EurJ Clin Pharmacol (1991) 41:273-283

PBGrQcOIog O Springer-Verlag 1991

Therapeutic monitoring of cyclosporin- an update* ** A. Lindholm Departments of Clinical Pharmacology and Transplantation Surgery,Karolinska Institute at Huddinge University Hospital, Huddinge, Sweden Received: December 3, 1990 / Accepted: November 22, 1990

Summary. The success of organ transplantation is closely related to clinical use of the immunosuppressive drug cyclosporin (CsA). The dosage of CsA is complicated by the large intra- and interindividual variability in its pharmacokinetics, as well as by the narrow concentration range between insufficient immunosuppression and toxicity. Potential sources of error in the sampling procedure and the advantages and disadvantages of the available analytical methods are discussed. Traditionally, 12 or 24 hour trough concentrations of CsA are monitored. Recently, peak concentrations or estimation of AUCs by a limited sampling strategy have been tried to improve the relatively weak concentration-effect and concentrationtoxicity relationships found with trough CsA concentration monitoring. Studies of the CsA concentration-effect relationships for various treatment indications are reviewed. Key words: Cyclosporin A; therapeutic monitoring, assay techniques, pharmacokinetics, dose-response relationships

Knowledge of the pharmacokinetics of cyclosporin (CsA) is essential for correct interpretation of the results of its therapeutic monitoring. This review focuses on certain factors of importance when monitoring CsA, namely the sampling procedure and the choice of analytical method. The optimal target concentrations for the various clinical indications are also considered. CsA is a highly lipophilic, neutral, cyclic polypeptide with a molecular weight of 1202 Da. It is derived from the * This review is based on parts of a PhD thesis presented by the author in April 1990. Reprints of the thesis may be requested from the author ** Metabolites 1, 17, 18, 21 and 26 (old nomenclature, used in text) correspond to M9, M1, Mlc, M4N, Mlc9 according to the new nomenclature proposed by Wenger (1990)

fungi Cylindrocarpon lucidum Booth and Tolypocladium inflatum Gams that grew in soil samples obtained from Hardanger Vidda, Norway, in 1970. CsA was purified from the fungal extracts in 1973 and it was structurally identified in 1975 [Borel 1982]. The principal effect of CsA is to block the production of interleukin-2 (IL-2) and other lymphokines by T-cells, while leaving the expression of IL-2 receptors unaffected [Larsson 1980; Bunjes et al. 1981; Palacios 1982]. The inhibition of IL-2 synthesis by CsA is the result of an action at the nuclear level, where the transcription of m R N A for IL-2 is prevented [Elliot et al. 1984; Kronke et al. 1984]. Recent data suggest that cyclophilin is the major intracellular receptor protein for CsA [Handschumacher et al. 1984; Foxwell et al. 1988]. It is identical to peptidyl-prolyl isomerase, an enzyme that catalyses the cis-trans isomerization of proline residues in proteins and peptides [Takahashi et al. 1989]. After the introduction of CsA in clinical transplantation in 1978 [Calne et al. 1978], results in all forms of organ transplantation have improved. However, several problems remain in the use of CsA. The drug has many adverse effects [Cockburn et al. 1988]. The most distressing is nephrotoxicity, which poses a special problem in renal transplantation, where it may be difficult to distinguish between graft rejection and CsA nephrotoxicity [Klintmalm et al. 1985]. Most studies on the correlation between the dose/concentration and effects of CsA have shown that there is a relationship between CsA concentration and its therapeutic effect [Shaw et al. 1987]. Considerable intra- and interindividual variability in the pharmacokinetics of CsA has been observed [Ptachcinski et al. 1986 b; Lindholm et al. 1988 a], so monitoring of CsA concentrations has been recommended [Keown et al. 1981; Shaw et al. 1987]. However, the use of several specific and non-specific assay methods for CsA and its metabolites, and the use of whole blood, plasma or serum separated at various temperatures, have complicated the evaluation of therapeutic monitoring of CsA and the search for an optimal therapeutic window.

274

Pharmacokinetics The pharmacokinetics of CsA has been extensively reviewed [Ptachcinski et al. 1986b; McMillan 1989] and is briefly described below. The absorption of CsA after oral administration is slow, incomplete and highly variable [Lindberg et al. 1986]. Marked interindividual variation in bioavailability has been observed, with a range of 2 % to 89 % (mean 30 % ) reported in recipients of various transplants [Ptachcinski eta. 1985; Burckart et al. 1986; Bertault-Pdras et al. 1985; Frey et al. 1988]. Peak blood concentrations are usually reached between 1 and 8 h after oral administration, but they may occur even later. Double peaks in the blood CsA concentration versus time curves are frequently encountered [Kahan et al. 1983; Phillips et al. 1988; Lindholm et al. 1988 a]. Since CsA is highly lipophilic, it is distributed throughout the body. The volume of distribution is reported to vary from 1.0 1. kg 1 in paediatric patients with congestive heart failure [Burckart et al. 1987] to 11.0 1-kg 1in uraemic patients [Lindberg et al. 1986]. In renal transplant recipients the mean volume of distribution was 2.9 to 4.5 1. kg- 1 [Morse et al. 1988, Ptachcinski et al. 1985]. In blood, 58 % of circulating CsA is bound to red blood cells, 4 % to granulocytes, 5 % to lymphocytes and 33 % is in plasma [Lemaire & Tillement 1982]. In plasma, 85 % to 98 % is bound to lipoproteins, 5 % to 15 % to other proteins and about 1% to 2 % of CsA is free [Lemaire & Tillement 1982; Mraz et al. 1986; Lindholm et al. 1989]. CsA is extensively metabolized (more than 99%) in the liver by several cytochrome P-450 isoenzymes and about 95 % is excreted in the bile [Venkataramanan et al. 1985]. The clearance is reported to range almost 40-fold from 0.63 ml. min 1. kg 1 t o 23.9 ml. min- 1. kg-i, with means of 5.7 ml. min- 1. kg- 1 (adult renal transplant patients; Ptachcinski et al. 1985) to 10.3ml-min-l.kg 1 (bone marrow transplant recipients; 'fee et al. 1988 a). The terminal half-life is reported to range between 4.3 and 53.4 h in renal transplant recipients (mean 10.7 h; Ptachcinski et al. 1985). Several factors may affect the pharmacokinetics of CsA [Ptachcinski et al. 1986b; Rodighiero 1989; Venkataramanan et al. 1989]. For example, the absorption of CsA is less after abdominal surgery (and early post-transplantation), if bile flow and liver function are impaired, or if the patient suffers from diarrhoea. The distribution of the drug in blood depends on the haematocrit and blood lipoprotein concentrations. The elimination is genetically determined and varies greatly between individuals. Elimination is faster in children than in adults and concomitant medication may block or induce the cytochrome P-450 metabolizing enzymes responsible for the metabolism of CsA [McMillan 1989]. The immunosuppressive potency of the major metabolites, especially that of metabolite 17, is debated. However, at a recent expert meeting there was general agreement that metabolites probably provide less than 20 % of the total immunosuppressive effect of CsA therapy [Holt 1990]. The most potent metabolite, number 17, is esti-

A. Lindholm: Therapeutic monitoring of cyclosporin mated to have 5 to 10 % of the immunosuppressive activity of CsA [Rosano et al. 1990, Copeland et al. 1990].

Sampling By using a correct sampling procedure severalpotential errors in the measurement of CsA concentrations may be avoided. CsA sampling usually relies on peripheral venesection, which is the only safe sampling procedure. However, for practical reasons, sampling is also frequently performed via indwelling lines and by capillary puncture, procedures which carry potential sources of error in CsA monitoring. Due to its high lipophilicity, CsA may adsorb onto plastics [Ptachcinski et al. 1986c] and an indwelling line will remain contaminated at least for two weeks after the has been administered through it [Blifeld and Ettenger 1987]. We recently found that topical skin contamination from finger sucking in children may give falsely high CsA concentrations in capillary samples [Lindholm et al. 1990 b]. Double-lumen central lines and capillary sampling from the ear to toe may be ways to eliminate these errors. Anticoagulation with ethylenediaminetetraacetic acid (EDTA) is preferred because with heparin the blood specimen may clot after refrigeration and freezing [Agarwal et al. 1985; van den Berg et al. 1985]. CsA is stable and may be stored at room temperature, dry or in solution, and it is not degraded by light [Ptachcinski et al. 1986 a]. Whole blood containing CsA may be sent by ordinary mail and stored at + 4 °C for more than a week without any loss of CsA. Blood samples containing CsA may also be dried on filter paper for easy transport and subsequent analysis [Lampe et al. 1987; Foradori et al. 1987]. In our experience, no CsA was lost from whole blood pool samples stored at -20°C for more than 6 months. The distribution of CsA between blood cells and plasma is highly temperature-dependent, which is important when separating plasma for CsA analysis. With a temperature decrease from + 37°C to + 20°C about 50 % of CsA partitions from plasma into erythrocytes [Niederberget et al. 1983; van den Berg et al. 1985]. Equilibration at room temperature occurs within 2 h, whereas at 37 °C it requires only 30 min [van den Berg et al. 1985]. Humbert et al. (1990) showed that plasma separation of + 36 °C gave a 15 % lower concentration of CsA than at + 37 °C, which further emphasizes the crucial nature of separation in plasma analysis of CsA. Because of the temperature-dependent distribution of CsA between whole blood and plasma, whole blood has been recommended as the sample matrix for analysis of CsA [Shaw et al. 1987]. Moreover, plasma concentrations are often below the limit of determination of the common analytical methods, in spite of clinically adequate immunosuppression [Klintmalm et al. 1985; Lindholm et al. 1990@ Plasma (or serum) was previously used as the sample matrix by most laboratories, but the number using plasma has rapidly decreased. From July 1987 to August 1988, the number of laboratories in Europe using plasma for CsA analysis decreased from 23 % to 16 % [Johnston et al. 1989], and by September 1989 it was only 10 %.

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Analytical methods CsA may be analyzed by methods specific or non-specific for the parent drug versus metabolites. As the metabolites so far isolated show only minor immunosuppressive potency, analysis by a specific method does seem preferable. Also, non-specific measurements of CsA concentrations do not correlate better with episodes of nephrotoxicity than measurement of the parent drug (see next section). Several reviews of different analytical methods, especially various HPLC techniques have lately been published [Furlanut et al. 1989; Shaw et al. 1987]. The most commonly used methods are summarised below.

HPCL

CsA is difficult to analyze by HPLC because it is lipophilic, lacks chromophores and lacks functional groups for derivatization. The maximum ultraviolet absorption is at 214 nm, part of the spectrum with which several other biological compounds may interfere. Extensive sample preparation is required, therefore, to remove all the interfering substances. This is usually performed by solvent extraction or two-phase chromatography (column switching). The initial pharmacokinetic studies of CsA were performed by gradient HPLC using solvent extraction [Niederberger et al. 1980]. One of the most frequently utilized methods is hexane extraction followed by isocratic HPLC, as described by Sawchuk & Cartier (1981) Column-switching was introduced by Nussbaumer et al. in 1982. With this technique the sample may be directly injected into the preparative column. A disadvantage of the technique is the need for advanced equipment and electronically controlled switching valves. Moreover, the large amount of blood proteins in the first preparative step frequently clog the filters. Because of its complexity. HPLC was used in only 15 of 124 laboratories which measured CsA in the UK CsA quality assessment scheme. Of two CsA-free samples circulated, 40 % of the laboratories analyzing CsA by HPLC reported false positive values compared to 25 % of the laboratories using other analytical methods [Johnston et al. 1989].

Specific monoclonal R I A with tritiated tracer

In order to find an antibody binding to parent CsA, but not to metabolites, Quesniaux et al. (1986, 1987) screened over 180 monoclonal antibodies to CsA. The antibody showing the least cross-reactivity with metabolites was chosen for inclusion in a RIA kit (Sandimmun kit, Sandoz Ltd, Basle, Switzerland). The antibody was reported to cross-react to 3 %, 4 % and 3 % with metabolites 1, 10 and 21, respectively (Instructions for Use). Clinical experience showed that analysis by specific monoclonal RIA gave 7% [Lindholm et al. 1990c] to 16% [Tredger et al. 1988] higher concentrations than analysis by HPLC. How-

ever, the correlation between the two methods is excellent (r > 0.97; Lindholm et al. 1990c).

Specific monoclonal R I A with iodinated tracer

In 1988 the specific monoclonal antibody to CsA described above was iodinated and introduced as a new kit (CYCLO-Trac SP; Sgoutas and Hammarstr6m 1989). Use of tritiated tracer requires a long counting-time in a liquid scintillation counter. By use of iodinated tracer the counting time was reduced from overnight to a few hours for a 30 sample series. In a quality control programme CsA concentrations measured by the latter method were higher than with the Sandimmun kit [Holt et al. 1990a]. However, there is an excellent correlation between the methods [Wong & Ma 1990]. Moreover, cross-reactivity with defined CsA metabolites did not differ between the two specific monoclonal RIA methods [Wallemacq et al. 1990].

Non-specific monoclonal R I A with tritiated tracer

As an alternative to the "original" polyclonal RIA antibody, a monoclonal antibody showing a high cross-reactivity with the metabolites was also chosen for inclusion in the new kit. It has been studied in detail, but no relevant application for this assay has been found [Lindholm et al. 1990a, 1990 c].

Polyclonal R I A with tritiated tracer

A polyclonal RIA method for the quantification of CsA was first described in 1981 by Donatsch et al. (Sandoz kit, Sandoz Ltd, Basel, Switzerland). Because it is simpler than HPLC, this RIA method was adopted for routine use by most laboratories, and it was employed from 1981 to 1988. The use of polyclonal antibodies has two major disadvantages. First, there are batch and species-dependent variations in the composition of the antibodies. Second, the antibodies cross-react with metabolites of CsA [Donatsch et al. 1981; Carruthers et al. 1983; Robinson et al. 1983; Copeland & Yatscoff 1988]. Rosano et al. (1986) compared CsA concentrations determined by HPLC and RIA in 177 blood samples from 11 renal transplant recipients and found that the true amount of CsA ranged from 6 % to 81% of the concentration measured by RIA. The R I A : H P L C ratio of CsA concentrations was especially high in paediatric liver transplant recipients during the first weeks after transplantation [Burckart et al. 1985]. In renal transplantation, the RIA :HPLC ratio of CsA concentrations declines with time after transplantation [Lindholm et al. 1990c]. Due to the disadvantages described above, the polyclonal RIA is no longer available, as it has been replaced by RIA with monoclonal tracers (see above).

276

Polyclonal RIA with iodinated tracer The "original" RIA was performed with a tritiated tracer. As for the monoclonal kit the turn-around time was shortened by use of an iodinated tracer instead of a tritiated marker [McBride et al. 1988]. Therefore, this RIA was introduced (polyclonal RIA, CYCLO-Trac, Immuno Nuclear Corporation, Stillwater, MN; Mahoney & Off 1985; Felder et al. 1986). This assay gave a slightly greater cross-reactivity with metabolites than that in the 'original' polyclonal assay (8 %, 46 %, 46 % and 11% cross-reactivity with metabolites 1, 17, 18 and 21, respectively; Package Insert), although the correlation between the two methods was high [Huang et al. 1987].

Polyclonal fluorescence polarization immunoassay (FPIA) Recently FPIA was introduced to quantify CsA [Sanghvi et al. 1988; Cyclosporine and metabolites TDx kit, Abbott Ltd, Irving, TX, USA]. The advantages include improved analytical precision, a short turn around time and prolonged time intervals between standard curve calibrations [Vanderbroucke 1988]. The main disadvantage is that the assay uses polyclonal antisera and cross-reacts to a greater extent with major metabolites than does the "original" polyclonal RIA method. Lensmeyer et al. (1990) found that FPIA had a cross-reactivity of 100 %, 57 % and 54 % with metabolites 17, 18 and 21.

Specific monoclonal FPIA The most recent development is FPIA with a monoclonal antibody (Wang et al. 1990). The antibody is different from the one in the monoclonal specific RIA assays. However, results with specific monoclonal FPIA correlates well with those by the monoclonal specific RIA and the percentage cross-reactivity is of the same magnitude (cross-reactivity of 15.3 %, 8.2 % and 3.7 % for metabolites 1, 17 and 21; Yatscoff et al. 1990).

Future developments Other analytical methods will soon be introduced on the market. One of these is an affinity column-mediated immunometric assay (ACMIA) specific for parent CsA [Hansen et al. 1990].

Therapeutic drugmonitoring Timing of sampling It is not known whether the trough, mean or peak concentration or the area through a dosing interval is the most important factor for optimal CsA treatment evaluation. In vitro, the interleukin production and release are almost totally blocked at a CsA concentration of 100 ng/ml [Bunjes et al. 1981]. It is tempting to speculate that the CsA concentration should be maintained somewhere above such threshold concentration throughout the dosing inter-

A. Lindholm: Therapeutic monitoring of cyclosporin val, thus avoiding triggering of the rejection process. In Europe CsA is dosed twice daily, while in some centers, especially on the American continent, CsA is given once daily. Much the commonest procedure is to monitor trough concentrations of CsA - i.e., 12 or 24 h after dosage, just before the next dose. However, in some centers CsA concentrations are obtained at an additional timepoint, commonly at 6 h after dosage. Cantarovich et al. (1988) suggest that measurements of CsA at 6 h after dosage are more useful than trough concentrations for the correlation to clinical events, especially to nephrotoxicity.

Trough concentrations or A UC measurements? Of great concern is how well CsA trough concentrations correlate with the A U C of CsA. Frey et al. (1988) measured the A U C of CsA in whole blood by HPLC after p.o. doses of CsA in 58 renal transplant recipients. They reported correlation coefficients between the A U C of CsA and 12- and 24-h trough blood concentrations of 0.73 and 0.86, respectively. Some studies of the relationship between A U C measurements and clinical events have been performed. However, this approach has its limitations because, for practical reasons, only a limited number of AUCs may be collected, and these cannot fully reflect the variable intrapatient kinetics of CsA after transplantation. Sommer and coworkers (1988) studied the 24-h AUC of CsA (as determined by polyclonal RIA in serum) 9 days after renal transplantation (4 days after initiation of CsA therapy) in 85 patients. They found no correlation between these AUCs and acute rejection or nephrotoxicity. On the other hand, Kasiske et al. (1988) studied 104 AUCs (as determined by HPLC in blood) in 45 patients at 1, 4 and 12 weeks after renal transplantation. Of the AUCs, 11 were performed within 2 weeks prior to acute rejection episodes. The pre-rejection concentration profiles had significantly lower maximum concentrations and dose-adjusted AUC's than the other concentration time profiles. However, there was no significant difference in 24-hour trough concentrations between the groups. Recently, Johnston et al. (1990) showed that measurement of CsA at 3 time points during a dosing interval accurately may predict the true AUC. This approach may prove useful in a clinical setting, especially regarding the possible relationship between the concentration of CsA/CsA metabolites and nephrotoxicity. However, in clinical practice so far, TDM of CsA has been performed by trough concentration measurements.

Patient selection for evaluation of CsA concentration-effect~toxicity relationship Most studies on the use of CsA and also of the concentration-effect relationship have been performed in renal transplant recipients. Some studies have also been carried out in bone marrow transplant recipients. However, liver and heart transplant recipients often receive as much CsA as they can tolerate. Thus, in these patients few evluations

A. Lindholm: Therapeutic monitoring of cydosporin

277

Table la. Studies of the correlation between CsA trough concentrations and episodes of acute rejection and acute nephrotoxicity after renal transplantation as determined by non-specific analytical methods (n. d. = not defined; 1.1. = lowest level recorded). Reference

Matrix

Plasma~Serum, polyclonal RIA Kahan Serum ( + 4 °C) et al. 1984

Weeks after transplantation

Group

No. of patients or episodes (pat. or ep.)

CsA conc. ng/ml (range or mean (SD)

P-value

0-4

Acute rejection

< 100 > 100 > 250 250-1300 100-500

P < 0.03

Klintmalm et al. 1985

Plasma ( + 20 °C)

04

Acute nephrotox, Acute nephrotox. Stable function

7/16 pat. 15/102 pat. n.d. 8/18 pat. 10/18 pat.

Lindholm et al. 1988b

Plasma ( + 20 °C)

2

Rej. patients Non-r@ pat.

104 pat. 180 pat.

n.d.

Acute rejection Acutenephrotox.

10/38 ep. 0/77 ep.

< 200 < 200

Acute rejection Acute nephrotox.

5/38 ep. 45/77 ep.

> 800 > 800

Acute rejection Acute nephrotox. Acute rejection Acute nephrotox.

26 ep. 30 ep. 36 ep. 40 ep.

Whole blood, polyclonal RIA Irschik et al. 1984

305 _+27 438 + 22

503 1005 471 891

P < 0.05 P < 0.01 P < 0.001

(216) (534) (248) (366)

P < 0.001

< 200 (1.1.) > 200 (1.1.)

P < 0.001

Taube et al. 1985 Holt et al. 1986

0- > 26

Rogerson et al. 1986 Martinez et al. 1989 Lindholm et al. 1990a

04

Acute rejection Acute rejection

23/28pat. 9/30pat.

0-39

Acute rejection Acute nephrotox. Rej. patients Non-r@ pat.

10ep. 11 ep. 44 pat. 22 pat.

Acute rejection Acute nephrotox.

10 ep. 11 ep.

529 (114) 1040 (382)

P 16 8 0-39

Spec. monocl. RIA

0-39

Polyclonal RIA Spec. monocl. RIA Spec. monocl. RIA Spec. monocl. RIA

0-104 0-104 0-4

Spec. monocI. RIA

0-4

Acute rejection Stable function Acute rejection Acute nephrotox. Acute rejection Acute nephrotox. Acute rejection Acute rejection Acute nephrotox. Acute rejection Acute rejection Rej. patients Non-r@ pat. Acute rejection Stable function Acute nephrotox.

Holt et al. 1989 Lindholm et al. 1990a

Sridhar et al. 1990

HPLC

125 ng.m1-1 2 months after transplantation (at which time renal function was stable in all patients). Holt et al. (1989) compared the blood CsA concentrations determined by polyclonal and specific monoclonal RIAs in 32 acute rejection episodes. Of the rejection episodes, 20 were associated with at least one specific measurement less than 200 ng-m1-1, while 13 were associated with at least one polyclonal measurement less than 400 ng. ml 1. A prospective study of CsA monitoring by different analytical techniques has recently been completed [Lindholm et al. 1990a]. The lowest CsA concentrations recorded during the first month after transplantation were significantly lower in patients with than in those without acute rejection episodes when the CsA concentrations were measured by polyclonal R I A in whole blood and plasma, and by specific and non-specific monoclonal R I A in whole blood, but not by H P L C in plasma or polyclonal FPIA in whole blood or plasma (Fig. 1). In plasma, the H P L C concentrations were often close to or below the limit of determination, making whole blood the preferable medium for CsA determination. In whole blood, analysis of CsA by specific monoclonal R I A showed the best correlation with rejection. Thus, using specific analysis of CsA, the results of Holt et al. (1989), Moyer et al. (1988) and Lindholm et al. (1990a) suggest that an acute rejection episode is more likely to occur if the 12 h trough concentration of CsA is below 125 to 200 ng.ml -~ during the first month after renal transplantation. Empirical experience has shown that lower trough concentrations of CsA are compatible with good graft function later after transplantation.

Relationship of CsA concentration to toxicity In the first reports using T D M of CsA an association was found not only with rejection but also between high

No. of patients or episodes (pat. or ep.) 14/17 ep. 38/39 pat. 10 ep. 11 ep. 8/10 ep. 2/11 ep. 6/32 ep. 20/32 ep. 10/25 ep. 31/44 pat. 6/22pat. 44 pat. 22 pat. 26 ep. 322 ep. 87 ep.

CsA conc. ng/ml (range or mean (SD) < 125 > 125 157 (43) 323 (127) < 200 < 400 < 200 > 400 < 140 > 140 108 183 156 174 213

P-value

P < 0.001 P < 0.01 P < 0.01 P < 0.05

(1.1.) (1.1.) (56) (113) (83) (91) (93)

P < 0.01 P < 0.001 P< 0.05 P< 0.05

trough concentrations of CsA and nephrotoxicity [Keown et al. 1981; Irschik et al. 1984]. However, there is a considerable overlap and acute nephrotoxicity may occur at any concentration of CsA [Klintmalm et al. 1985; Taube et al. 1985; Holt et al. 1989]. It is difficult to propose an upper therapeutic limit. There is no doubt that high concentrations of CsA are associated with an increased risk of acute and chronic nephrotoxicity, as well as other adverse effects and risk of infection [Kahan et al. 1984; Klintmalm et al. 1985]. However, most such studies have employed non-specific analysis of CsA. By polyclonal analysis, Kahan et al. (1984) found that serum CsA concentrations (separation at + 4 °C) above 200 ng. ml- I were associated with an increased incidence of hepatotoxicity and concentrations above 250 ng. ml-1 with ne~ phrotoxicity (P < 0.05; Table 1). In a comparison of specific and non-specific determinations of CsA, Holt et al. (1989) found that 10 of 25 episodes of acute nephrotoxicity were associated with specific monoclonal blood CsA concentrations above 400ng.m1-1 compared to 11 of 25 episodes with polyclonal blood CsA concentrations above 800 ng. ml-1. The recent studies by specific analysis of CsA have not succeeded in demonstrating a highest acceptable concentration. This may be due to the fact that acute nephrotoxicity is uncommon with the low doses of CsA in c u r r e n t u s e [Kasiske et al. 1988, Lindholm et al. 1990a]. In a prospective study, in agreement with Holt et al. 1989, it was found that the correlation between CsA concentrations and the occurrence of nephrotoxicity was no weaker with analysis by specific monoclonal R I A than by polyclonal R I A [Lindholm et al. 1990 a]. On the one hand, these results do not support the proposition that the major metabolites are responsible for nephrotoxicity. On the other hand, Sewing et al. (1990) recently reported a correlation between nephrotoxicity and the concentration of the double hydroxylated metabolites of CsA and cyclized

279

A. Lindholm: Therapeutic monitoring of cyclosporin d 100

80

~=

-£

p 250 ng. ml- :, respectively.

Autoimmune disease In most autoimmune indications CsA therapy has not yet been fully evaluated. CsA is often given in a fixed dosage. The dose is high in indications where blockade of an antibody response is desired (e.g. diabetes melli-

A. Lindholm: Therapeutic monitoring of cyclosporin

280 Table 2. Currently (1990) recommended trough whole blood con-

centrations of CsA (determined by specific monoclonal RIA) for various indications at Huddinge University Hospital Indication Renal transplantation

Pancreatic transplantation Liver transplantation

Bone marrow transplantation (reduction when s-creatinine 5(~100 % above baseline) Psoriasis

Months after transplantation Month 1 2-3 >3 Month 1-3 >3 Month 1-3 4-6 >6 Month 1- 3 4-12

Concentration 16(~240 100-160 60-120 150-200 100-150 250-300 200-250 150.200 > 200 > 150 < 185

tus), but low in T-cell mediated diseases (e. g. psoriasis). In a recent multicentre-single laboratory investigation no relationship was found between specific CsA whole blood concentrations and efficacy in the treatment of psoriasis [Lindholm et al. 1990d]. Although 10 of the 54 patients had CsA concentrations below 60 ng.ml t, there was excellent improvement of the psoriatic lesions. The nephrotoxic effect of CsA limits its use in autoimmune disease, especially in indications were high concentrations are needed. The severity of renal dysfunction is of the same degree as in bone marrow transplantation.

Free fraction and concentration of CsA For drugs with a high degree of plasma protein binding, pharmacological theory predicts that the free concentration in plasma should be more closely related to the clinical efficacy than the total concentration. CsA is highly bound to plasma lipoproteins and therefore the free fraction and free concentration of CsA in plasma were studied in renal transplant recipients [Lindholm et al. 1989; Lindholm 1990]. There was an overall eightfold variation in the free fraction of CsA in plasma, with up to fivefold intraindividual variability, and 2.3-fold interindividual variability in the mean free fraction between individuals [Lindholm et al. 1989]. The free fraction of CsA was dependent on the content of lipoproteins (estimated by determinations of HDL-cholesterol and apolipoprotein A1), other plasma proteins (s-albumin and related non-lipoproteins), the time after transplantation (higher shortly after transplantation) and the diagnosis (higher in diabetics than in nondiabetics). Furthermore, the free fraction of CsA was lower at the time of rejection as compared to one week earlier. The correlation between the total and free concentrations of CsA was high (overall r = 0.90) [Lindholm 1990]. Both the total and the free concentrations of CsA in plasma were decreased to a similar extent prior to acute rejection and they were increased to a similar extent during nephrotoxic episodes. Thus, determination of the free

concentration of CsA in plasma gave no additional information or guidance compared to specific measurement of total plasma CsA concentrations. Due to its complexity with two consecutive analyses, routine monitoring of free CsA in renal transplant recipients is, at present, not recommended.

Conclusions

In organ transplantation, CsA monitoring is especially important in the early postoperative period, when the risk of acute graft rejection is highest and when absorption may be erratic. I recommend CsA monitoring at least every second day during first two weeks after transplantation. Clinical decisions should not be based on just a single determination as the trend should be studied. In case of a single CsA determination outside the desired range, a fresh sample should be collected on the following day. If two or more samples point in the same direction, then dose adjustment may be justified. In autoimmune disease it is usually adequate to analyse the CsA concentration weekly during the first 4 to 8 weeks of treatment. It is more important to make frequent determinations of serum creatinine during this period. Target concentration of CsA depends on the indication for treatment, time after initiation of therapy and concurrent immunosuppressive therapy. For the reasons mentioned, analysis in whole blood does seem preferable. Several groups and expert meetings have strongly suggested analysis by a specific method [Shaw et al. 1987; Holt 1990]. Whichever method used, it is wise to monitor a patient by one method only, and to use the same analytical methods at the transplant unit and in the local hospital. In Sweden and Norway there is agreement on using one of the specific analytical methods and on the assay of whole blood, thus simplifying patient follow up as well as inter-centre evaluations. The analytical precision is frequently checked within the Cyclosporine Quality Assessment Schedule [Johnston et al. 1986, 1989; Holt et al. 1990]. As an example of target trough CsA concentrations, Table 2 shows the currently recommended trough concentrations in various indications at the Huddinge University Hospital. In renal transplantation it is suggested that the trough whole blood concentration of CsA should be no less than 150 ng- ml- 1at any time during the first month after transplantation, as determined by a specific method in whole blood. After the first post-transplantation months empirical experience has shown that the dose of CsA and the concentrations may be reduced, as it appears that whole blood concentrations down to 50 ng. ml-1 may be compatible with long term renal graft function.

Acknowledgements. The study was supported by grants from the Swedish Societyof Medicine (no. 86-372, 87-394, 88421), the Karolinska Institute, the Swedish Medical Research Council (no.3902) and Sandoz Ltd.

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mance liquid chromatography whole blood trough levels. Transplant Proc 20 [Suppl 2]: 394-401 Van den Berg JWO, Verhoef ML, de B oerh AJH, Schalm SW (1985) Cyclosporin A assay: conditions for sampling and processing of blood. Clin Chim Acta 147:291-297 Vandebroucke AC (1988) Evaluation of the TDx R method for cyclosporine and a comparison to CYCLO-Trac R RIA in renal transplant patients. Clin Biochem 21:307-309 Venkataramanan R, Starzl TE, Yang S, Burckart G J, Ptachcinski R J, Shaw BW, Iwatsuki S, Van Thiel DH, Sanghvi A, Seltman H (1985) Biliary excretion of cyclosporine in liver transplant patients. Transplant Proc 17:286-289 Venkataramanan R, Habucky K, Burckart GJ, Ptachcinski RJ (1989) Clinical pharmacokinetics in organ transplant patients. Clin Pharmacokinet 16:134-161 Wallemacq PE, Lee SC, LhoSst G, Hassoun A (1990) Cross-reactivity of cyclosporine metabolites in two different radioimmunoassays in which the same specific monoclonal antibody is used. Clin Chem 36:385 Wang E Meucci V, Simpson E, Morrison M, Lunetta S, Zajac M, Boeckx (1990) A monoclonal antibody fluorescent polarization immunoassay for cyclosporine. Transplant Proc 22:1186-1188 Wenger RM (1990) Structures of cyclosporine and its metabolites. Transplant Proc 22:1104-1108 Wong PY, Ma J (1990) Specific and nonspecific monoclonal t25-I-Incstar assays. Transplant Proc 22:1166-1170 Yatscoff RW, Copeland KR, Faraci CJ (1990) Abbott TDx monoclonal antibody assay evaluated for measuring cyclosporine in whole blood. CIin Chem 36:1969-1973 Yee GC, McGuire TR, Gmur DJ, Lennon TR Deeg HJ (1988a) Blood cyclosporine pharmacokinetics in patients undergoing marrow transplantation. Transplantation 46:399-402 Yee GC, Self SG, McGuire TR, Carlin J, Sanders JE, Deeg HJ (1988b) Serum cyclosporine concentration and risk of acute graftversus-host disease after allogeneic marrow transplantation. N Engi J Med 319:65-70 Dr. A. Lindholm Department of Transplantation Surgery Huddinge Hospital S-14186 Huddinge Sweden