Clin Biochern. Vol.24, pp. 81-87. 1991 Canada. All righte reserved.
0009-9120/91$3.00 + .00 Copyright©1991The CanadianSocietyof Clinical Chemists.
Printedin
Therapeutic Monitoring for Cyclosporine: Difficulties in Establishing a Therapeutic Window LARRY D. BOWERS Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN, USA Despite the fact that cyclosporine (CsA) has been used clinically for a number of years, there is still uncertainty about the efficacy of monitoring its blood levels. Few if any studies have documented clear differentiation of rejection, immunosuppression, and toxicity on the basis of CsA concentrations alone. The issues in CsA monitoring include selection of sample matrix, analytical method, dosing interval and the timing of trough measurements, the temporal relationship between measurements and physiological events such as toxicity, the concurrent presence of multiple other immunosuppressive agents, and the lack of "gold standards" for determining rejection, adequate immunosuppression, and toxicity. In contrast to using CsA levels for the differential diagnosis of rejection and toxicity, there is evidence that maintenance of CsA concentrations within a therapeutic window results in a lower prevalence of toxicity and rejection.
KEY WORDS: cyclosporine; therapeutic range; therapeutic window; rejection; toxicity; transplantation. Introduction rug concentration measurements are useful when dosage and clinical outcomes are not D well correlated. This can occur if there is variable bioavailability or clearance of the drug, if there is significant prevalence of noncompliance, or if there is variable drug handling due to disease or physiological state, such as age or pregnancy. The basis of therapeutic drug monitoring is the presence of a relationship between either a beneficial outcome or a toxic event and the concentration of the drug or its metabolites in an accessible biological fluid. Establishing a therapeutic range for cyclosporine (CsA) (1-4) has presented a formidable task. The lack of "gold standards" for diagnosing toxicity and rejection is a major limitation. Unlike phenytoin, where there is rapid equilibration between the drug and its receptor which reflects its action, there is a delay between CsA concentration measured in body fluids reaching a peak and resultant immunosup-
Correspondence: Larry D. Bowers, Ph.D., D e p a r t m e n t of Laboratory Medicine and Pathology, U n i v e r s i t y of Minnesota, 420 Delaware Street, SE, Minneapolis, MN 55455, USA. M a n u s c r i p t received May 25, 1990; revised and accepted October 9, 1990. CLINICAL BIOCHEMISTRY, VOLUME 24, FEBRUARY 1991
pression or toxicity. Useful measures of immunosuppressive activity of CsA other than overt rejection have been elusive. The only readily repeatable test for nephrotoxicity has been serum creatinine concentration, which is insensitive to small changes in glomerular filtration rate. In addition, there is no consensus on the magnitude of the change in creatinine concentration which represents a toxic event. There may also be additional risk factors that predispose certain patients to toxicity, and we have no clear indication of the time delay between a toxic concentration and the resultant effect. The complex metabolism of CsA and its distribution in blood and tissues complicates the situation further. Analytical considerations A number of factors influence the drug concentration measured, including the choice of matrix, and analytical method. In addition, a number of pharmacological variables may affect the observed toxicity of CsA. CHOICE O F M A T R I X
The choice of serum, plasma, or whole blood influences the concentration of CsA measured, because of the partitioning of CsA and its metabolites between erythrocytes and the plasma (2,3). Metabolites AM1, AM9, and others are found predominantly in the erythrocytes, and their distribution is different from that of CsA (5). The binding protein in erythrocytes has been identified as cyclophilin (6). Its binding avidity is temperature dependent, and the temperature of the whole blood influences the distribution of drug between cells and plasma. Furthermore, reequilibration of whole blood to 37 °C does not reliably restore the in vivo state. Plasma concentration could be construed as representing the "free" drug concentration, although there is significant binding to lipoproteins. Use of plasma would also be favoured by the observation that the binding of CsA to erythrocytes is saturable, and therefore not representative of free concentrations. However, the saturation has been shown to be an artifact of binding at 25 °C, and at 37 °C no saturable binding has been demonstrated (7). The 81
BOWERS TABLE 1 Cross-Reactivity (%) of Various Immunoassays with
Cyclosporine Metabolites Metabolite
New Zealand Rabbit a
Sheep a
Selective Monoclonal ~
Nonselective Monoclonal a
AM1 AMlc AM9 AM4N AM19 (M8) (M9) (M10) (MI3) (M16)
60 -13.5 1.4 2 -----
32 20 13 7 12 -20 4 --
0009-9120/91$3.00 + .00 Copyright©1991The CanadianSocietyof Clinical Chemists.
Printedin
Therapeutic Monitoring for Cyclosporine: Difficulties in Establishing a Therapeutic Window LARRY D. BOWERS Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN, USA Despite the fact that cyclosporine (CsA) has been used clinically for a number of years, there is still uncertainty about the efficacy of monitoring its blood levels. Few if any studies have documented clear differentiation of rejection, immunosuppression, and toxicity on the basis of CsA concentrations alone. The issues in CsA monitoring include selection of sample matrix, analytical method, dosing interval and the timing of trough measurements, the temporal relationship between measurements and physiological events such as toxicity, the concurrent presence of multiple other immunosuppressive agents, and the lack of "gold standards" for determining rejection, adequate immunosuppression, and toxicity. In contrast to using CsA levels for the differential diagnosis of rejection and toxicity, there is evidence that maintenance of CsA concentrations within a therapeutic window results in a lower prevalence of toxicity and rejection.
KEY WORDS: cyclosporine; therapeutic range; therapeutic window; rejection; toxicity; transplantation. Introduction rug concentration measurements are useful when dosage and clinical outcomes are not D well correlated. This can occur if there is variable bioavailability or clearance of the drug, if there is significant prevalence of noncompliance, or if there is variable drug handling due to disease or physiological state, such as age or pregnancy. The basis of therapeutic drug monitoring is the presence of a relationship between either a beneficial outcome or a toxic event and the concentration of the drug or its metabolites in an accessible biological fluid. Establishing a therapeutic range for cyclosporine (CsA) (1-4) has presented a formidable task. The lack of "gold standards" for diagnosing toxicity and rejection is a major limitation. Unlike phenytoin, where there is rapid equilibration between the drug and its receptor which reflects its action, there is a delay between CsA concentration measured in body fluids reaching a peak and resultant immunosup-
Correspondence: Larry D. Bowers, Ph.D., D e p a r t m e n t of Laboratory Medicine and Pathology, U n i v e r s i t y of Minnesota, 420 Delaware Street, SE, Minneapolis, MN 55455, USA. M a n u s c r i p t received May 25, 1990; revised and accepted October 9, 1990. CLINICAL BIOCHEMISTRY, VOLUME 24, FEBRUARY 1991
pression or toxicity. Useful measures of immunosuppressive activity of CsA other than overt rejection have been elusive. The only readily repeatable test for nephrotoxicity has been serum creatinine concentration, which is insensitive to small changes in glomerular filtration rate. In addition, there is no consensus on the magnitude of the change in creatinine concentration which represents a toxic event. There may also be additional risk factors that predispose certain patients to toxicity, and we have no clear indication of the time delay between a toxic concentration and the resultant effect. The complex metabolism of CsA and its distribution in blood and tissues complicates the situation further. Analytical considerations A number of factors influence the drug concentration measured, including the choice of matrix, and analytical method. In addition, a number of pharmacological variables may affect the observed toxicity of CsA. CHOICE O F M A T R I X
The choice of serum, plasma, or whole blood influences the concentration of CsA measured, because of the partitioning of CsA and its metabolites between erythrocytes and the plasma (2,3). Metabolites AM1, AM9, and others are found predominantly in the erythrocytes, and their distribution is different from that of CsA (5). The binding protein in erythrocytes has been identified as cyclophilin (6). Its binding avidity is temperature dependent, and the temperature of the whole blood influences the distribution of drug between cells and plasma. Furthermore, reequilibration of whole blood to 37 °C does not reliably restore the in vivo state. Plasma concentration could be construed as representing the "free" drug concentration, although there is significant binding to lipoproteins. Use of plasma would also be favoured by the observation that the binding of CsA to erythrocytes is saturable, and therefore not representative of free concentrations. However, the saturation has been shown to be an artifact of binding at 25 °C, and at 37 °C no saturable binding has been demonstrated (7). The 81
BOWERS TABLE 1 Cross-Reactivity (%) of Various Immunoassays with
Cyclosporine Metabolites Metabolite
New Zealand Rabbit a
Sheep a
Selective Monoclonal ~
Nonselective Monoclonal a
AM1 AMlc AM9 AM4N AM19 (M8) (M9) (M10) (MI3) (M16)
60 -13.5 1.4 2 -----
32 20 13 7 12 -20 4 --
