Cyclosporine lymphocyte versus whole blood pharmacokinetic monitoring: correlation with histological findings

Cyclosporine Lymphocyte Versus Whole Blood Pharmacokinetic Monitoring: Correlation With Histological Findings A. Barbari, M.A. Masri, A. Stephan, J. Mokhbat, H. Kilani, S. Rizk, G. Kamel, and N. Joubran

C

YCLOSPORINE (CyA) blood trough level (BTL), CyA dose pharmacokinetics, area under the curve (AUC), blood Cyclophylin (CyP) binding, and exposure index (EI) are often used as indicators of CyA efficacy and dose adjustment,1–3 despite conflicting results regarding their correlation with graft rejection and CyA nephrotoxicity.4 –10 CyA immunosuppressive effect is initiated by its binding to its lymphocyte (Lc) receptor, the CyP.11,12 It would be, therefore, more advantageous to monitor CyA pharmacokinetics at the site of action with the Lc by measuring intra-lymphocytic CyA trough level (LTL). Recent studies have shown a good correlation between CyA-CyP binding and its in vitro immunosuppressive activity.13,14 We hypothesized that drug-receptor interaction measurement might reflect more accurately the concentrations of biologically relevant compounds and hence may provide a better correlation with clinical events. The aim of our study was to compare CyA LTL to CyA BTL and dose in relation to 1) histological findings in renal transplant patients with graft dysfunction and 2) total lymphocyte count, a reflector of the state of immunosuppression.

be acute CyA nephrotoxicity. Acute rejection was diagnosed according to the Banff criteria.16 At the time of the biopsy, the following CyA pharmacokinetic parameters BTL and LTL were obtained in addition to a CBC and total Lc count. CyA/BTL was measured in whole blood using monoclonal antibodies (Abbott TDx method). The CyA LTL expressed in picog/Lc, was determined according to the method of Masri et al.7 Results were expressed as mean ⫾ SD and were compared using the Student’s t test and chi-square test. Differences were considered to be statistically significant for P ⬍ .05.

MATERIALS AND METHODS

Significant interpatient variability was observed in relation to the LTL in all patients and within each study group. Three levels were identified as level 1 (⬍10 picog/Lc) with a mean of 3.6 picog/Lc in 38% of the patients (27), level 2 (between 10 and 20 picog/Lc) with a mean of 12.9 picog/Lc in 31.5% of the patients (23), and level 3 (⬎20 picog/Lc) with a mean of 39.9 picog/Lc in the remaining 30.5% of the patients (22).

Forty-six patients with graft dysfunction (mean serum creatinine Scr of 2.2 mg/dL) were included in this study. Thirty-five underwent 43 graft biopsies, and the remaining eleven patients had isolated CMV disease. A historical control group of 26 patients with uneventful posttransplant course and normal graft function (mean Scr 1.2 mg/dL) was chosen for comparison. All groups were on CyA-based triple therapy and were matched for age, gender, weight, and donor age and type. Patients who underwent graft biopsies (35) were divided into Group A: CyA toxicity; Group B: Acute rejection; and Group C: CMV-associated acute rejection. Control patients with normal graft function were assigned to Group D. Twenty patients had their graft biopsies within the first year posttransplantation, and the remaining 15 underwent kidney biopsies after the first year following transplantation. Graft dysfunction was defined as persistent fluctuation in Scr and/or continuous rise in Scr (ⱖ30% from baseline) in the absence of ultrasonographic abnormalities. The graft biopsy was performed if no improvement or continuous deterioration in renal function were noted despite the initiation of Cymevan therapy in CMV-PCR positive patients or the tapering of CyA dose in CMV-PCR negative patients. Acute and chronic CyA toxicity were diagnosed according to previously described criteria.15 Normal biopsy findings in the context of an unexplained graft dysfunction was presumed to

RESULTS Histological Findings

Among the forty-six patients who were diagnosed with graft dysfunction, 35 underwent a total of 43 biopsies. CyA nephrotoxicity (Group A) was diagnosed in 16 biopsies (37%). Acute rejection was observed in a total of 27 biopsies (63%) either isolated (Group B) in 19 biopsies (44%) or associated with CMV disease (Group C) in eight biopsies (19%). Cyclosporine Lymphocyte Trough Level (LTL)

Cyclosporine Monitoring Parameters and Histological Findings

The CyA monitoring parameters including results of CyA dose, BTL, LTL, and Scr are summarized in Table 1. The rejection groups (group B and C) have significantly reduced (P ⬍ .02) CyA LTL (8.6 ⫾ 7.4 and 9.5 ⫾ 5.2 picog/Lc) when compared to either the CyA toxicity group A (18.8 ⫾ 20.7 From the Department of Transplantation and Nephrology, Rizk Hospital, Beirut, Lebanon. Address reprint requests to Antoine Barbari, MD, Transplantation and Nephrology Unit, Rizk Hospital, PO Box 11-3288, Beirut, Lebanon.

0041-1345/01/$–see front matter PII S0041-1345(01)02190-X

© 2001 by Elsevier Science Inc. 655 Avenue of the Americas, New York, NY 10010

2782

Transplantation Proceedings, 33, 2782–2785 (2001)

CYCLOSPORINE LYMPHOCYTE

VS

WHOLE BLOOD

2783

Table 1. Comparison Among Study Groups

Patient groups

CyA Dose mg/kg

CyA BTL ng/mL

CyA LTL Picog/Lc

Lymphocyte Count

Serum Creatinine mg/dL

CyA Toxicity (A) Rejection (B) CMV ⫹ Rejection (C) Normal function (D)

3.8 ⫾ 2.1 3.8 ⫾ 2.0 4.1 ⫾ 1.9 4.7 ⫾ 2.2

134 ⫾ 108 105 ⫾ 71 126 ⫾ 71 187 ⫾ 100

18.8 ⫾ 20.7 8.6 ⫾ 7.4* 9.5 ⫾ 5.2* 16.5 ⫾ 12.6

1227 ⫾ 794 1232 ⫾ 593 1557 ⫾ 763 1562 ⫾ 831

2.3 ⫾ 0.9 2.2 ⫾ 1.1 2.2 ⫾ 0.6 1.2 ⫾ 0.2†

*P: ⬍ .02 when compared with group A and D. † P: ⬍ .001 when compared with group A, B and C.

picog/Lc) or the normal function Group D (16.5 ⫾ 12.5) who had similar results. While CyA Lc binding (LTL) was significantly lower in the rejection groups, no difference was observed between the four groups in any of the CyA dose or the BTL, suggesting poor correlation between CyA dose, BTL, and LTL, and these two former monitoring parameters and CyA toxicity and acute rejection. In contrast, the majority of biopsy-proven acute rejections (95% of group B and 100% of group C) was associated with an LTL ⬍ 20 picog/Lc, as compared to only 62% and 65% in group A and D respectively, as shown in Fig 1. These results were statistically highly significant (P ⬍ .005), indicating strong association between an acute rejection state and low CyA lymphocyte binding (LTL) and poor correlation between this monitoring parameter and CyA nephrotoxicity. Cyclosporine Monitoring Parameters and Total Lc Count

All CyA monitoring parameters were analyzed within each CyA binding level according to low (ⱕ4 mg/kg) or high (⬎4 mg/kg) CyA dose as shown in Table 2. Regardless of the CyA dose, both LTL and total Lc count were similar within each of the three CyA Lc binding levels despite significant differences in CyA BTL, suggesting poor correlation between CyA dose, CyA BTL, and both CyA LTL and total Lc count. In contrast, when the LTL was plotted against the total Lc count (Fig 2), a strong inverse correlation was observed that was highly significant, indicating a solid

association between CyA Lc binding and the total Lc count (a rough indicator of the immune state). DISCUSSION

Our results clearly indicate a wide range of variation in CyA LTL suggesting an important interpatient difference in CyA-CyP binding, independent of CyA dose, and BTL as shown in Table 2. This could be responsible for the variable degrees of calcineurin activity inhibition in different CyA treated renal transplant patients.14 Furthermore, we have shown like others,4 –7 a poor correlation between CyA monitoring parameters (dose, BTL), and clinical events such as rejection episodes and nephrotoxicity (Table 1). These findings may be explained by the recent observations by Batiuk14,18 and Pai19 regarding the weak correlation between CyA BTL and the degree of calcineurin activity inhibition, and by our own observation regarding the poor correlation between the standard monitoring parameters and the CyA Lc binding, an indicator of LTL and a strong inverse correlate of in vitro13,20 and in vivo14 CyA immunosuppressive activity. While our results failed to show any relation between LTL and CyA nephrotoxicity (Table 1, Fig 1), it did clearly demonstrate, however, a strong association between rejection episodes and a low CyA Lc binding state where the majority of patients (96%) with a biopsy-proven acute rejection had a LTL ⬍ 20 picog/Lc with a mean of 8.6 picog/Lc. According to a previous recent report14 on the

Fig 1. Distribution of biopsy results according to CyA Lc binding (LTL ⬍ 20 or ⱖ 20 picog/Lc) P: ⬍.02 Acute rejection vs others; P: ⬍.04 Rejection ⫹ CMV vs others.

2784

BARBARI, MASRI, STEPHAN ET AL Table 2. Correlations

CyA Lc Binding Level

CyA Dose mg/kg

BTL ng/mL

LTL (Picog/Lc)

Lymphocyte (Lc) Count

Level 1 (0 –10) Level 2 (10 –20) Level 3 (⬎20)

ⱕ4 (2.3) ⫾ 0.7 ⬎4 (6.1) ⫾ 1.3* ⱕ4 (2.9) ⫾ 0.7 ⬎4 (5.7) ⫾ 1.3* ⱕ4 (2.7) ⫾ 0.6 ⬎4 (6.0) ⫾ 1.0*

84 ⫾ 44 168 ⫾ 73* 93 ⫾ 24 193 ⫾ 82* 110 ⫾ 45 269 ⫾ 96*

3.2 ⫾ 3.0 4.6 ⫾ 3.5 13 ⫾ 2.6 13 ⫾ 2.3 36.6 ⫾ 15.6 41.6 ⫾ 18.9

1608 ⫾ 542 1603 ⫾ 977 1279 ⫾ 277 1257 ⫾ 522 739 ⫾ 363 849 ⫾ 447

*P ⬍ .001 when compared to the low (ⱕ4 mg/kg) CyA dose.

inverse correlation between CyA Lc binding and the degree of calcineurin activity inhibition, a low CyA Lc binding state could negate the immunosuppressive effect of CyA13,18,20 through the lack of calcineurin inhibition, and would potentiate the risk for acute rejection as was observed in our rejecting patients. Interestingly, our results clearly demonstrate a strong inverse correlation between LTL and the total Lc count at any CyA binding level (Fig 2); whereas, level 3 and level 1 binding were associated with the lowest and the highest total Lc count respectively. Likewise, similar correlations were observed within each study group (data not shown). Since total Lc count may be considered as a rough indicator of the immune state, these findings seem to suggest a good correlation between LTL and the degree of CyA immunosuppressive activity and are in agreement with previous reports.13,14,18,20 We also have shown a lack of significant difference in the total Lc count among the study groups (Table 1), suggesting that Lc count may not be a good monitoring parameter if used alone but may be helpful, however, in predicting clinical events if used in combination with LTL. Our results are not in accordance with those of Shen21 where graft biopsies were performed in the context of graft dysfunction when deemed necessary

rendering the clinical correlation a presumed one rather than biopsy-proven as was the case in our patients. CONCLUSION

Our data clearly demonstrated a significant interpatient variability in LTL (CyA-CyP binding), unrelated to either CyA dose or BTL and probably partially responsible for the poor correlation between the standard CyA monitoring parameters (dose, BTL) and its immunosuppressive effect. Furthermore, acute rejection episodes seem to be associated with a rather low CyA Lc binding (LTL) state, which appears to inversely correlate with the total Lc count, a marker of the immune state. The lack of correlation of CyA nephrotoxicity with either BTL or LTL suggests that nephrotoxicity may be a donor and not a recipient related problem. We propose a new CyA monitoring technique using a receptor-based approach, that is rapid, reliable, and cost-effective. It definitely correlates with CyA pharmacodynamic effect and hence represents a better indicator than the routine monitoring parameters for CyA efficacy since it provides useful indices to establish links between drug concentration and immunosuppressive effect, thereby yield-

Fig 2. Correlation between Lc trough level (LTL) and lymphocyte count according to CyA Lc binding level in all patients. P ⬍ .002 for 1 vs 2 & 3 and for 2 vs 3.

CYCLOSPORINE LYMPHOCYTE

VS

WHOLE BLOOD

ing a reliable insight into CyA dosing.22 Further studies are underway to determine whether LTL in conjunction with total Lc count could effectively be used to provide good therapeutic guidelines in patients with graft dysfunction. REFERENCES 1. Barbari A, Stephan A, Kamel G, et al: Transplant Proc 29:2941, 1997 2. Lorber MI, Paul K, Harding MW, et al: Transplant Proc 22:1240, 1990 3. Paul K, Harding MW, Marks WH, et al: Transplant Proc 23:974, 1991 4. Klintmalm G, Sawe J, Ringden O, et al: Transplantation 39:132, 1985 5. Albercht K, Niebel W, Marggraf G, et al: Transplant Proc 19:1719, 1987 6. Ferguson RM, Canafax DM, Sawchuk RJ, et al: Transplant Proc 183:113, 1985 7. Kasike BL, Heim-Duthoy K, Venkateswara R, et al: Transplantation 46:716, 1988 8. Lindholm A, Kahan BD: Clin Pharmacol Ther 54:205, 1993 9. Meyer MM, Munar M, Udeaja J, Bennett W: J Am Soc Nephrol 4:1306, 1993

2785 10. Kahan BD, Welsh M, Schoenberg L, et al: Transplantation 62:599, 1996 11. Takahaski N, Hayano T, Suzuki M: Nature 337:473, 1989 12. Emmel E, Verweij CL, Durand DB, et al: Science 246:1617, 1989 13. Durette PL, Bager J, Dumont F, et al: Transplant Proc 20:51, 1988 14. Batiuk TD, Pazderka F, Enns J, et al: J Clin Invest 96:1254, 1995 15. Danovitch GM: Handbook of Kidney Transplantation. Boston: Little Brown; 1992, p 272 16. Solez K, Axelsen RA, Benediktsson H, et al: Kidney Int 44:411, 1993 17. Masri MA, Barbari A, Stephan A, et al: Transplant Proc 30:3561, 1998 18. Batuik TD, Pazderka F, Halloran P: Transplantation 59: 1400, 1995 19. Pai SY, Fruman DA, Leong T, Neuberg D, et al: Blood 84:3974, 1994 20. Handschumacher RE, Harding MW, Rice J, Drugge RJ, et al: Science 226:544, 1984 21. Shen SY, Weir MR, Kosenko A, Revie DR, et al: Transplantation 40:620, 1985 22. Kahan BD, Napoli KL: Transplant Proc 30:2189, 1998