Chao, J

McKaveney, T

Starzl, T

Todo, S

Venkataramanan, R

Warty, V

Zendehrouh, P

PubMed

Name not available

Distribution of FK 506 in plasma lipoproteins in transplant patients

D-Scholarship@Pitt

Distribution of FK 506 in Plasma Lipoproteins in Transplant Patients V. Warty, R. Venkataramanan, P. Zendehrouh, T. McKaveney, J. Chao, S. Todo, and T. Starzl FK 506, a powerful. new immunosuppressive agent, is currently undergoing clinical trials in transplant pa-tients. Like cyclosporine (CyA). FK 506 suppresses inter-leukin-2 production and receptor expression on T cells. 1.2 Additionally, both FK 506 and CyA are highly lipophilic. 3 CyA is mainly transported by lipoproteins in plasma.4 The objective of the present study is to characterize the distri-bution of FK 506 in various plasma components. MATERIALS AND METHODS Materials FK 506 powder drug. FK 5{)6..peroxidase conjugate (FK-PODl. and anti-FK 506 monoclonal antibody was a gift from Fujisawa Pharmaceutical Co .. Ltd. Osaka. Japan. Goat anti-mouse poly-clonal antibody was obtained from Atlantic Antibodies (Stillwa-ter. MN). All necessary reagents were prepared with analytical grade chemicals. Sample extractions were performed using the Sep-Pak Cartridge Column (Waters, Milford, MA) method with an ALL TECH Manifold Assembly. Extracted samples were plated on a Linbro EIA-II Plus 96-well microtiter plate and absorbance readings were made using a Titertek Multiskan Plus microtiter plate reader (Flow Laboratories, McLean, V A) at 492 nm. Ultra-centrifugations were performed in a Beckman LS-SO ultracentri-fuge using a Ti80 rotor. Methods The distribution of FK 506 in plasma lipoproteins in vivo was studied in 13 transplant patients receiving an oral dose of FK 506 twice a day. Blood specimens, representing trough levels in these individuals, were warmed to 37°C for I hour, the erythrocytes were then separated from the plasma by centrifugation for 15 minutes at 1500 g and 37"C. The plasma samples from each patient were pooled and the lipoproteins were separated by ultracentrif-ugation using a sequential density gradient. Plasma very 101.1.'-density lipoprotein ([VLDLJ 1.006-1.019 glmL), low-density lipo-protein ([LDL) 1.019-J.()63 glmL), and high-density lipoprotein «HDLJ 1.063-1.21 glmLl were separated by standard differential ultracentrifugation techniques using appropriate density gradi-entsK~ The remaining fraction was termed lipoprotein deficient serum (LPDSl. The distribution of FK 506 among each fraction was determined using a two-step enzyme-linked immunoabsorp-tion assay (ELISA) that utilizes a double antibody reaction.6 A standard curve was produced by the addition of FK 506, in methanol. to blood bank plasma to give final concentrations of 0, 0.1.0.5. 1.0. 2.0, 5.0. and 10.0 ngimL. The standards and patient samples were extracted using Sep-Pak columns. The bound FK 506 was eluted with methanol and then evaporated to dryness under nitrogen. The dried samples were then analyzed by the method described by Tamura et al.6 Plasma lipoprotein choles-terol in each of the patient lipoprotein fractions was measured by the Cobas Bio Chemical Analyzer (Roche. Nutley, NJ). RESULTS The distribution of FK 506 (Table I) in plasma VLDL was II :!: 3%; LDL. 3 :!: 3%; HDL. 21 :!: 8%; and the remaining in lipoprotein deficient (LPDS) fraction. 64 :!: 8%. The plasma lipoprotein fractions were also analyzed for the relative percent distribution of cholesterol (Table 2). VLDL contained 12 :!: 7%; LDL, 57 :!: 8%; HDL, 27 :!: 10%; and LPDS. 4 :!: 2%. The FK 506 plasma concentra-From the Departments of Pathology p./.W., P.Z., T.M., J.e.), Pharmacy (R.V.), and Surgery (S.T .• T.S.), University of Pitts-burgh, Pittsburgh, Pennsytvania. Address reprint requests to V, Warty, Department of Pathology, University of Pittsburgh, Pittsburgh. PA 15213. r&. 1991 by Appleton & Lange 0041-1345191'$3.00, + 0 Table 1. Distribution of FK 506 In Plasma Upoprotelns In Transplant Patients FK 506 (nglml) Percentage of Distribution of Plasma Concentration Patien1 No. Plasma VlOl LDl HDl LPDS 1 12.8 9 7 21 63 2 19.0 12 9 19 60 3 1.1 10 7 26 57 4 2.1 9 3 29 59 5 1.4 9 5 30 56 6 7.9 15 5 20 60 7 0.4 8 NO 26 66 8 1.8 9 NO 28 61 9 0.5 11 NO 15 72 10 0.3 8 NO 20 72 11 1.0 16 NO 21 62 12 0.7 11 NO NO 84 13 1.9 11 5 19 65 Mean = SO 11 = 3 3=3 21 = 8 64 = 8 Abbreviation: NO. nondeIedIbIe. 954 Transplantation Proceedings, Vol 23, No 1 (February), 1991: pp 954-955 lipo.. olein ~nliag :radi-cien! :tion '011>-I." A 1. in )fO, ient FK less the les-I by ·/as Ing he he 2). :t a-.), s-y, FK 506 IN PLASMA LIPOPROTEINS 955 Table 2. Distribution of Cholesterol In Plasma Lipoproteins In Transplant Patients --Cholesterol (mgllt.) PII*1I No Plasma VLDl -103 13 1 2 127 18 3 117 11 4 128 7 5 99 11 6 107 10 7 122 8 8 150 14 9 112 10 10 133 5 11 162 28 12 169 22 13 76 2 Mean ~ SO 12 ~ 7 tion ranged from 0.25 to 12.8 ngimL. However, there was no systemic change in the percent distribution of FK 506 into various plasma components indicating linear uptake over this concentration range. DISCUSSION Using conventional differential ultracentrifugation meth-ods with density gradients. we separated the plasma lipo-proteins from the pooled serum of 13 transplant patients. The plasma lipoprotein fractions were then analyzed for the average percent distribution of both FK 506 and cholesterol. The distribution of FK 506 was highest in the LPDS fraction of the plasma (Fig I). In contrast. the C Q j ·c ~ is 1 t~ __________________________ --. 60 40 20 o ~ CyA • Cbolesterol o FKS06 Fig 1. Average percentage of distribution of FK 506, CyA, and cholesterol. Percentage of 0istrb..4I0n of Plasma Concentration LDL HDL LPOS 62 . 22 3 67 13 2 51 34 4 58 32 3 62 24 3 73 14 3 44 45 3 55 29 2 50 35 5 51 41 3 48 22 2 52 18 B 63 30 5 57 ~ B 27 ~ 10 4~O average percent dist:-;bution of cholesterol was found to be lowest in this fracti..-n (Fig I). This finding is particularly interesting in view o-f the belief that FK 506 is similar to CyA in its lipophilic ;:>roperties as well as its mechanism of immunosuppressive activity. The majority of plasma CyA is associated with LDL and HDL where the average percent distribution of cholesterol is highest (Fig 1).4 These observations show that FK 506 and CyA have significantly differen: distributions in plasma; additionally. this suggests that the mechanism of transport in plasma may be different for the two compounds. We propose that plasma FK 506 is rn..llnly associated with plasma proteins, such as albumin and or alpha-I acid glycoprotein (AAG). These results indJo.;ate that changes in lipoprotein con-centrations in transplant patients are not likely to alter FK 506 distribution in plasma. while those of albumin and AAG are more likel~ to change the extent of binding of FK 506 to plasma protems. ACKNOWLEDGMEf'.. TS This project was funded in part by the Pathology Education and Research Foundation. REFERENCES I. Zeevi A. Eiras G. Burckart G. et al: Transplant Proc 22:60. 1990 (suppl t) 2. Bennett WM. ~Cfrnlan OJ: Ann Rev Med 37:215. 1986 3. Venkataramanan R. Jain AB. Cadolf E. et at: Transplant Proc 22:52. 1990 Esu~l I) 4. Gureki J. Wart! V. Sanghvi A: Transplant Proc /7:1997. 1985 5. Havel RJ. Eder H ..... Bragdon JH: J Clin Invest 34: 1345. 1955 6. Tamura K. Kobayashi M. Hashimoto K. et al: Transplant Proc 19:23. 1987 (suppl 61 

Distribution of FK 506 in plasma lipoproteins in transplant patients.

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Distribution of FK 506 in plasma lipoproteins in transplant patients.

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