Distribution and concentration of cyclosporin in human blood

Transcription

1 J Clin Pathol 1984;37: Distribtion and onentration of ylosporin in hman blood KERRY ATKINSON, KATHRYN BRITTON, JAMES BIGGS From the Department ofhaematology, St Vinent's Hospital, Sydney, New Soth Wales 21, Astralia SUMMARY In patients reeiving ylosporin to minimise graft verss host disease after allogenei bone marrow transplantation, whole blood ylosporin onentration was roghly twie the serm onentration when blood was separated at 37 C. In trn, blood separation at 37 C reslted in a dobling of serm ylosporin onentration ompared with separation at room. In vitro stdies showed that the latter phenomenon was de to a dependent partitioning of ylosporin between plasma and red ells, sh that inreased ylosporin was taken p from the serm into red ells at room. Inreasing delay in separation of patient blood (at either ) reslted in a gradally inreasing ylosporin serm onentration. Frther in vitro stdies showed that a distribtion eqilibrim between blood omponents was reahed within 3 min inbation. Red ell ptake of ylosporin was satrable at an inbation onentration of >4,g/ml, while plasma and mononlear ells showed a linear ptake to 7 g/ml. The elllar ylosporin ontent of a mononlear ell was roghly 1 times greater than that of an erythroyte. For linial monitoring we reommend the measrement of ylosporin onentration either in whole blood or in serm separated at 37 C withot delay after venepntre. Cylosporin, with its niqe ability to inhibit T ell ativity,' 2 has beome an important drg in hman organ transplantation. It is effetive in reding the inidene of solid organ graft rejetion34 as well as bone marrow graft rejetion.5 It is as effetive as methotrexate in minimising ate graft verss host disease after marrow transplantation.6 Unfortnately, it is assoiated with nephrotoxiity,'-9 hepatotoxiity,' " and entral nervos system toxiity.'2 Despite mh effort aimed at measring drg onentrations in serm and whole blood, relatively little information is available on either toxi or therapeti onentrations. We report on the onentration and distribtion of the drg in whole blood and blood omponents in man and on a nmber of fators that inflene the onentration of ylosporin in these omponents. Material and methods PATIENTS STUDIED All patients were reeiving ylosporin immnosppression to minimise graft verss host disease after allogenei bone marrow transplantation as treat- Aepted for pbliation 28 Jne 1984 ment for severe aplasti anaemia or haematologial malignany. The protool for marrow transplantation has been desribed previosly.'3 Blood samples were olleted from patients reeiving ylosporin immediately before a reglar 12 horly dose of ylosporin (trogh onentration). For measrement of ylosporin serm onentration blood was allowed to lot at either room or 37 C. The lotted blood was entrifged at 1 g for 1 min at the respetive s. All serm samples were stored at - 2 C before radioimmnoassay.'4 In or laboratory the interassay variation was 8-4% and the intra-assay variation 5.6%." To evalate the effet of the at whih the blood was separated on sbseqent ylosporin serm onentration, aliqots of lotted blood were allowed to stand for 1 h at room or 37 C before separation. To investigate the effet of delayed blood separation, aliqots of lotted blood were allowed to stand at room or 37 C for 1, 4, 7, or 22 h intervals before separation. For measrement of whole blood ylosporin onentration, EDTA blood was olleted and stored frozen at - 2 C ntil assayed. On the day of assay the sample was thawed, broght to room, and mixed thoroghly. No intat 1167

2 1168 red ells were detetable by light mirosopy after this proedre. Cylosporin onentration was then measred by radioimmnoassay.'4 Again, this was standardised sh that a ylosporin onentration as low as 3 ng/ml old be measred. The interassay variation was 8-9% and the intra-assay variation 5*8%. IN VITRO STUDIES Cylosporin pre sbstane was dissolved in ethanol and Tween 8 and dilted with -9% sodit.. hloride soltion to give onentrations in the range -625,g/ml to 8,g/ml. Fresh blood (antioaglated with EDTA) from normal individals was sed. The desired amont of ylosporin was added to whole blood at 37 C and inbated with gentle mixing for 1 h. Isolation of plasma, the mononlear ell fration, and the red ell/granloyte fration was performed on a Fioll gradient. Two millilitres of inbated blood was dilted 1/3 with phosphate bffered saline (PBS) and the dilted blood layered areflly on to 4 ml Fioll-Paqe (Pharmaia, Sydney); the samples were then entrifged at 4 g for 35 min. The top layer of dilted plasma was drawn off and retained for ylosporin assay. The mononlear ell layer was then removed, leaving the red ell pellet. Both the mononlear layer and the red ell pellet were resspended separately in PBS and entrifged at 1 g for 1 min. After two frther washes, the ells were lysed to reover the ylosporin. The mononlear ells were resspended in 5:5 ethanol-pbs and lysed by rapid freezethawing in liqid nitrogen and hot water. After entrifgation, the spernatant was retained for ylosporin assay. The red ells were resspended in 5:5 distilled water-ethanol and vortexed. The lysed ells were entrifged and the spernatant was retained for ylosporin assay. To evalate the effet of the at whih the blood was separated, dilted blood was separated on Fioll at either room or 37 C. To evalate the effet of an inrease in ylosporin onentration dring inbation, inreasing onentrations were added to the inbation tbe. Con- Table 1 Effet of ofblood separation on serm ylosporin onentration Temperatre at whih blood was separated 37 C Room Trogh serm ylosporin onentration (ng/ml) (n = 12) 283 ± Vales represent mean 1 SD. Differene is signifiant (p <.1). Atkinson, Britton, Biggs Table 2 Effet on serm ylosporin onentration of reheating blood to 37 C after initial ooling to room Blood reheated to Blood separated at 37'C after ooling to 37'C withot ooling room * Trogh serm ylosporin onentration (ng/ml) (n = 26) *Vales represent mean + 1SD. Differene is not signifiant. entrations sed were -62, -125, -25, -5, 1, 2, 3, 4, 6, 7, and 8,g ylosporin/ml blood. To evalate the effets of the dration of inbation, the inbation period was varied: blood was inbated with ylosporin for 3, 6, 9, or 12 min before separation on Fioll. STATISTICAL ANALYSIS This was performed sing the two tailed Stdent's t test. Reslts EFFECT OF TEMPERATURE AT WHICH BLOOD WAS SEPARATED Trogh serm ylosporin onentration was signifiantly inreased when blood from patients reeiving ylosporin was separated at 37 C ompared with separation at room (Table 1). When patient blood was reheated to 37 C before serm separation, after it had initially been allowed to ool to room, the ylosporin serm onentration was the same as that obtained in parallel samples that were separated at 37 C withot ooling (Table 2). These findings were frther investigated sing blood from normal sbjets inbated with ylosporin in vitro (Fig. 1). The proportion of ylosporin in plasma was lower, and the proportion of ylosporin in red ells higher, at room ompared with 37 C. Ths, partitioning of ylosporin between plasma and red ells was dependent, and it appeared that some ylosporin was taken p from the serm into red ells at room. EFFECT OF DELAYING BLOOD SEPARATION With inreasing delay in separation of patient blood after venesetion there was a gradal inrease in the ylosporin serm onentration, regardless of whether the blood was separated at room or at 37 C (Fig. 2).

3 Distribtion and onentration of ylosporin in hman blood C) in 3 o _z = Plasm a= Red blood elli Mononlear ell 1I 1 - I Lem Room 37'C Fig. 1 Effet of at whih blood was separated on distribtion ofylosporin in blood omponents. Blood from normal sbjets inbated with ylosporin in vitro was sed (n = 11). Vales represent mean + ISD. The differene between the two s was signifiant for eah omponent (p <.1). SATURABILITY OF BLOOD COMPONENTS WITH CYCLOSPORIN The satrability of the three blood omponents (plasma, red ells, and mononlear ells) is shown in Fig. 3. Red blood ells showed an apparent linear ptake p to an inbation onentration of 4,tg/ ml, after whih their ptake appeared to be satr- E -S 3 a2.o 21 a. 1* I.) ;: 1 Room..~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1 7 Dration of delay (h) Fig. 2 Effet on serm ylosporin onentration ofdelay in separation ofblood (at either room or 37 C). Blood from patients reeiving ylosporin was sed (n = 5). Vales represent mean + ISD. Differenes between 1 h and 7 h vales and I h and 22 h vales were signifiant (p < 5 for room vales and p < 1 for 37 C vales). 22 -o D 7-6- a. C.- 5- d) ml- 4. o > 3-2- a 1. L. _ Red blood ell Mononlear ell --4I Inbation onentration of ylosporin (,g/ml blood) Fig. 3 Effet on onentration of ylosporin in blood omponents of inreasing onentrations of ylosporin dring inbation. Blood from normal sbjets inbated with ylosporin in vitro was sed (n = 4). Vales represent mean + ISD. able. Both the plasma and the mononlear ell omponents showed an apparent linear ptake p to 7,g/ml. Plasma ylosporin onentration inreased more rapidly than red ell ylosporin onentration with inreasing onentrations of ylosporin dring inbation. 37 2* 1 t) (DC.) ) C C 1 U) ni.1 Mononlear ell Inbation onentration of ylosporin (,g/ml blood) Red blood ell Fig. 4 Cylosporin ontent of mononlear ells and erythroytes at inreasing inbation onentrations of ylosporin. Blood from normal sbjets inbated with ylosporin in vitro was sed (n 4). Vales = represent mean + lsd.

4 _C~ 4 to o i i I Plasma Red blood ell Mononlear ell Dration of inbation (min) Fig. 5 Effet on ylosporin ontent of blood omponents of inreasing dration of inbation ofblood with ylosporin. Blood from normal sbjets was sed (n = 3). Vales represent mean ± ISD. Differenes for different drations ofinbation not signifiant. CELLULAR CYCLOSPORIN CONTENT Mononlear ells had a ylosporin ontent roghly 1 times greater than that of erythroytes at all onentrations of ylosporin (Fig. 4). EFFECT OF DURATION OF INCUBATION There was no hange in the distribtion of ylosporin between blood omponents when ylosporin and blood were inbated for inreasing periods of time (3-12 min) (Fig. 5). A distribtion eqilibrim had ths been reahed within 3 min of inbation. WHOLE BLOOD CYCLOSPORIN CONCENTRATION Estimation of ylosporin onentration in whole Table 3 Atkinson, Britton, Biggs blood and in the orresponding serm separated at 37 C was performed in 2 marrow transplant reipients. Mltiple samples, most of whih were drawn on onsetive days from the day of transplant, were analysed from eah patient (Table 3). The proportion of ylosporin deteted in serm ompared with that deteted in whole blood ranged from 44% to 72% (mean ± 1 SD, %). Disssion Cylosporin ontent ofserm separated at 37C and of whole blood Althogh ylosporin is an effetive immnosppressive agent in hman organ transplantation, it also has onsiderable toxiity. In marrow transplant reipients there have been few stdies showing a orrelation between trogh serm ylosporin onentrations and toxiity. Barrett'6 showed a orrelation between trogh serm ylosporin onentration and blood rea onentration, and Hows8 showed a orrelation between trogh serm ylosporin onentration and serm reatinine vales. In the latter stdy, however, patients with other ases of nephrotoxiity or potential nephrotoxiity (sh as aminoglyoside antibioti treatment) were inlded in the analysis and make interpretation of the data diffilt. No stdies have yet been reported - orrelating serm ylosporin vales and effiay in preventing graft verss host disease. Diffilties in the tehniqes for measring ylosporin onentration in hman serm or blood may be a reason for the lak of sh stdies. Lemaire'7 and Hows'8 have previosly shown that the onentration of ylosporin in hman serm is inreased roghly twofold if the blood is separated at 37 C rather than at room before analysis. We have onfirmed Patient no No ofsamples Serm ylosporin onentration Whole blood ylosporin Serm: whole blood ylosporin (nglml) onentration (nglml) onentraton ratio ± ± ± ± ± ± ± ± 12 * 369 ± ± ± 172 * ± ± ± ± ± 135 * ± ± ± ± ± ± ± * Mean 33 ± ± ± 9 Vales represent mean 1SD.

5 Distribtion and onentration of ylosporin in hman blood this in or stdy and have shown that this is de to ptake of ylosporin by red ells at room. We have also shown that inreasing time before separation of the blood reslts in an inreased serm ylosporin onentration, probably de to release of ylosporin from haemolysed red ells dring the delay.'9 In ontrast to previos reported pratie,'9 however, we wold reommend either that hman blood be separated at 37 C withot delay after venepntre (rather than storing it for 2-3 h at room ) before separation and measrement of the serm ylosporin onentration, or that whole blood ylosporin onentration be measred. Separation at 37 C withot delay minimises the ptake of ylosporin by red ells, ths allowing a loser approximation to in vivo serm vales. Sh a poliy wold at least lessen onfsion ased by two fators-the dependene of serm ylosporin onentration and the inreased ylosporin onentration de to delay in separation. If sh pratie is not possible, similar reslts an be obtained by rewarming the whole blood sample to 37 C before separation. One separated, the serm an be stored at - 2 C with impnity for several months ntil the time of assay.'9 The measrement of whole blood ylosporin onentrations wold avoid all these diffilties, bt, again, no orrelation between whole blood onentrations, toxiity, and effiay is available. In vitro stdies showed that the distribtion of ylosporin between the three major omponents of the blood (plasma, erythroytes, and mononlear ells) depends on the at whih the blood was separated, the onentration of ylosporin present dring the inbation, bt not on the dration of inbation. Of the three omponents, only the erythroytes appeared satrable in the onentration range tested and only then at a onentration higher than wold normally or in vivo. A mononlear ell ontained mh more ylosporin than an erythroyte. Work is in progress to determine the ylosporin ontent of the T ell, T ell sbsets, and non-t ell omponents of mononlear ells in order to determine if any of these parameters are sefl for linial monitoring. This work was spported by grants from the National Health and Medial Researh Conil of Astralia and the New Soth Wales State Caner Conil. We thank Deirdre Nix for typing the mansript and the Adiovisal Department of St Vinent's Hospital for help in the preparation of the illstrations. Referenes 'Andrs L, Lafferty KJ. Inhibition of T-ell ativity by ylosporin A. Sand J Immnol 1982;15: Bnjes D, Hardt C, Rollinghof M, Wagner H. Cylosporin A mediates immnosppression of primary ytotoxi T responses by impairing the release of Interlekin I and interlekin II. Er JImmnol 1981;11: Cylosporin A as sole immnosppressive agent in reipients reeiving allografts from adaver donors. Preliminary reslts of a Eropean mltientre trial. Lanet 1982;ii: Reitz BA, Wallwork JL, Hnt SA, et al. Heart-lng transplantation; sessfl therapy for patients with plmonary vaslar disease. N Engl J Med 1982;36: 'Hows JM, Palmer S, Gordon-Smith EC. Use of vlosporin A in allogenei bone marrow transplantation for severe aplasti anaemia. Transplantation 1982;33: h Deeg HJ, Storb R, Thomas ED, et al. Marrow transplantation for ate non lymphoblasti lekaemia in first remission. Preliminary reslts of a randomised trial omparing ylosporine and methotrexate for prophylaxis of graft-verss-host disease. Transplant Pro 1983;15: Atkinson K, Biggs JC, Hayes J, et al. Cylosporin A-assoiated nephrotoxiity in the first I () days after allogenei bone mairow transplantation; 3 distint syndromes. Br J Haematol 1983;54: Hows JM, Chipping PM, Fairhead S, Smith J, Baghan A, Gordon-Smith EC. Nephrotoxiity in bone marrow transplant reipients treated with ylosporin A. Br J Haematol 1983; 54: Shlman H, Striker G, Deeg HJ, Kennedy M, Storb R, Thomas ED. Nephrotoxiity of ylosporin A after allogenei marrow transplantation. Glomerlar thromboses and tblar injries. N Engl J Med 1981;35: ' Atkinson K, Biggs J, Dodds A, Conannon A. Cylosporineassoiated hepatotoxiity after allogenei marrow transplantation in man; differentiation from other ases of post transplant liver disease. Transplant Pro 1983; 15: Klintmalm GBG, Iwatski S, Starzl TE. Cylosporin A hepatotoxiity in 66 renal allograft reipients. Transplantation 1981;32: Atkinson K, Biggs J, Darveniza P, Boland J, Conannon A, Dodds A. Cylosporine-assoiated entral nervos system toxiity after allogenei bone marrow transplantation. Transplantation (in press). 3 Biggs J, Atkinson K, Conannon A, et al. Bone marrow transplantation in 33 patients with malignant blood diseases and severe aplasti anaemia. Med J Ast 1983;2:12-5. Donatsh P, Abish E, Homberger M, Traber R, Trapp M, Voges R. A radioimmnoassay to measre ylosporin A in plasma serm samples. J Immnoassay 1981; 2: Atkinson K, Biggs JC, Britton K, et al. Oral administration of ylosporin A for reipients of allogenei marrow transplants; impliations of linial gt dysfntion. Br J Haematol 1984;56: Barrett AJ, Kendra JR, Las CF, et al. Cylosporin A as prophylaxis against graft-verss-host disease in 36 patients. Br Wed J 1982;285: '' Lemaire M, Niederberger W, Nssbamer K. Fators inflening the distribtion between blood ells and plasma. Conseqene for the monitoring of plasma onentrations. Sandoz doment 1982; Hows JM, Smith JM. In vitro stability of ylosporin A. J Clin Pathol 1983;36:72-1. Smith JM, Hows JM, Gordon-Smith EC. Stability of ylosporin A in hman serm. J Clin Pathol 1983;36:41-3. Reqests for reprints to: Dr K Atkinson, Department of Haematology, St Vinent's Hospital, Sydney, New Soth Wales 21, Astralia.