Like others, we have shown a weak correlation between drug blood levels and clinical outcome and immune response. We recently established that in contrast to blood levels, drug lymphocyte levels are strongly associated with therapeutic efficacy. The discordance between the 2 methodologies regarding clinical outcome and immune response is related mainly to the weak association between drug blood level and target-cell content. This weak association explains the intra- and interindividual variabilities of the therapeutic response. These variations are related mainly to genetic and environmental factors including age, sex, body mass index, organ function, food and subsequent drug absorption, drug interactions, and the availability of extra–target-cell binding sites. These factors seem to influence the modes of action of immunosuppressive agents including drug absorption, metabolism, elimination, transport, extra–target-cell sites, as well as target-cell receptor expression and its binding affinity and specific enzyme baseline activity. Therefore, the cellular fraction of a drug at a fixed dosage is the result of the integration of out-fluxing and in-fluxing forces that are affected by genetic and environmental factors. Any redistribution of the drug between the different binding sites will ultimately affect its bioactivity with no change to its extracellular bioavailability (which is currently determined by pharmacokinetic measurements). Compared with whole-blood or plasma-level measurements, monitoring immunosuppression therapy at the site of action appears to be not only more clinically and immunologically relevant (since it measures the free fraction of the drug at its effector site), but this method also bypasses the potentially complex extracellular factors that affect bioactivity. 

Since the intracellular content of a drug strongly correlates with its biological effect, monitoring immunosuppression therapy at the site of action is comparable to pharmacodynamic monitoring. It is cost effective and easy to perform in clinical practice and could be used for all immunosuppressive drugs. Since it allows maximal reduction of adverse effects through dosage reduction while maintaining an optimal level of immunosuppression, it should offer a new alternative for immunosuppressive therapy monitoring and tailoring to the individual patient.

Key words : Immunity, Bioavailability, Bioactivity, Pharmacokinetic, Pharmacodynamic

The lack of a reliable immune response assay has been a significant hurdle for clinicians in their attempts to monitor the immune state. Despite the enormous amount of literature published over the last 2 decades, the clinical relevance of most monitoring techniques for cyclosporine therapy and other immunosuppressive agents in solid organ transplant remains controversial (1-5). Unfortunately, short of graft biopsies, all currently used noninvasive pharmacokinetic therapeutic monitoring techniques have proven unreliable (6-9). Monitoring the efficacy of immunosuppressive therapy in most trials is accomplished mainly by measuring target-drug blood or plasma levels, which differ among trials and among immunosuppressive protocols within each trial. Additionally, surveillance of renal function is accomplished by performing graft biopsies according to clinical indications.

We and others have shown a weak correlation between drug blood dosages and levels and clinical outcome and immune response (3-11). We established that in contrast to drug blood levels, drug lymphocyte levels are strongly associated with therapeutic efficacy as demonstrated by clinical outcome (mainly acute rejection [Figure 1] and immune responsiveness [11-17] [Figure 2]). These differences are due to the significant discordance between bioavailability and bioactivity in relation to immune sensitivity (6, 7, 8, 11, 12, 13, 18), acute rejection, and adverse effects.

Bioavailability, as measured by pharmacokinetic assay, represents the fraction of an administered amount of a drug that reaches the peripheral circulation; it reflects the effect of the body on the drug. In contrast, bioactivity, which is a major pharmacodynamic marker that reflects the effect of the drug on the body, correlates strongly with the amount of the active ingredient of a drug that is available at the site of its effector (18, 19). As we and others have demonstrated (11-16, 20), the discordance between bioavailability (drug levels in blood and plasma) and bioactivity (biological effect) is related mainly to the weak association between drug levels in blood or plasma and target-cell content and tissue concentration (Figure 3). The poor correlation between drug blood level and drug lymphocyte level is explained by the striking interindividual variability that we have shown recently regarding drug lymphocyte levels for cyclosporine, tacrolimus, and sirolimus within the same ranges of drug blood levels and dosages (12, 14, 17) (Figure 4). These observations are in agreement with the works of Londono (21) and Hartmann (22) regarding the poor relationship between tacrolimus and sirolimus blood levels and target enzyme (ie, calcineurin and p70 S6 kinase) activities, respectively.

This poor relationship clearly indicates that bioavailability and bioactivity are not bioequivalent. Variations between these 2 factors are related mainly to genetic, ethnic, and environmental parameters such as age, sex, body mass index, organ function, food and subsequent drug absorption, drug interactions, and the availability of extra–target-cell binding sites (23-25). These parameters seem to influence all the steps involved in the mode of action of any immunosuppressive agent (including drug absorption, metabolism, elimination, transport, extra–target-cell binding sites) as well as target-cell P-glycoprotein efflux pump expression, receptor affinity, and specific enzyme inhibition (Figure 5), which explains the existence of several recipient profiles with regard to drug absorption, metabolism, transport, and cellular binding. Therefore, the intracellular fraction of a drug depends not only on the amount of free drug available in the blood (extracellular compartment) but also on the availability of extra-lymphocytic binding sites (cyclophilin B and red blood cell mass for cyclosporine), the activity of the lymphocyte P-glycoprotein pump, and receptor expression and its drug-binding affinity, which are all affected by genetic and environmental factors (Figure 5). Any variation in lymphocyte P-glycoprotein activity and/or redistribution of the drug between the different binding sites (eg, red blood cell mass [Figure 6] and cyclophilin B and lymphocyte mass) will ultimately affect the cellular concentration of the drug and hence, its bioactivity, with no change in its extracellular bioavailability (12, 26-29). 

Any increase in cyclophilin B and/or a decrease in hematocrit and/or lymphocyte P-glycoprotein activity will enhance cyclosporine entry into the lymphocyte and hence, its bioactivity, with no change in its extracellular bioavailability. Conversely, any decrease in cyclophilin B and/or increase in red blood cell mass and/or lymphocyte P-glycoprotein activity will facilitate extrusion of cyclosporine from the lymphocyte and hence, will reduce its bioactivity with no change in its extracellular bioavailability. Other genetic and environmental combinations are possible as well and may influence the intracellular concentration of a drug in one way or another. Therefore, the ultimate net intracellular amount of a drug at a fixed dosage is the net result of the integration of both out-fluxing and in-fluxing forces that are influenced by genetic (50%) and environmental factors (50%). 

We recently suggested that cyclosporine-induced nephrotoxicity is predominantly donor dependent with a partial recipient influence related to drug bioavailability and exposure (8, 12, 13, 30). Hauser and associates recently confirmed this hypothesis by establishing that the ABCB1 genotype of the donor but not of the recipient is a major risk factor for cyclosporine nephrotoxicity after renal transplant (7). Expression of the TT genotype in the donor at the ABCB1 3435C→T polymorphism, which is associated with decreased expression of the P-glycoprotein pump in renal tissue that extrudes cyclosporine from renal cells, becomes a major risk factor for developing cyclosporine nephrotoxicity. Evidently, this nephrotoxicity is amplified and exaggerated by higher drug exposure and hence, a greater bioavailability that is solely recipient dependent. Interestingly, the effect of this specific genotype expression on cyclosporine lymphocyte content and hence, on immune responsiveness in the recipient, remains unknown. These findings are in agreement with our own observations and those of others (3, 7, 8, 12, 13, 16) on the poor correlation between cyclosporine nephrotoxicity, which is donor dependent, and cyclosporine monitoring tools (ie, blood and lymphocyte levels), which are recipient related. These interesting observations explain the intra- and interindividual variabilities in therapeutic response and adverse reactions and hence, the poor correlation between pharmacogenetics assessing recipient bioavailability and chronic allograft nephropathy, which is both donor and recipient related.

It is currently estimated that genetic factors account for nearly half of the individual variations in the efficacy and toxicity of drugs (7, 31-33). These genetic factors could explain the differences (despite their similar designs) between the 2- and 3-continent trials, in which calcineurin inhibitors were withdrawn and sirolimus was added (34, 35), in the reported incidences of biopsy-proven acute rejection in the calcineurin-inhibitor–sparing groups. These genetic factors also may explain the higher incidence of hyperlipidemia and the use of lipid-lowering agents in sirolimus-maintenance groups observed mainly in 2 other calcineurin-inhibitor–sparing studies conducted in Spain and Italy (36, 37). These findings are in agreement with our own observations (38) on the relatively high rate of adverse events (mainly mixed hyperlipidemia) in our sirolimus-treated patients. Given the importance of these genetic and environmental factors and their significant potential impact on clinical outcome, using the newly emerging disciplines of pharmacogenetics and pharmacogenomics, it is essential to account for them when designing future trials (39) (31-33). This also should caution against extrapolating universal recommendations based on studies conducted in a specific geographic area or in a population of a particular ethnic background. Conducting similar studies in different areas of the world is crucial in today’s era of applied clinical practice according to evidence-based medicine. Given the strong correlation between drug lymphocyte content and clinical outcome (mainly acute rejection and immune responsiveness and the potential impact of extra–target-binding sites and the P-glycoprotein pump expression on drug lymphocyte content in the recipient that could significantly affect bioactivity without changing extracellular bioavailability), monitoring immunosuppressive drugs at their target cells becomes that much more clinically relevant and hence, more appropriate than monitoring the drugs in blood or plasma.

This type of specific monitoring at the site of action is easily applicable in clinical practice, cost-effective, and could be used for all immuno­suppressant medications. Since it allows maximal reduction of adverse effects through dosage reduction, while maintaining an optimal level of immunosuppression, it offers a new alternative for immunosuppressive therapy monitoring and tailoring to the individual patient. The validity of this novel approach should be confirmed through larger, multicentered, multinational trials.

References:

1. Grevel J, Welsh MS, Kahan BD. Cyclosporine monitoring in renal transplantation: area under the curve monitoring is superior to trough-level monitoring. Ther Drug Monit. 1989;11(3):246-248.
2. Barbari A, Stephan A, Kamel G, Kilany H, Masri MA. Experience with new cyclosporine formulations: Consupren and Neoral in renal transplant patients. Transplant Proc. 1997;29(7):2941-2944.
3. Mahalati K, Belitsky P, Sketris I, West K, Panek R. Neoral monitoring by simplified sparse sampling area under the concentration-time curve: its relationship to acute rejection and cyclosporine nephrotoxicity early after kidney transplantation. Transplantation. 1999;68(1):55-62.
4. Thervet E, Pfeffer P, Scolari MP, et al. Clinical outcomes during the first three months posttransplant in renal allograft recipients managed by C2 monitoring of cyclosporine microemulsion. Transplantation. 2003;76(6):903-908.
5. Marcén R, Pascual J, Tato A, et al. Comparison of C0 and C2 cyclosporine monitoring in long-term renal transplant recipients. Transplant Proc. 2003;35(5):1780-1782.
6. Groth CG, Bäckman L, Morales JM, et al. Sirolimus (rapamycin)-based therapy in human renal transplantation: similar efficacy and different toxicity compared with cyclosporine. Sirolimus European Renal Transplant Study Group. Transplantation. 1999;67(7):1036-1042.
7. Hauser AI, Schaeffeler E, Gauer S, et al. ABCB1 genotype of the donor but not the recipient is a major risk factor for cyclosporine-related nephrotoxicity after renal transplantation. J Am Soc Nephrol. 2005;16:1501-1511.
8. Barbari A, Masri MA, Stephan A, et al. Cyclosporine lymphocyte versus whole blood pharmacokinetic monitoring: correlation with histological findings. Transplant Proc. 2001;33(5):2782-2785.
9. Kuypers DR, Vanrenterghem Y, Squifflet JP, et al. Twelve-month evaluation of the clinical pharmacokinetics of total and free mycophenolic acid and its glucuronide metabolites in renal allograft recipients on low dose tacrolimus in combination with mycophenolate mofetil. Ther Drug Monit. 2003;25(5):609-622.
10. Yatscoff RW, Aspeslet LJ, Gallant HL. Pharmacodynamic monitoring of immunosuppressive drugs. Clin Chem. 1998;44(2):428-432.
11. Barbari A, Stephan A, Masri MA, et al. Mycophenolic acid plasma trough level: correlation with clinical outcome. Exp Clin Transplant. 2005;3(2):355-360.
12. Barbari A, Masri M, Stephan A, et al. Cyclosporine lymphocyte maximum level: a new alternative for cyclosporine monitoring in kidney transplantation. Exp Clin Transplant. 2005;3(1):293-300.
13. Barbari A, Masri M, Stephan A, et al. Cyclosporine lymphocyte maximum level monitoring in de novo kidney transplant patients: a prospective study. Exp Clin Transplant. 2006;4(1):400-405.
14. Barbari A, Masri M, Stephan A, et al. Tacrolimus whole blood versus lymphocyte trough level: Correlation with immune response. [MESOT abstract]. Exp Clin Transplant. 2006;4(2):58.
15. Barbari A, Masri M, Stephan A, et al. Cyclosporine A whole blood vs lymphocyte maximum concentration: correlation with immune response. [MESOT abstract]. Exp Clin Transplant. 2006;4(2):58.
16. Barbari A, Masri M, Stephan A, et al. Tacrolimus lymphocyte vs blood trough level monitoring in de novo kidney transplant patients: Clinical relevance. [MESOT abstract]. Exp Clin Transplant. 2006;4(2):36.
17. Masri M, Rizk S, Barbari A, Stephan A, Kamel G, Rost M. An assay for the determination of sirolimus levels in the lymphocyte of transplant patients. Transplant Proc. 2007;39(4):1204-1206.
18. Batiuk TD, Pazderka F, Enns J, DeCastro L, Halloran PF. Cyclosporine inhibition of calcineurin activity in human leukocytes in vivo is rapidly reversible. J Clin Invest. 1995;96(3):1254-1260.
19. Batiuk TD, Kung L, Halloran PF. Evidence that calcineurin is rate-limiting for primary human lymphocyte activation. J Clin Invest. 1997;100(7):1894-1901.
20. Podder H, Stepkowski SM, Napoli KL, et al. Pharmacokinetic interactions augment toxicities of sirolimus/cyclosporine combinations. J Am Soc Nephrol. 2001;12(5):1059-1071.
21. Londono MC, Cirera I, Brunet M, Millan O, Martorell J, Rimola A. Calcineurin inhibitors (CNI) Pharmacodynamics in liver transplantation. [ESOT abstract No. 243]. Transplant Int. 2005:109.
22. Hartmann B, Schmid G, Graeb C, et al. Biochemical monitoring of mTOR inhibitor-based immunosuppression following kidney transplantation: a novel approach for tailored immunosuppressive therapy. Kidney Int. 2005;68(6):2593-2598. 
23. Srinivas TR, Meier-Kriesche HU, Kaplan B. Pharmacokinetic principles of immunosuppressive drugs. Am J Transplant. 2005;5(2):207-217.
24. Wilkinson GR. Drug metabolism and variability among patients in drug response. N Engl J Med. 2005;352(21):2211-2221.
25. Christians U, Strom T, Zhang YL, et al. Active drug transport of immunosuppressants: new insights for pharmacokinetics and pharmacodynamics. Ther Drug Monit. 2006;28(1):39-44.
26. Rosano TG. Effect of hematocrit on cyclosporine (cyclosporin A) in whole blood and plasma of renal-transplant patients. Clin Chem. 1985;31(3):410-412.
27. Denys A, Allain F, Masy E, Dessaint JP, Spik G. Enhancing the effect of secreted cyclophilin B on immunosuppressive activity of cyclosporine. Transplantation. 1998;65(8):1076-1084.
28. Denys A, Allain F, Foxwell B, Spik G. Distribution of cyclophilin B-binding sites in the subsets of human peripheral blood lymphocytes. Immunology. 1997;91(4):609-617.
29. Barbari A, Stephan A, Masri M, et al. Parameters affecting temporal variations of CsA lymphocyte maximum levels in stable de novo kidney Transplant patients. [MESOT abstract]. Exp Clin Transplant. 2004; 2(2): 12.
30. Barbari A, Stephan A, Masri MA, Kamel G, Kilani H, Barakeh A. Chronic graft dysfunction: donor factors. Transplant Proc. 2001;33(5):2695-2698.
31. Meyer UA. Pharmacogenetics and adverse drug reactions. Lancet. 2000;356(9242):1667-1671.
32. Ingelman-Sundberg M. Pharmacogenetics: an opportunity for a safer and more efficient pharmacotherapy. J Intern Med. 2001;250(3):186-200.
33. Fredericks S, Holt DW, MacPhee IA. The pharmacogenetics of immunosuppression for organ transplantation: a route to individual­ization of drug administration. Am J Pharmacogenomics. 2003;3(5):291-301.
34. Johnson RW, Kreis H, Oberbauer R, Brattström C, Claesson K, Eris J. Sirolimus allows early cyclosporine withdrawal in renal transplantation resulting in improved renal function and lower blood pressure. Transplantation. 2001;72(5):777-786.
35. Gonwa TA, Hricik DE, Brinker K, Grinyo JM, Schena FP; Sirolimus Renal Function Study Group. Improved renal function in sirolimus-treated renal transplant patients after early cyclosporine elimination. Transplantation. 2002;74(11):1560-1567.
36. Grinyo JM, Campistol JM, Paul J, et al. Pilot randomized study of early tacrolimus withdrawal from a regimen with sirolimus plus tacrolimus in kidney transplantation. Am J Transplant. 2004;4(8):1308-1314.
37. Stallone G, Di Paolo S, Schena A, et al. Early withdrawal of cyclosporine A improves 1-year kidney graft structure and function in sirolimus-treated patients. Transplantation. 2003;75(7):998-1003.
38. Stephan A, Barbari A, Kamel G, et al. Sirolimus side effects. The different experience of a single centre. [MESOT abstract]. Exp Clinic Transplant. 2004;2(2):38. 
39. Barbari A, Stephan A, Masri M. Calcineurin Inhibitors-free immunosuppression: Risks and benefits. Saudi J of Kid Dis Transplant. March 2007;18(1):1-23.