Clinical Chemistry 43: 104-108, 1997;
(Clinical Chemistry. 1997;43:104-108.)
© 1997 American Association for Clinical Chemistry, Inc.
Effect of assay methodology on pharmacokinetic differences between cyclosporine Neoral® and Sandimmune® formulations
Launa J. Aspeslet1,
Donald F. LeGatt1,
Gerard Murphy2 and
Randall W. Yatscoff1,a
1
Department of Laboratory Medicine & Pathology, University of Alberta, Edmonton, AB, Canada.
2
Sandoz Canada Inc.
a Address correspondence to this author at: Department of Laboratory Medicine & Pathology, University of Alberta Hospitals, 8840–112 St., Edmonton, AB, Canada T6G 2B7. Fax 403-492-8599; email randy.yatscoff{at}ualberta.ca
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Abstract
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The new oral formulation of cyclosporine (CsA), Neoral® (CsA-N),
results in increased area under the curve (AUC) and decreased intra-
and interindividual variation in blood concentrations and other
pharmacokinetic (PK) parameters when compared with the current
Sandimmune® (CsA-S) formulation. The present study
examines the effect of assay methodology on variability in blood
concentrations and PK parameters for renal transplant patients
receiving CsA-N and CsA-S and whether this variation is reduced with
CsA-N. The results show that interindividual variations in PK
parameters for patients receiving CsA-N were less than those for
patients receiving CsA-S. Both blood concentrations and dose of CsA
better correlated with abbreviated (4-h) AUC after administration of
CsA-N. For both CsA-S and CsA-N, blood concentrations at 4 h
postdose exhibited the best correlation with AUC. All samples were
analyzed by three common procedures: HPLC, RIA, and fluorescence
polarization immunoassay (FPIA). There were no significant differences
observed in blood concentrations or PK parameters obtained from FPIA
and RIA. HPLC results, however, were lower because of specificity of
this method for the parent drug. The assay methodology did not have an
effect on interindividual variability, indicating that the
cross-reactivity of metabolites in commonly used immunoassays for CsA
does not contribute to the PK variability observed in renal transplant
patients.
Key Words: indexing terms: pharmacokinetics • therapeutic drug monitoring • metabolite cross-reactivity • renal transplants • immunosuppression • radioimmunoassay • high-performance liquid chromatography • fluorescence polarization immunoassay
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Introduction
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Therapeutic drug monitoring (TDM) of cyclosporine (CsA) is
required for dosing of the drug to optimize immunosuppressive efficacy,
while minimizing its side effects
(1).1
The majority of experience with the therapeutic
monitoring and pharmacokinetics (PK) of CsA has been when CsA is
administered orally with olive or corn oil vehicles, more commonly
referred to as the Sandimmune® (CsA-S) preparation. These
formulations must be digested by pancreatic enzymes and emulsified by
bile into hydrophilic particles before absorption (2). A
new oral formulation of CsA, Neoral® (CsA-N), incorporates
the drug into a microemulsion, which readily emulsifies upon contact
with aqueous fluids without the requisite action of bile, enzymes, or
small intestinal secretions (3).
Recent reports have shown that the CsA-N formulation results in
increased bioavailability, more rapid absorption, and decreased intra-
and interindividual variation in blood concentrations and PK parameters
(4)(5). The data also indicate an improved
correlation between trough concentrations and area under the curve
(AUC), as compared with the CsA-S formulation (6). CsA-N
is expected to improve initial immunosuppression with CsA, since there
is a good correlation between higher CsA AUC and freedom from rejection
in the critical posttransplant period (7).
TDM of CsA is most commonly performed by one of the following three
procedures: HPLC, RIA, and fluorescence polarization immunoassay
(FPIA). These assays differ in their precision and specificity for the
parent drug. The FPIA, in general, exhibits better precision than HPLC
and RIA (8), whereas both the RIA and FPIA procedures
exhibit cross-reactivity with CsA metabolites (9)(10)(11)(12)(13).
Both of these assay methodology issues may influence the intra- and
interpatient variation for CsA-N. In this study, we report on the
effect of assay methodology on variability in blood concentrations and
PK parameters for patients receiving CsA-N and CsA-S.
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Methods
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patient selection and specimen collection
Fifty stable renal transplant patients participating in a
multicenter trial on CsA-N were randomly selected for the study. All
patients were receiving triple immunosuppressive therapy consisting of
CsA (CsA-S), azathioprine, and prednisone. Specimens were collected
before conversion to CsA-N (month 0) and 3 months after conversion. At
these intervals, whole-blood specimens (EDTA anticoagulant) were
collected at the following times: trough, 1, 2, and 4 h postdose.
Specimens were transported to the central laboratory site in Edmonton
where they were stored at 4 °C until analysis. Use of human subjects
received approval from each participating institution's Ethics
Committee on use of human subjects in research.
method of analysis and data calculation
All specimens were analyzed by HPLC (13), RIA
(Cyclotrac; IncStar, Stillwater, MN), and FPIA (Monoclonal Whole Blood
Assay, Abbott TDx; Abbott Diagnostics, Abbott Park, IL). The two
immunoassays were performed according to the manufacturers'
instructions. The interassay CVs at 100 and 300 µg/L were: HPLC, 12%
and 8%; RIA, 11% and 7%; FPIA, 6% and 4%, respectively.
Noncompartmental PK parameters were calculated by using PC NONLIN 4.2
(Scientific Consulting, Apex, NC). AUC were calculated for the complete
12-h dosing interval by extrapolation of the 4-h PK profile to a 12-h
profile by using the t = 0 as the t = 12 time point. These
are referred to as abbreviated AUC. For dose-normalized calculations,
all parameters were normalized to a CsA dosage of 100 mg/day. We, as
well as others (14), have previously shown that this
provides an appropriate standardization. Standard linear regression
analyses to determine correlation coefficients (r) were
performed for comparison of time points postdose with AUC.
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Results
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analytical validation
To ensure that blood concentration results for parent CsA as
monitored by HPLC were comparable with those generated by FPIA and RIA,
the calibrators from both assays were analyzed by HPLC. All assays
provide similar results for the parent drug (data not shown) and one
can therefore assume that any difference among assays will be due to
cross-reactivity with metabolites.
effect of assay and formulation on csa pk parameters
A comparison of PK parameters for the 50 patients receiving the
CsA-S and CsA-N formulations as determined from HPLC, RIA, and FPIA
blood concentration data is shown in Table 1
. For all three assays, significant (P 
0.01)
increases in 1- and 2-h postdose concentrations,
Cmax concentrations,
Cmax/trough ratio, and abbreviated AUC were
observed for patients receiving CsA-N vs CsA-S. No significant
differences in trough (t = 0) or 4-h postdose concentrations were
observed. Similar results were obtained when the concentrations were
normalized for dose (data not shown). The averaged HPLC concentration
vs time profiles (0–4 h) for CsA-N and CsA-S with both raw and
dose-normalized concentrations are shown in Fig. 1
.
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Figure 1. CsA blood concentration profiles from renal transplant
patients receiving either CsA-N or CsA-S.
Concentrations were obtained by the HPLC method; both raw and
dose-normalized values are shown. Results are expressed as mean ±
SE (n = 50).
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For each formulation, there was no significant difference (P
>0.05) in the blood concentrations or the PK parameters obtained with
FPIA and RIA. In contrast, the concentrations obtained by FPIA and RIA
were significantly (P 0.01) higher than those obtained by
HPLC. For CsA-N, RIA, and FPIA, mean concentrations were 10–41%
higher than those obtained by HPLC, whereas for CsA-S, mean
concentrations were 12–51% higher. Values for abbreviated AUC
calculated from RIA and FPIA data were 27–32% higher than values
derived from HPLC data for CsA-N and 28–36% higher than HPLC-derived
values for CsA-S. The PK profiles for CsA-N constructed from
concentrations generated by the three assays are shown in Fig. 2
.
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Figure 2. CsA blood concentration profiles of patients receiving
CsA-N determined by FPIA, RIA, and HPLC.
Results are expressed as mean ± SE (n = 50).
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The variations in the PK parameters were determined by comparison of
the standard deviation intervals (SDI =
SD/
) obtained from each analytical method
for both the CsA-S and CsA-N formulations. Fig. 3
A and B demonstrate that the variation in PK parameters was less
for CsA-N as compared with CsA-S for all assays. This was true when
both raw and dose-normalized data were considered. Variations in
concentrations and PK parameters (SDI) among assays for a given
formulation were similar (Fig. 3A
and B).
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Figure 3. . Interindividual variations in PK parameters
among assays as shown by SDI.
CsA blood concentrations are from patients receiving CsA-N
(A) or CsA-S (B). Values represent the mean of 50
patients.
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correlation of time points with bioavailability (auc)
Regression analyses of abbreviated AUC vs [CsA] at the specified
time points (trough, 1, 2, and 4 h postdose) for each assay (HPLC,
FPIA, RIA) were performed for both CsA-N and CsA-S. Two representative
analyses are depicted in Fig. 4
(A, B). The correlation coefficients are presented in Fig. 5
. For both the CsA-N and CsA-S formulations, the 4-h postdose
concentration provided the best correlation with abbreviated AUC,
regardless of the analytical assay used (r =
0.92–0.95), followed by, in order of decreasing r value:
trough = 2 h >1 h. For a given time point and analytical
method, CsA-N generally gave more favorable correlations to abbreviated
AUC than CsA-S. When the linear regression analyses were reanalyzed,
based on dose-normalized data, 71% of the r values averaged
16% less than the corresponding original r values derived
from nonnormalized data (data not shown), while no change was observed
for the remaining values.
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Figure 4. Correlation between CsA trough concentrations and AUC in
patients receiving CsA-N (A) or CsA-S (B).
Blood concentrations were determined by the FPIA method.
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Figure 5. Correlation of AUC with blood concentrations at various
time points for both CsA-N and CsA-S.
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correlation of dose with bioavailability (auc)
Correlations of abbreviated AUC vs CsA doses were performed with
data from the three assays for both CsA-N and CsA-S (Fig. 6
). Regardless of assay, correlations of AUC with CsA-N dose were
significantly (P 0.01) greater than those for CsA-S dose.
There was negligible difference in the correlations between assays for
both CsA-S and CsA-N.
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Figure 6. Correlation between CsA dose and AUC in patients receiving
CsA-N or CsA-S as determined by FPIA, RIA, or HPLC.
Results represent a mean of 50.
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Discussion
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The current formulation of CsA, CsA-S, exhibits high intra- and
interindividual variations in absorption, distribution, metabolism, and
elimination, which complicates its use
(1)(15). A new microemulsion formulation of
CsA, CsA-N, has shown increased bioavailability (4) and a
less variable PK profile (5). This more consistent
absorption profile has led to an improved correlation between the
trough concentrations and AUC as compared with CsA-S (6).
This is important since the systemic exposure to CsA in patients is
typically predicted on the basis of the trough CsA blood concentration
(16)(17). The increase in drug exposure or
bioavailability observed with CsA-N may also lead to improved
immunosuppression with CsA, since there is good correlation between
higher CsA AUC and freedom from rejection in the critical
posttransplant period (7).
The present study confirms previous findings that CsA-N exhibits an
increased bioavailability over that found with CsA-S
(6)(18)(19). The abbreviated AUCs
with CsA-N were significantly higher (P <0.01) than with
CsA-S, both with and without dose normalization of the data. The dose
normalization was not expected to have a great effect on the PK
parameters, since CsA follows first-order kinetics and the CsA
clearance and Vd are independent of dosage
(1). Also consistent with previous findings is the
significant increase in the Cmax with CsA-N and
no significant difference in trough concentrations between the two
formulations
(5)(6)(18)(19). Also,
the concentrations of CsA were higher with CsA-N than with CsA-S at 1
and 2 h postdose, but after 4 h the concentrations were
similar. The interindividual variations in the PK parameters in this
study were less with CsA-N than with CsA-S. This allows for better
predictability of PK profiles and thus makes blood concentration
monitoring of more value when CsA-N is prescribed. The study design did
not allow evaluation of the diurnal variation of the PK of CsA with the
various formulations. All trough specimens were collected in the
morning, with the PK studies being performed during the afternoon.
In the present study, better correlations of blood concentrations with
AUC at most time points after administration of CsA-N compared with
CsA-S demonstrates the enhanced clinical usefulness of TDM for the
former formulation. Furthermore, we found that the blood concentrations
best correlated to abbreviated AUC at 4 h postdose for both CsA-N
and CsA-S. The 4-h time point may therefore provide a more accurate
prediction of total drug exposure; however, it may not be as practical
or realistic to obtain as a trough concentration in the clinical
situation (20). TDM of CsA was further justified by poor
correlations (P <0.50) between CsA dose and abbreviated AUC
for both formulations.
For both formulations of CsA, there were no significant differences in
the observed blood concentrations or PK parameters obtained from either
the FPIA or RIA methods. This finding has previously been reported by
other investigators (21). Also, in good agreement with
previous reports, values obtained with the HPLC method were lower than
those obtained by either FPIA or RIA methods (22).
The discrepancy between the immunoassays and the HPLC method is due to
the specificity of the HPLC method for the parent drug. Both the FPIA
and RIA methods exhibit some cross-reactivity with numerous CsA
metabolites (9)(10)(11)(12). However, these two assays do not
exhibit the same cross-reactivity patterns, which has led to some
concern over the interpretation of the results obtained by the
immunoassay procedures. There is some suggestion that this
cross-reactivity with metabolites could result in misleading and
variable estimates of parent drug because of the interindividual
variation in rates of metabolite formation and clearance
(23).
The primary focus of the present study was to examine the effect that
assay methodology has on variability of CsA blood concentrations and
calculated PK parameters. However, we found that the interindividual
variability in the blood concentrations and the PK parameters was
comparable between all three methods. This suggests that the
metabolites do not have a strong influence on the interindividual
variability observed in patients receiving CsA and, in turn, the
cross-reactivity seen with both the FPIA and RIA methods does not
negate the validity of the results obtained by these assays.
One should take into account, however, that all the results reported
here are from renal transplant patients. For heart and liver transplant
patients, an increase is seen in the variability in absorption and
clearance, which leads to an increase in variability in metabolite
concentrations (24). In these situations, assays such as
FPIA and RIA, which cross-react with CsA metabolites, may show greater
variability in CsA concentrations and PK parameters as compared with
the more parent-drug-specific HPLC method.
In conclusion, the data presented here suggest that the new
formulation of CsA, CsA-N, offers PK advantages over CsA-S. Variations
in CsA concentration and PK parameters observed for CsA-N and CsA-S are
equivalent among the assays studied, confirming the clinical validity
of immunoassay procedures for the routine TDM of CsA in the clinical
setting.
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Acknowledgments
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This work was supported by a grant from Sandoz Canada.
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Footnotes
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1 Nonstandard abbreviations: TDM, therapeutic drug monitoring; CsA-S, -N, cyclosporine Sandimmune, Neoral; PK, pharmacokinetics; AUC, area under the curve; FPIA, fluorescence polarization immunoassay; and SDI, standard deviation intervals. 
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Abstract
Introduction
