Burckart, G

Duquesnoy, RJ

Eiras, G

Fung, J

Jain, A

Kragack, A

Starzl, TE

Todo, S

Venkataramanan, R

Zeevi, A

English

PubMed

D-Scholarship@Pitt

Bioassay of Plasma Specimens From Liver Transplant Patients on FK 506 Immunosuppression A. Zeevi, G. Eiras, G. Burckart, A. Jain, A. Kragack, R. Venkataramanan, S. Todo, J. Fung, T.E. Starzl, and R.J. Duquesnoy L IVER transplantation has been increasingly used to treat end-stage liver diseases since the introduction of Cy A in combination with other immunosuppressive agents. 1 Although Cy A has a powerful immunosuppres-sive effect on allograft rejection, many transplant recipi-ents still experience rejection episodes that are often irreversible. The nephrotoxicity of Cy A in some recipients has necessitated the reduction of CyA dosage below effective levels. With liver recipients refractory to conven-tional immunosuppression or with CyA-induced nephro-toxicity, a first-phase clinical trial of a new immunosuppres-sive drug, FK 506. has been initiated at our institution. Z FK 506 was shown by in vitro and in vivo studies to be considerably more immunosuppressive than CyA. J•7 The successful outcome of the initial clinical studies on 14 liver transplant recipients2 has led to a formal clinical trial of FK 506 as the primary immunosuppressive agent in combina-tion with low doses of steroids.8 Appropriate dosages of FK 506 are being determined to provide optimal immunosuppression without significant adverse side effects. The monitoring of blood levels of FK 506 is essential in these studies. This monitoring has become possible with an enzyme immunoassay (ELISA) using anti-FK 506 MoAbs. This assay was developed by Tamura et al9 and modified by Cadoff et al. 10 FK 506 is a highly lipophilic macrolide (molecular weight 822). II Although little is known about the metabo-lites of FK 506, recent studies have implicated the liver as the primary site of FK 506 metabolism. 12 The objective of this study was to determine the rela-tionship between plasma levels of FK 506 as measured by ELISA and by a bioassay based on the proliferative response of an alloreactive T cell line. MATERIALS AND METHODS Plasma Five milliliters of heparinized blood was collected from four liver transplant recipients during lhe lirst month following transplanta· tion. Each sample was drawn Just prior to FK 506 administration. All blood samples were incubated at 37"C for I hour prior to centrifugation. The plasma was analyzed for FK 506 trough levels using an indirect ELISA 10 and a bioassay described in detail below. Drug Source FK 506 was supplied in crystalline form by the Fujisawa Pharma-ceutical Co Ltd. Osaka. Japan. A stock solution of 100 !L8iml FK 506 was prepared in methanol and kept at -4°C. Bioassay of FK 506 Levels in Plasma Inhibition Effect of FK 506 on Lymphocyte Proliferation. This was determined in a secondary proliferation assay using a DQwI-specific alloreactive T cell clone 082913 as responder cells. Frozen cloned cells were thawed and washed in RPM I 1640 (Gibcol and resuspended at 10-' cells/ml in tissue culture medium (TCM) consisting of RPMI 1640 supplemented with 25 mmoVL Hepes buffer. 100 U/ml gentamicin. and 10% nontransfused human male serum. The inhibitory effect of FK 506 on the primed lymphocyte (PLTI response of D829 cells was measured at different concentrations of the drug. ranging from 0.003- t nglml. In these 3-day PL T assays. 10' responder cells were incubated in triplicate with IW DQw I-positive stimulator cells irradiated (2.000 rad) in 200 IA-i of TCM. During the final 20 hours of incubation. each culture was labeled with IILCi ofJH-thymidine. The cultures were harvested and counted in a liquid scintillation counter (LKB). The inhibitory effect of FK 506 at various concentrations was determined with the following formula: ( cpm with drug ) percent inhibition = I -. x 100 cpm wtthout drug Effect of Plasma Samples on Lymphocyte Proliferation. Plasma samples were diluted (V2 to V .. ) in TCM. The inhibitory effect of different plasma dilutions on the PL T response of 0829 cells was measured as described for FK 506. In these assays. 10" responder cells (50 IA-il and 10-' stimulator cells (50 IA-i) were incubated with 100 ILl of plasma. A pretransplant plasma sample from each patient was used as a control. The results were calculated with the following formula: percent mhibition in plasma = I - x 100 ( cpm with test PlaSma) cpm control plasma, Calculation of FK 506 Levels From Bioassay Results. The plasma concentrations of FK 506 were calculated from the inhi-bition curve observed with various plasma dilutions in comparison with a standard curve for FK 506. These calculations were made using a nonlinear sigmoidal model curve litting program (Statisti-cal Analysis System ISASllnstitute. release 5.18. Cary. Nc). The From the Division of Clinical Immunopathology, Schools of Pharmacy and Surgery. Presbyterian-University Hospital, Univer· sity of Pittsburgh, Pittsburgh. PA. Supported by Grants No. HL36416 and A123467 from the National Institutes of Health. Bethesda. MD. Address repnnt requests to A. Zeevi. PhD. University of Pitts-burgh, Clinical Immunopathology-CLSI, Presbyterian-University Hospital. DeSoto & O'Hara Streets, Pittsburgh, PA 15213-2582. !:l 1990 by Appleton & Lange 0041-13451901$3.001 + 0 60 Transp/alltat1Orl Proceealngs, Vol 22. No 1, Suppj t (February), 1990: pp 6().63 FK 506 BIOASSAY IN TRANSPLANT PATIENTS IC50 from the standard curve was determined as the concentration of FK 506 (ng/m!) which caused 50% inhibition of the PLT response of 0829. From the plasma Inhibition curve. the 10'0 was determined as the dilution factor for which plasma induced 50% inhibition of PLT response of 0829. From these values. the plasma concentration of FK 506 was calculated as fC~o x 1050, RESULTS Initial studies showed highly consistent dose response curves of PLT inhibition of D829 celis by FK 506. Fig 1 shows representative results of three separate experi-ments. These data yielded a mean fC~o value of 0.063 :!: 0.001 ngtmi. Examples of PLT inhibition of D829 by patient plasma are shown in Table I. These bioassays were done on a liver allograft recipient at 7. 12. and 14 days posttransplant. The plasma FK 506 levels were calculated by multiplying the fa~o values with the fC~o value of 0.063 nglml. Companson Between FK 506 Levels Measured by ELISA and Bioassay In all plasma samples tested. the values for FK 506 levels obtained by ELISA were generally much higher than those determined in the bioassay. Fig 2 compares FK 506 levels determined by ELISA and bioassay for two liver trans-plant recipients. Patient P2 was originally diagnosed with sclerosing cholangitis and had lost five previous liver allografts while on CyA treatment. Following the sixth transplant. 6 mgld of FK 506 was administered intravenously for 7 days. Methylprednisolone (I g) was given on the second day posttransplant and was tapered daily to a maintenance dose of 10-20 mgld. Although FK 506 levels determined by bioassay were always lower than those measured in St.nd.rd curve 100 !:; ... 75 -l!: °111 c: Q IC 50 • 00S3t 0.001 nglmI FK50e ,g! ~! .5 : 50 _c: ~CK ... • ! 0.. 25 . 01 . 1 FKSII nglml Fig 1. Standard curves of the FK 506 bioassay. The concentra-tion of FK 506 is plotted versus percent inhibition of PL T response of DB29 cells. The ICso was calculated uSIng a nonlinear sigmoIdal model fitted to the points. 61 Table 1. Bioassay of FK 506 Levels In Plasma From a Liver Transplant Recipient Dilutions ("to Inhibitionl FK 506t Days Posttransolanl 1:4 1:8 1:16 1:32 ID",' (ng/mll 7 99 89 48 0 15 0.94 12 93 40 0 0 7 0.44 14 99 98 76 16 21 1.32 'ID5O represents the plasma dilUtion factor causing 50"1. inhibition of PLT response of DB29 cells. tCalculated trv multiplying ID", by \he IC50 of 0.063 nglml. ELISA (0.2-2 nglml versus 0.5-4.5 nglm!). they showed a significant correlation (P < 0.001. r = 0.77). Following the first bolus of methylprednisolone (day 2 postsurgeryl, the FK 506 concentrations measured by ELISA were four- to fivefold higher than the bioassay levels. FK 506 was discontinued for 4 days; the ELISA concentrations of FK 506 dropped sharply to 0.5-1 nglml. and no detectable immunosuppressive activity was found in the bioassay in those plasma samples. On day II postsurgery. the patient showed clinical symptoms of graft dysfunction as determined by changes in liver enzymes. On the same day. FK 506 (6 mgld) and a bolus of methylprednisolone (I g) were administered. Conse-quently. there was a rapid increase in the ELISA plasma concentrations of FK 506 and a more gradual rise in the bioassay-determined FK 506 levels (Fig 2a). Similar findings are shown for patient P14. who was I •. E ~ . '" ... P •• , OP.'IU..! J....! -, -,-• ......::.::::::.,' .. 0-,"', ;...,-.-,-.-'-.-I-• ...::-I7 ......... '--I1....:<f-0., FKIOI •••• ""/~Dv E 0' c ... :;t • '" ... b. oL---------------P ••• Op., ........ , .121,,.15,.'71.1. 0., FK'" _ •••• ,,--- '.5.2 ---+ .. -Fig 2. FK 506 levels In plasma measured by the bioassay and by the ELISA for lIver transplant patIents. (a) P2 and (b) P14. 62 diagnosed with hepatitis B and juvenile diabetes mellitus. He received kidney, pancreas. and liver allografts. FK 506 (4.5 mg) was administered intravenously two times per day, and this dose was maintained for 19 days posttrans-plantation. Steroids were given on days 2, 5, and 19 post surgery . A significant correlation (P < 0.001. r = 0.75) was found between the two assays for plasma levels of FK 506 (Fig 2b). Following steroid administration, FK 506 levels deter-mined by bioassay were still much lower than ELISA plasma concentrations of FK 506, Relationship Between FK 506 Dose and the Plasma Concentration There was no apparent correlation between the dose of FK 506 given and the concentration of FK 506 in the patient's plasma. Although patients P14. P16. and P32 received the same dose of FK 506. the plasma levels of FK 506 (ELISA and bioassay) were much higher in patients PI4 and PI6 than in the patient P32 (Fig 2 and Table 2). Furthermore. in three of four plasma samples tested from patient P32. the bioassay levels of FK 506 were <0.1 ng/ml. whereas the ELISA values ranged from 0.3-\.6 ng/ml. This patient had a histologically confinned cellular rejection. Patient P16. on a similar dose of FK 506. had a stable posttransplant course. His plasma concentrations of FK 506 as deter-mined by ELISA and bioassay were 1.2-6 ng/ml and 1-4 ng/ml. respectively. DISCUSSION This study was designed to assess the correlation between FK 506 levels measured by bioassay and ELISA. The bioassay was based on the inhibition of PL T reac-tivity of an alloreactive T cell clone. Low concentrations of FK 506 significantly inhibited the proliferative re-sponses of mixed lymphocyte reaction (MLRl-derived Table 2. Correlation Between Blo .... y .nd ELISA Drug Dose Days PosttranSJ)lant (mglday) Patient P32 2 4.5 x 2 IV" 5 4.5 x 2 IV" 8 9 x 2 PDt 12 9 x 2 PDt Patient P16 2 9 x 2 PDt 3 9 x 2 PDt 4 9 x 2 PDt 5 9 x 2 PDt 6 9 x 2 PDt 7 9 x 2 POt 'Intravenous dose 01 4 5 mg FK S06 twice • day. tOraI dose 01 9 ITIgId twICe • day. Bioassay (ng/mI) < 0.1 1.7 < 0.06 < 0.06 2.4 1.7 1.0 1.3 ·4 2.5 ELISA Ing/ml) 1.6 6.4 0.9 0.3 2.6 2.3 1.2 1.8 6 3 ZEEVI, EIRAS, BURCKART ET AL alloreactive T cell clones! and this inhibition was repro-ducible when the same T cell line was used (Fig I), The sensitivity of the ELISA for FK 506 was 0.1 ng/ml lo whereas in the bioassay. drug levels of 0.063 :!: 0.001 ng/ml had a significant inhibitory effect on the responder cells, The levels of FK 506 determined in bioassay were consistently lower than those measured by ELISA. This suggests that the blood may contain inactive metabolites of FK 506 which could be detected by the ELISA. Since the ELISA is an immunoassay, it may measure biologically active and non-active metabolites that might cross-react with the anti-FK 506 antibody. Similarly. Cy A metabolites are measured by monoclonal and polyclonal radioimmu-noassay (RIA )used for monitoring trough levels ofCyA in transplant recipients. 14 FK 506-treated patients received a bolus of high-dose steroids the second day posttransplant and when acute allograft rejection was suspected. Steroids could affect the determination of FK 506 levels by the bioassay since this assay does not discriminate between various Immunosup-pressive drugs. Nevertheless. the FK 506 levels measured by bioassay poststeroid treatment were not much affected in contrast to the marked increases in immunoassayed FK 506. It is unclear how steroids influence ELISA assay. Corticosteroids have been shown to be inducers of hepatic drug metabolism. 12 Experience with CyA immunosuppres-sion has demonstrated that RIA levels of CyA increased after steroid treatment while high-pressure liquid chroma-tography-measured CyA levels appeared unatfectedKl~ Further studies are needed to elucidate the potential effect of steroids on FK 506 metabolism. Biologic assays have previously been used for the mea-surement of CyA in patient's plasma based on the inhibi-tion by the plasma of a third-party MLR. No attempt was made. however. to express the inhibitory activity as a concentration of biologically active CyA. Both Kahan l4 and Reisman et al lo suggested that biologic assay has clinical relevance: patients with continually low plasma immunosuppressive activity are prone to rejection of their kidney allografts. Our preliminary observations also indi-cated that the absence of immunosuppressive activity in patient's plasma was associated with liver allograft rejec-tion (patient P321. Recently. Jain et al 17 have shown that moderate to severe hepatic dysfunction induced an increase in trough levels of FK 506 measured by ELISA. Also. the Cy A RIA levels were higher than the specific measurements of Cy A during rejection episodes following liver transplantation. 1M Studies are currently in progress to analyze the relation-ship between ELISA and bioassay levels during acute allograft rejection. In conclusion, a bioassay based on the inhibition of an alloreactive T cell clone proliferation was used to deter-mine FK 506 levels in patient's plasma. A positive corre-lation was demonstrated between drug levels measured by the bioassay and ELISA. However, the FK 506 levels were lower for the bioassav than for ELISA. Since FK 506 FK 506 BIOASSAY IN TRANSPLANT PATIENTS cannot yet be assayed accurately in plasma using specific chromatographic techniques. the bioassay is important for assisting in the interpretation of an immunoassay (ELISA) with unknown specificity for biologically non-active forms of FK 506. Furthennore. biologic assay may be useful for patient management if active metabolites are induced. or to detect the interaction between FK 506 and other drugs (high-dose steroids) in the immunosuppression of allograft rejection. REFERENCES I. Starzl TE: Transplant Proc 20:356. 1988 (suppl 3) 2. Starzi TE. Todo S. Fung J. et al: Lancet 2: 1000. 1989 3. Ochiai T. Nakajima K. Nagata M. et al: Transplant Proc 19: 1284. 1987 4. Zeevi A. Duquesnoy RJ. Eiras G. et al: Transplant Proc 19:40. 1987 (suppl 6) 5. Sawada S. Suzuki G. Kawase Y. et al: J Immunol138: 1797. 1897 6. Todo S. Ueda Y. Demetns JA. el al: Surgery 104:239. 1988 63 7. Todo S. Demetris JA. Ueda Y. et al: Surgery 106:444. 1989 8. Fung J. Todo S. Demetris JA. et al: Transplant Proc (this issue) 9. Tamura K. Kobayaski M. Hashimoto K. el al: Transplant Proc 19:23. 1987 (suppl 6) 10. Cadotf EM. Venkataramanan R. Krajack A. et al: Trans-plant Proc (this issue) II. Tanaka H. Kuroda A. Marusawa H. et al: J Am Chem Soc 109:5031. 1987 12. Venkataramanan R. Jain A. Cadotf E. et al: Transplant Proc (this issue) 13. Zeevi A. Duquesnoy RJ: J Immunogenet 12:17. 1985 14. Kahan DB: Transplantation 40:457. 1985 15. Ptachcinski RJ. Venkataramanan R. Burckart GJ. et al: Transplant Proc 19: 1728. 1987 16. Reisman L. Lieberman KV. Martinelli G. et a1: Am J Kidney Dis 12: 104. 1988 17. Jain AB. Venkataramanan R. Cadoff E. el al: Transplant Proc (this issue) 18. Burckart GJ. Ptachcinski RJ. Venkataramanan R. et al: Transplant Proc 18: 188. 1986 (suppl 5) 

Bioassay of plasma specimens from liver transplant patients on FK 506 immunosuppression

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Bioassay of plasma specimens from liver transplant patients on FK 506 immunosuppression

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