Cyclosporin A: Analytical Methodology and Factors Affecting Therapeutic Drug Monitoring
David W.Holt and *Atholl Johnston
Analytical Unit,St.George’s Hospital Medical School,and*Clinical Pharmacology,St.Bartholomew’s Hospital, London,England
Summary:The majority of centres measuring cyclosporine have followed the advice of previous consensus meetings. Although most measurements are made using assays with a high specificity for the parent compound,there are still some between-method differences that can be attributed to methodological variables,such as antibody specificity and assay calibration. With experience of cyclosporine therapeutic drug monitoring (TDM) now spanning 15 years,it would be reasonable to infer that the subject is incapable of growth.However, continued interest in the pharmacokinetics of cyclosporine,the introduction of a new formulation of the drug, and the development of new immunosuppres-sive agents all combine to emphasise the still incomplete extent of our knowl-edge in this field. Key Words: Cyclosporine-High-performance liquid chromatography-EMIT-Radioimmunoassay-Fluorescence polarization immunoassay.
Since the first consensus document on cyclospor-ine measurement (1),subsequent reports have been in agreement on the choice of sample matrix and the need for specificity in the analytical technique to be used (2-6).The basic guidelines for routine clinical monitoring have also remained the same,the differ-ences in the documents reflecting changes in ana-lytical methodology,a greater understanding of the pharmacokinetics of the drug, changes in prescrib-ing patterns, and the introduction of the Neoral for-mulation.
We aim to set in context some of the methodolog-ical factors that may influence the reliability of a cyclosporine concentration measurement and the pharmacological factors that should be taken into account when the measurements are used as a guide to therapy.
Address correspondence and reprint requests to Dr. D. W. Holt at Analytical Unit,St George’s Hospital Medical School, London SW17 0RE, U.K.
Data from quality-assessment schemes in Europe and North America show that most centres measur-ing cyclosporine have now opted for semiauto-mated immunoassay techniques ratherthan high-performance liquid chromatography (HIPLC).Data from the UK Cyclosporin Quality Assessment Scheme,which is predominantly European based, show that most laboratories are using one of three immunoassays: the INCSTAR CYCLO-Trac SP radioimmunoassay (RIA), the Abbott TDx monoclonal antibody fluorescence polarization im-munoassay (FPIA), and the Syva enzyme multi-plied immunoassay technique (EMIT). Nonspecific measurements are being made, almost exclusively, using the Abbott TDx polyclonal antibody FPIA. Results from other schemes based in Germany, France,Italy,the United States,and Canada are in broad agreement with these findings.
Although the methods most commonly in use have a high specificity for the parent compount,it is
D.W.HOLT AND A. JOHNSTON
well established that they do not give identical re-sults for patient samples, in particular if the sample contains a relatively high concentration of cyclo-sporine metabolites (4,7,8).The difference between values obtained using HPLC and the high-specificity immunoassays is, in part, due to the spectrum of cross-reactivity towards cyclosporine metabolites displayed by the antibodies used in the immunoassays.Data from the UK Cyclosporin Quality Assessment Scheme suggest that,for the measurement of cyclosporine in a pooled blood sample from patients receiving the drug after kidney transplantation, the results using specific assays dif-fer by up to 22%;the mean result using the TDx Polyclonal antibody assay is ~2.5-fold higher. These differences cannot be attributed to antibody specificity alone.
Reports have suggested that there are between-method differences in the absolute values of the cal-ibrators supplied with commercial assay kits and calibrators prepared in house (9,10). Figure la illus-trates the results for the measurement of cyclospor-ine in a blood sample spiked with the drug to a nominal concentration of 180 μg/L;the sample was circulated to participants in the scheme in Septem-ber 1993. There is a clear difference,of~12%,be-tween the median results produced by participants using the INCSTAR RIA with calibrators supplied with the kit,compared with those using the same kit with calibrators prepared in house. This difference has been maintained over a prolonged period of ob-servation, as shown in Fig. 1b, in which the results for another sample,spiked with the drug to a nom-inal concentration of 200 μg/L and circulated in Oc-tober 1994, are illustrated. It is interesting to note that the results for the second sample also show a shift in the relative values obtained by the TDx monoclonal antibodyassay, both in comparison with the specific assays and the polyclonal antibody TDx kit produced by Abbott Laboratories.This rel-ative change became apparent early in 1994 and has been monitored closely since then.
The differences resulting from both antibody specificity and variations in calibration are gener-ally not very large, on the order of 20-30%,for blood samples from kidney-transplant patients. Such differences are unlikely to cause substantial changes in patient management, especially if the same analytical method is always used in an insti-tution.However,there are some circumstances in which between-method differences can bed of im-portance.
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FIG.1. a: Box and whisker plots for the measurement of cy-closporine in a blood sample spiked with cyclosporine to a nom-inal concentration of 180 μg/L. The sample was analysed in Sep-tember 1993. The methods are (number of results): HPLC, high-performance liquid chromatography (8); EMIT, Syva enzyme multiplied immunoassay technique (27);INCSTAR-IHS, INCSTAR CYCLO-Trac SP radioimmunoassay using calibra-tors prepared in house (11); INCSTAR-KS, INCSTAR CYCLO-Trac SP radioimmunoassay using calibrators supplied with the kit (52);TDx Mono, Abbott fluorescence polarization immuno-assay with monoclonal antibody (83); TDx Poly, Abbott fluores-cence polarization immunoassay with polyclonal antibody(25). Data:UK Cyclosporin Quality Assessment Scheme.b:Box and whisker plots for the measurement of cyclosporine in a blood sample spiked with cyclosporine to a nominal concentration of 200 μg/L.The sample was analysed in October 1994. Method details are as in (a); the number of results was 9,43,11,50,103, 25,for the respective methods. Data: UK Cyclosporin Quality Assessment Scheme.
Antibody specificity becomes an issue when sam-ples containing relatively high concentrations of cy-closporine metabolites are analysed. In practice, the problem is most pronounced in samples from
CYCLOSPORIN A:METHODOLOGY AND TDM
liver-transplant patients in the early period after transplantation but can be encountered in samples from any patient with hepatic dysfunction. Of the three high-specificity immunoassays indicated pre-viously,the TDx monoclonal antibody assay is the most affected, as the antibody used in this kit shows more cross-reactivity with cyclosporine metabo-lites,especially AM9, than the antibodies used in the other two kits (4,11).As a result, there is a bias between TDx and the other immunoassays on the order of 5-50% but generally not >20%(4). In a recent study comparing the EMIT assay and the TDx monoclonal antibody assay for the measure-ment of cyclosporine in 59 samples from liver-transplant patients, a median bias of +30% was found using the TDx assay, but the difference was as high as 69% (Holt DW, Johnston A, Tredger JM, unpublished data).
Accuracy and Sensitivity
There is increasing interest from regulatory au-thorities in the use of randomised concentration-controlled trials as an aid to the clinical assessment of new drugs (12). Such trials are usually based at a number of clinical centres, and the accuracy with which the drug is measured is of particular rele-vance to the proper conduct of such studies (13). Even the differences inherent in the use of current methodology for the measurement of cyclosporine are sufficient to obscure predefined target concen-tration ranges and may necessitate the use of a cen-tral laboratory to reanalyse samples, using a single method,rather than analysis on an intention-to-treat basis.
The three immunoassays vary in their suitability for pharmacokinetic studies. For a full profile of samples, drawn over a period of 48-72 h after a single dose of cyclosporine, a sensitivity of at least 10 μg/L is required.This can be achieved using the CYCLO-Trac RIA, modified by use of a larger sam-ple size (Analytical Unit, data on file).The ability to measure cyclosporine at such low concentrations, with acceptable precision, has an important impact on the calculation of pharmacokinetic parameters. For instance,the elimination half-life will vary sub-stantially,depending on the time of blood sampling. For instance,based on the blood cyclosporine con-centration profiles from 24 volunteer subjects given an 800-mg oral dose of the Neoral formulation,t1/2 ranged from 3.4 h,if the sampling period was only 0-12 h, to 11.3 h if sampling was continued up to 48
h after dosing (Analytical Unit, data on file). The sensitivity of the cyclosporine assays may become a significant issue if some of the newer immunosup-pressive agents, such as rapamycin or mycopheno-late mofetil,are prescribed with cyclosporine.The limit of accurate measurement may need to be suf-ficient to distinguish compliant and noncompliant patients receiving very low doses of cyclosporine.
The assays also vary in their dynamic range.The EMIT assay is particularly limited, with a top cali-brator of only 500 μg/L,resulting in the need for the dilution of samples containing higher concentra-tions. Although this is infrequently a problem for the measurement of trough concentrations,it is a disadvantage when measuring cyclosporine in sam-ples collected to determine the area under the curve (AUC),either from a full profile of samples or by means of a limited sampling strategy.
For pharmacokinetic studies involving the analy-sis of blood samples from species other than hu-mans, it is important to assess the effect of sample matrix with respect to accuracy against the cali-brators supplied with the assay, sensitivity, and re-producibility. Our own findings suggest that the CYCLO-Trac RIA can be used to measure cyclo-sporine in blood samples from rat and dog using calibrators prepared in human blood (Analytical Unit,data on file).
Any laboratory that has been measuring cyclo-sporine since the early 1980s is likely to have changed methodology at least once. This can lead to problems of continuity when there is a need to re-late data collected some years previously to current findings.We have been approached on a number of occasions to supply information on the relative dif-ferences between current and obsolete assays and, indeed,matrices.Imperfect as the conversion fac-tors we calculate are,they are often the only way to draw some meaning from clinical data carefully col-lected over some years.
External Quality Assessment
All the reports of consensus groups on cyclospor-ine monitoring have recommended that laboratories should participate in external quality-assessment programmes.Whilst the growth in membership of such schemes is encouraging, it is surprising to find some countries poorly represented. In particular,
D.W.HOLT AND A. JOHNSTON
Third World countries,in which the access to cur-rent literature may be beyond the financial reach of many laboratories, are often badly informed of the need for internal quality control and external qual-ity assessment. In this respect, multinational man-ufacturers of reagents and suppliers of equipment should be encouraged to educate their customers about current developments in the field of cyclo-sporine monitoring. Even in well-developed coun-tries,there is evidence of some complacency with regard to quality-assessment issues. For instance,a recent survey of Australian laboratories measuring cyclosporine showed that only nine of 30 laborato-ries were members of an external quality-assess-ment scheme (the UK scheme; 14). After publicity of these findings, the number of participants has increased to 21.
The interpretation of any single blood-cyclo-sporine concentration is complicated by a number of variables, some of which relate to the method-ological issues described previously.Others,how-ever, relate to pharmacological variables,all of which contribute to the disparity in the concentra-tion ranges individual centres consider acceptable as a guide to therapy. Previous consensus docu-ments have recorded the between-centre differ-ences in reference concentration ranges,most of which vary both with the indication for cyclospor-ine and the time since transplantation. It could be anticipated that, with fewer methods in use,there would be a convergence of these ranges,but the cyclosporine consensus document from this meet-ing does not suggest this to be the case (15). What, then,are the factors that affect the routine thera-peutic monitoring of cyclosporine and that can be investigated by concentration measurements in the preclinical or clinical setting?
Poor oral absorption of cyclosporine has been shown to influence the incidence of graft rejection in kidney-transplant patients. Patients with an oral bioavailability of <25% had a lower graft survival at 1 year and were less likely to have a rejection-free year than patients absorbing a larger proportion of the dose (16).Thus there is a need to identify pa-tients who are poor absorbers of the drug.Some groups, such as those with poor bile flow and pa-
Ther Drug Monit, Vol.17,No.6,1995
tients with cystic fibrosis, have a well-established risk.Less well defined are ethnic differences,which could be due either to genetic factors or social in-fluences such as diet. It has been established that black patients absorb cyclosporine less well than do whites(17).However,the ethnoparmacokinetics of other racial groups, such as Indian or Chinese Asians or Mediterranean groups, have yet to be documented.
Oral absorption of cyclosporine from the Sand-immun formulation is very erratic, and the poorpre-dictability of the extent of absorption has led to the development of a new formulation, Neoral (18).The impact of this formulation on the need for routine monitoring has still to be established,but the early volunteer and clinical studies suggest that it should increase the relevance of cyclosporine concentra-tions with respect to clinical events. For a start, using Neoral, there is a near-linear relationship be-tween dose and exposure (measured by area under the concentration-time curve,AUC; 19). There is also a closer relationship between the trough con-centration and AUC (20).Whether the latter finding will result in more optimal prescriptionof the drug has yet to be established,but if the trough concen-tration is a better reflection of exposure using Neoral,then there may be less need for costly and inconvenient multiple determinations of cyclospor-ine concentrations to estimate AUC.
Sample Timing and Dosing Frequency
It should be remembered that the trough concen-tration is influenced by dosing frequency. Thus whether the trough concentration represents a C12h, rather than a C24h, value should be recorded.If twice-daily dosing is used, a diurnal effect on clear-ance has been noted,such that the trough concen-tration measured after the daytime dose tends to be lower than the trough concentration measured after the nighttime dose(21).
Estimates of either the pharmacological activity or toxicity of cyclosporine metabolites are limited by the use of in vitro studies that may not reflect adequately the clinical situation in humans. The ef-ficacy of the metabolites appears to be small, no more than 10%,although it could be significant for some metabolites that are found in blood in higher concentrations than in the parent compound (22).
CYCLOSPORIN A: METHODOLOGY AND TDM
Only one study has addressed the in vivo toxicity of the three principal cyclosporine metabolites in the rat (23). Although some caveats must be placed on the interpretation of the data, only the parent com-pound could be shown to induce characteristic signs of cyclosporine nephrotoxicity.
These findings do not help in deciding the value of monitoring the concentrations of cyclosporine metabolites as a guide to therapy.The measurement of cyclosporine using both specific and nonspecific assays has been advocated, on the basis that the difference between the two results gives some mea-sure of cyclosporine metabolite concentrations(24). This approach seems fraught with difficulties,be-cause the extent of cross-reactivity exhibited by a nonspecific antibody towards each of the metabo-lites varies substantially and, for many metabolites, has not been characterised fully. Thus the numeric value obtained using the TDx polyclonal antibody assay is influenced mostly by the concentration of metabolite AM1(25).Changes in the concentrations of other metabolites present at much lower concen-trations may be masked by changes in AM1 con-centration.
Although one group has suggested an association between nonspecific measurements made using a nonspecific RIA (CYCLO-Trac NS) and signs of nephrotoxicity,the data could not establish wheth-er high nonspecific measurements were the cause or the consequence of diminished renal function (26). A definitive answer on the value of metabolite-concentration measurements could result only from an extensive study in which individual metabolites were measured using HPLC and the concentrations related to clinical signs. In the absence of such data, it seems more appropriate to follow the guidelines of the consensus documents by making measure-ments with one of the high-specificity assays.
Many of the pharmacokinetic drug interactions between cyclosporine and coprescribed drugs have been identified after routine monitoring of cyclo-sporine.Patient and volunteer studies are often re-stricted to small numbers of participants, and al-though they may indicate the magnitude of an in-teraction,there is often substantial between-patient variation in the effect a particular drug exerts on cyclosporine pharmacokinetics. For instance, Brockmöller et al. (27) showed that the mean in-crease in cyclosporine concentration when diltia-
zem was taken with cyclosporine was 45%. How-ever,four of 22 patients showed no effect of diltia-zem on trough cyclosporine concentrations. Recently,there has been interest in the effect of grapefruit juice on the oral absorption of cyclospor-ine. One group showed an average increase of >50% in cyclosporine AUC when the drug was taken with 250 ml grapefruit juice (28). The authors suggested that coadministration with grapefruit juice could be an inexpensive and nontoxic means of reducing cyclosporine dosing requirements. However,although superficially attractive,such an approach would be ill advised, because it is compli-cated by a number of variables, including between-batch differences in the concentration of the active flavonoid,naringenin (29).The potential for pharma-cokinetic interactions remains an important reason for monitoring cyclosporine.
Monitoring blood-cyclosporine concentrations has become an accepted aid in the optimal prescrip-tion of the drug,but reference concentration ranges vary quite widely between centers.Some of the fac-tors leading to the disparity in reference ranges quoted by individual centers have been considered here.
Although the main technical issues relating to monitoring blood-cyclosporine concentrations have been resolved, it is clear that there is a continuing need to be aware of methodological factors that may limit the value of routine monitoring.How-ever, in addition to analytical considerations, the interpretation of cyclosporine-concentration mea-surements requires close attention to a broad spec-trum of factors,which,undoubtedly,contributes to the array of concentration ranges in common use.
Finally,the introduction of the Neoral formula-tion and the potential for using cyclosporine in combination with some of the newer immunosup-pressive agents have implications for the routine monitoring of cyclosporine that have yet to be es-tablished.
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