abstract Cyclosporine is metabolized in the liver by the Cytochrom P450 system. Many of the metabolites have been identified and thoroughly studied. However the sulphate metabolite is the least known. The metabolite is hydrophilic and thus it is not easily detectable by HPLC and it is not available in pure form for analysis. We have isolated the metabolite from transplant patients receiving CYA as part of their immunosuppressive therapy. The amino acid sequence was determined and the molecule was synthesised in our laboratories. We have tested the molecule in vitro using cell culture to determine its activity. 1/2 ml of blood + 1/2 ml of RPMI was incubated with the following concentration of either CSA or CSS: 0, 25, 50, 100, 250, 500, 750, 1000, 1250, 1500, 2000 ng/ml. for 2 and 1/2 h at 37 ◦ C with 5% CO2 . The blood was then stimulated with Ionomycine and PMA (phorpol 12 myristate 13-acetate) for additional 2 h. Supernatant was collected and assayed for the concentration of the following cytokines: IL-1a, IL-2, Interferon (IFNa), IL-6, IL-12, TNFa. The cells were used to evaluate the expression of cell activation marker CD69. Blood was assayed for CYA concentration using Abbott TDx assay. Results the level of the metabolite was actually higher than the parent compound indicating that the monoclonal antibodies detected the S form preferentially. The results with other CYA monoclonal was also similar. These results suggest that blood level monitoring of CYA many not be accurate as all the monoclonal antibodies currently used cross react with the metabolites. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: CYA; Monitoring; Cross-reactivity; Metabolite
1. Introduction Cyclosporine (CYA) is metabolized in the liver by the Cytochrome P450 system. Many of the metabolites have been identified and thoroughly studied (Burckart et al., 1986; Freed et al., 1987; Zeevi et al., in press; Ryffel et al., in press). Blood level monitoring of CYA became a necessity to differentiate between side effects and under-immunosuppression (rejection). The assays that are currently used to monitor CYA are the high performance liquid chromatography assays (HPLC), radioimmunoassay (RIA), fluorescence polarization immuno assay (FPIA), enzyme multiplied immunoassay technique (EMIT), and CEDI (Masri, 1992; Masri et al., 1992; Lindholm and Henricsson, 1989). With the exception of HPLC, all these methods are based on the use of anti-CYA monoclonal antibodies. These antibodies are supposedly very ∗
Corresponding author. Fax: +961-1-203-48. E-mail address: [email protected] (M. Masri).
specific with minimal cross-reactivity with the metabolites. It is also generally accepted that these metabolites are not abundant and are not immunosuppressive. Thus almost 90% of the transplant centers world wide have opted to use the monoclonal technique for CYA monitoring. The remaining 10% are using the HPLC which is designed for the routine detection of the parent compound only. However, one of the least known of these metabolites (CYA-S) is a sulphuric acid conjugate of CYA and is abundant in the blood of CYA-treated patients (Johansson et al., 1990). Although this molecule was first described in 1990, it was described as a non-active CYA metabolite (Henricsson et al., 1989). The metabolite is hydrophilic and thus it is not easily detectable by HPLC and it is not available in pure form for analysis. We have isolated the metabolite from transplant patients receiving CYA as part of their immunosuppressive therapy. The amino acid sequence was determined and the molecule was synthesised in our laboratories. In order to differentiate between the activities of the two compounds,
0161-5890/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0161-5890(03)00079-8
1060
M. Masri et al. / Molecular Immunology 39 (2003) 1059–1060
we have tested their activity in experimental adjuvant arthritis in rats and in cultured lymphocytes. The experimental design required establishing a monitoring procedure for CYA-S which can be used in these procedures. Rather than developing new assay to monitor CYA-S in the blood of animals and in cell culture, we opted to examine the currently used CYA assays mainly FPIA, EMIT and CEDIA.
2. Materials and methods Both CYA and CYA-S were obtained in powder form (gift from IVAX Corporation, Opava, Czech Republic) and dissolved in a solution of 1/9 ethanol PBS. Blood was collected from a healthy volunteer in EDTA vacutainer tubes and aliquoted into 22 tubes. The blood was then cultured in vitro with either CYA or CYA-S to determine the molecular activity. 0.5 ml of blood and 0.5 ml of RPMI was incubated with the following concentration of either CYA or CYA-S: 0, 25, 50, 100, 250, 500, 750, 1000, 1250, 1500, 2000 ng/ml for two and a half hours at 37 ◦ C with 5% CO2 . The concentration “recovery” of the added CYA or CYA-S was determined using the commercially available kits (FPIA, Abbott laboratories USA, EMIT, Syvia or the CDIA).
The level of the metabolite CYA-S was actually higher than the parent compound (Table 1) indicating that the monoclonal antibodies detected the S form preferentially especially, at higher concentration.
Table 1 Difference in the recovery of CYA and CYA-S at different concentrations
0 25 50 100 250 500 750 1000 1250 1500 2000
The results with the other CYA monoclonal were also similar. These results suggest that blood level monitoring of CYA many not be accurate as all the monoclonal antibodies currently used cross-react with the metabolites. Although the HPLC is very specific, however, by measuring only the parent compound, the immunosuppressive effects of the CYA-S are ignored and its contribution to graft outcome is not highlighted. On the other hand it may also be argued that the CYA-S has no immunosuppressive effects and thus, the monoclonal based assays measure both compounds and the relevance of CYA blood level monitoring to therapeutic regiment becomes minimal. These results may explain the poor correlation between the CYA blood levels and the clinical events. 5. Conclusion The CYA-S activity is being assessed in vitro as well as in vivo and if it is proven to be immunosuppressive, the assays should be able to differentiate between CYA and CYA-S. Finally, it would be also beneficial to design new assays that are based on measuring at the site of action, thus eliminating the need to use blood level monitoring. References
3. Results
Concentration added (ng/ml)
4. Discussion
Recovery CYA-S Blood
CYA
0 14 19 53 113 261 408 493 669 857 1245
0 15 31 73 89 183 331 427 489 583 776
Burckart, G.J., Starzel, T.E., Venkataramanan, R., 1986. Excretion of cyclosporine and its metabolites in human bile. Transplant. Proc. 18, 46. Freed, B.M., Rosano, T.G., Lempert, N., 1987. In vitro immunosuppressive properties of cyclosporine metabolites. Transplantation 43, 123. Henricsson, S., Lindholm, A., Johansson, A., 1989. Identification of a sulphate conjugate of cyclosporine. Transplant. Proc. 21, 837. Johansson, A., Henricsson, S., Moller, E., 1990. A novel sulphate conjugate of Cyclosporine occurring in high concentration in vivo. Transplantation 49, 619. Lindholm, A., Henricsson, S., 1989. Simultaneous monitoring of Cyclosporine in blood and plasma with four analytical methods, a clinical evaluation. Transplant. Proc. 21, 1472. Masri, M.A., 1992. Cyclosporine blood level monitoring by three specific methods, RIA H3, RIA I125, and fluorescence polarization: comparison of accuracy, cost, reproducibility and percent recovery. Transplant. Proc. 24 (5), 1716–1717. Masri, M.A., Dhawan, V.S., Hayes, K., et al., 1992. Cyclosporine dosage according to pharmacokinetic profiles leads to better graft and patient survival rates and a decrease in cyclosporine consumption. Transplant. Proc. 24 (5), 1718–1720. Ryffel, B., Foxwell, B.M.J., Mihatsch, M.J., Donatsch, P., Maurer, G., in press. Biological significance of cyclosporine metabolites, Transplant. Proc. 20, 575. Zeevi, A., Eiras, G., Burckart, G., in press. Immunosuppressive effect of cyclosporine metabolites from human bile on alloreactive T-cells, Transplant. Proc.
Short communication
Cyclosporine blood level monitoring Cross-reactivity of anti-Cyclosporine A monoclonal with its sulphate metabolite: an in vitro study Marwan Masri a,∗ , Sylvana Rizk a , Tomas Andrysek b , Vladimir Matha b b
a Rizk Hospital, P.O. Box 11 2388, Beirut, Lebanon IVAX-CR, Ostravska 29, 74770 Opava 9, Czech Republic
Abstract Cyclosporine is metabolized in the liver by the Cytochrom P450 system. Many of the metabolites have been identified and thoroughly studied. However the sulphate metabolite is the least known. The metabolite is hydrophilic and thus it is not easily detectable by HPLC and it is not available in pure form for analysis. We have isolated the metabolite from transplant patients receiving CYA as part of their immunosuppressive therapy. The amino acid sequence was determined and the molecule was synthesised in our laboratories. We have tested the molecule in vitro using cell culture to determine its activity. 1/2 ml of blood + 1/2 ml of RPMI was incubated with the following concentration of either CSA or CSS: 0, 25, 50, 100, 250, 500, 750, 1000, 1250, 1500, 2000 ng/ml. for 2 and 1/2 h at 37 ◦ C with 5% CO2 . The blood was then stimulated with Ionomycine and PMA (phorpol 12 myristate 13-acetate) for additional 2 h. Supernatant was collected and assayed for the concentration of the following cytokines: IL-1a, IL-2, Interferon (IFNa), IL-6, IL-12, TNFa. The cells were used to evaluate the expression of cell activation marker CD69. Blood was assayed for CYA concentration using Abbott TDx assay. Results the level of the metabolite was actually higher than the parent compound indicating that the monoclonal antibodies detected the S form preferentially. The results with other CYA monoclonal was also similar. These results suggest that blood level monitoring of CYA many not be accurate as all the monoclonal antibodies currently used cross react with the metabolites. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: CYA; Monitoring; Cross-reactivity; Metabolite
1. Introduction Cyclosporine (CYA) is metabolized in the liver by the Cytochrome P450 system. Many of the metabolites have been identified and thoroughly studied (Burckart et al., 1986; Freed et al., 1987; Zeevi et al., in press; Ryffel et al., in press). Blood level monitoring of CYA became a necessity to differentiate between side effects and under-immunosuppression (rejection). The assays that are currently used to monitor CYA are the high performance liquid chromatography assays (HPLC), radioimmunoassay (RIA), fluorescence polarization immuno assay (FPIA), enzyme multiplied immunoassay technique (EMIT), and CEDI (Masri, 1992; Masri et al., 1992; Lindholm and Henricsson, 1989). With the exception of HPLC, all these methods are based on the use of anti-CYA monoclonal antibodies. These antibodies are supposedly very ∗
Corresponding author. Fax: +961-1-203-48. E-mail address: [email protected] (M. Masri).
specific with minimal cross-reactivity with the metabolites. It is also generally accepted that these metabolites are not abundant and are not immunosuppressive. Thus almost 90% of the transplant centers world wide have opted to use the monoclonal technique for CYA monitoring. The remaining 10% are using the HPLC which is designed for the routine detection of the parent compound only. However, one of the least known of these metabolites (CYA-S) is a sulphuric acid conjugate of CYA and is abundant in the blood of CYA-treated patients (Johansson et al., 1990). Although this molecule was first described in 1990, it was described as a non-active CYA metabolite (Henricsson et al., 1989). The metabolite is hydrophilic and thus it is not easily detectable by HPLC and it is not available in pure form for analysis. We have isolated the metabolite from transplant patients receiving CYA as part of their immunosuppressive therapy. The amino acid sequence was determined and the molecule was synthesised in our laboratories. In order to differentiate between the activities of the two compounds,
0161-5890/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0161-5890(03)00079-8
1060
M. Masri et al. / Molecular Immunology 39 (2003) 1059–1060
we have tested their activity in experimental adjuvant arthritis in rats and in cultured lymphocytes. The experimental design required establishing a monitoring procedure for CYA-S which can be used in these procedures. Rather than developing new assay to monitor CYA-S in the blood of animals and in cell culture, we opted to examine the currently used CYA assays mainly FPIA, EMIT and CEDIA.
2. Materials and methods Both CYA and CYA-S were obtained in powder form (gift from IVAX Corporation, Opava, Czech Republic) and dissolved in a solution of 1/9 ethanol PBS. Blood was collected from a healthy volunteer in EDTA vacutainer tubes and aliquoted into 22 tubes. The blood was then cultured in vitro with either CYA or CYA-S to determine the molecular activity. 0.5 ml of blood and 0.5 ml of RPMI was incubated with the following concentration of either CYA or CYA-S: 0, 25, 50, 100, 250, 500, 750, 1000, 1250, 1500, 2000 ng/ml for two and a half hours at 37 ◦ C with 5% CO2 . The concentration “recovery” of the added CYA or CYA-S was determined using the commercially available kits (FPIA, Abbott laboratories USA, EMIT, Syvia or the CDIA).
The level of the metabolite CYA-S was actually higher than the parent compound (Table 1) indicating that the monoclonal antibodies detected the S form preferentially especially, at higher concentration.
Table 1 Difference in the recovery of CYA and CYA-S at different concentrations
0 25 50 100 250 500 750 1000 1250 1500 2000
The results with the other CYA monoclonal were also similar. These results suggest that blood level monitoring of CYA many not be accurate as all the monoclonal antibodies currently used cross-react with the metabolites. Although the HPLC is very specific, however, by measuring only the parent compound, the immunosuppressive effects of the CYA-S are ignored and its contribution to graft outcome is not highlighted. On the other hand it may also be argued that the CYA-S has no immunosuppressive effects and thus, the monoclonal based assays measure both compounds and the relevance of CYA blood level monitoring to therapeutic regiment becomes minimal. These results may explain the poor correlation between the CYA blood levels and the clinical events. 5. Conclusion The CYA-S activity is being assessed in vitro as well as in vivo and if it is proven to be immunosuppressive, the assays should be able to differentiate between CYA and CYA-S. Finally, it would be also beneficial to design new assays that are based on measuring at the site of action, thus eliminating the need to use blood level monitoring. References
3. Results
Concentration added (ng/ml)
4. Discussion
Recovery CYA-S Blood
CYA
0 14 19 53 113 261 408 493 669 857 1245
0 15 31 73 89 183 331 427 489 583 776
Burckart, G.J., Starzel, T.E., Venkataramanan, R., 1986. Excretion of cyclosporine and its metabolites in human bile. Transplant. Proc. 18, 46. Freed, B.M., Rosano, T.G., Lempert, N., 1987. In vitro immunosuppressive properties of cyclosporine metabolites. Transplantation 43, 123. Henricsson, S., Lindholm, A., Johansson, A., 1989. Identification of a sulphate conjugate of cyclosporine. Transplant. Proc. 21, 837. Johansson, A., Henricsson, S., Moller, E., 1990. A novel sulphate conjugate of Cyclosporine occurring in high concentration in vivo. Transplantation 49, 619. Lindholm, A., Henricsson, S., 1989. Simultaneous monitoring of Cyclosporine in blood and plasma with four analytical methods, a clinical evaluation. Transplant. Proc. 21, 1472. Masri, M.A., 1992. Cyclosporine blood level monitoring by three specific methods, RIA H3, RIA I125, and fluorescence polarization: comparison of accuracy, cost, reproducibility and percent recovery. Transplant. Proc. 24 (5), 1716–1717. Masri, M.A., Dhawan, V.S., Hayes, K., et al., 1992. Cyclosporine dosage according to pharmacokinetic profiles leads to better graft and patient survival rates and a decrease in cyclosporine consumption. Transplant. Proc. 24 (5), 1718–1720. Ryffel, B., Foxwell, B.M.J., Mihatsch, M.J., Donatsch, P., Maurer, G., in press. Biological significance of cyclosporine metabolites, Transplant. Proc. 20, 575. Zeevi, A., Eiras, G., Burckart, G., in press. Immunosuppressive effect of cyclosporine metabolites from human bile on alloreactive T-cells, Transplant. Proc.