Chronopharmacokinetics of cyclosporine A following a single i.v. dose in the Wistar rat

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ELSEVIER European Journal of Pharmaceutical Sciences 3 (1995) 49-56

PHARMACEUTICAL SCIENCES

Chronopharmacokinetics of cyclosporine A following a single i.v.

dose in the Wistar rat

M.F. Malmary*, I. Houti, C. Labat, A. Batalla, S. Moussamih, D. Bouguettaya, J. Oustrin, G. Houin

Laboratoire Cin~tique des X~nobiotiques, Facuft~ des Sciences Pharmaceutiques, Universit~ Paul Sabatier, 35 chemin des Marafhers, F31062 Toulouse Cedex, France

Received 22 March 1994; accepted 21 October 1994

Abstract

The chronopharmacokinetics of cyclosporine A (CsA) injected at one of four circadian stages, 02, 08, 14 or 20 H A L O (hours after light on), were studied in Wistar rats (i.v., 5 mg kg-1). Plasma samples were analyzed for CsA using a specific H P L C method. The kinetic profiles were best described by a three-compartment open model. A two-way A N O V A of the data showed significant dosing time variations. The terminal half-life (22.5 - 2 h) was dosing time-independent. The mean half-lives of the two distributive phases were 13 --- 3 min and 4 --. 1.5 h, respectively. Small but significant secondary peaks were noticed during the late distributive phase. There were substantial time-dependent differences in area under the concentration-time curve A U C : 35275---1185 vs 29087---752 /~g 1-1 h -1 and clearance CL: 0.142-+0.005 vs 0.172-+0.004 1 h -1 kg -1, at 08 and 20 H A L O , respectively. These data indicate an apparent circadian influence on CsA pharmacokinetics after i.v. dosing, drug exposure being greater during the resting span.

Keywords: Cyclosporine A; Chronopharmacokinetics; Rat; HPLC; Area under the curve; Compartment-dependent or independent approaches

1 . I n t r o d u c t i o n

Cyclosporine A (CsA), a cyclic polypeptide of fungal origin, is a potent immunosuppressive agent used in organ transplantation (Wolf et al., 1990) and autoimmune diseases (Feutren and Bach, 1987). The concentrations of CsA in blood

- - or plasma - - of the patients must be closely

monitored because of the relatively narrow therapeutic range of this drug (Mihatsch e t al., 1989).

Overdosage causes renal toxicity but these toxic effects do not correlate well with the dose administered, and the pharmacokinetics display extensive inter- and intrapatient variations (Freeman, 1991). It has been reported that one of the causes of such variability is circadian influence (Bowers et al., 1986). Chronokinetics are defined as dosing time-dependent and pre- dictable changes in parameters such as Cmax, tma x, A U C and half-life (Reinberg, 1992). Previously,

* Corresponding author. Tel. ( + 33) 61 25 21 40; Fax ( + 33) 62 26 26 33.

we have developed experiments to investigate the chronopharmacokinetics of plasma CsA in the male Wistar rat, after a single oral dose (40 mg kg-1): the drug was monitored using radioimmunoassay (Malmary et al., 1992). Several methods are available to measure CsA in biological fluids. Routinely, high performance liquid chromatography, radioimmunoassay and fluorescence polarization immunoassay are used (Blick et al., 1990). The HPLC assay offers specificity, thus permitting the determination of parent compound levels, largely supposed to be the main active compound (Mihatsch et al., 1989). We were confronted with analytical difficulties in measuring CsA concentrations in blood, plasma and tissues of the rat. The first purpose of the work reported here was to validate a sensitive, precise and accurate HPLC method which is suitable for such a study. This method was then applied to a chronokinetic study of plasma data following intravenous injection (5 mg kg -1) at one of four equally-spaced circadian dosing stages. The aim of this study was to investigate

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circadian changes in concentration-time profiles and pharmacokinetic parameters.

2. Experimental procedures

Chemicals. Cyclosporine A and cyclosporine D (powders) used as an internal standard were kindly supplied by Sandoz France. All chemicals were of analytical reagent grade, all solvents were of HPLC grade. Aqueous solutions were prepared using Milli-Q water.

Experimental design. Two-hundred male Wistar rats (CREJ, France) were maintained for at least 3 weeks before experimentation, in cages ar- ranged in a rack, with 12 h light (L) alternating with 12 h dark (D), the L onset being at 07.00 h local time. The illumination level across all cages was about 280 lux. Water and food were supplied freely, The animals were randomly divided into f o u r groups differing by the temporal dosing stages, referred to as time elapsed since the light onset, i.e. "hours after light on: H A L O " (02, 08, 14 and 20 H A L O ) . The i.v. administration of CsA (Sandimmun solution, ~ 50 mg m1-1 diluted with 0.9% saline NaC1, for a final concentration of 1.25 mg m1-1) was performed by rapid injection (dose ( D o ) = 5 mg kg -1) via the tail vein of the rats (250--+ 20 g). Such an i.v. dose given to rats led to blood levels roughly comparable to those obtained after a 20 mg kg -1 o r a l dose, which was used in a bioavailability study (un- published results). Blood samples were obtained by cardiac puncture on animals slightly anaesthet- ized with ether at 0.25, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 10,: 12, 16, 20, 24, 48 and 72 h after drug administration. Each rat was punctured only once or twice at an interval of at least 6 h. T h e samples were collected into vials containing a solution of E D T A (50/xl), vortex.mixed and the plasma was separated by centrifugation at 37°C and frozen until analyzed.

HPLC conditions. The HPLC system consisted of an automatic sampler (Spectra Physics SP 8100) with a 50 /zl loop injection, an isocratic pump (SP 8800), a variable U V detector (SP 100) and a PC-controlled integrator (Chrom-Jet) for data acquisition and storage. The C8 Supelco (250 m m x 4 . 6 mm ID, 5 /zm particle size) column was maintained at a temperature of 70°C with a column heater (Kontron Instrument). The

mobile phase was acetonitrile:methanol:water (50:33:17, v/v/v). The mixture was filtered and degassed. The flow rate was 1.3 ml min -1. The detection wavelength was 210 nm.

Sample preparation. Frozen samples were re- equilibrated to room temperature by incubating for 30 min in a 30°C water bath. When possible, two 1-ml aliquots were taken and submitted to the extraction procedure. Internal standard, cyclosporin D (50 /zl of 10 mg 1-1 solution in methanol) and double-distilled water (1 ml) were added to a 1-ml sample of plasma. This mixture was vortexed for 1 min, then sodium hydroxide (2 ml of 1 mol 1 -~ solution) and diethylether (5 ml) were added. After vortexing for 1 min and centrifugation at 1000g for 8 min, the ether layer was decanted and diethylether (5 ml) was added again to the pellet. Following a second vortexing and centrifugation for 8 min, the ether layer was transferred together with the first one and hydro- chloric acid was added (1 ml of 1 mol 1-1 solution). After vortexing and centrifugation, the total ether layer was transferred into a clean tube and evaporated to dryness under nitrogen at 40°C. The residue was redissolved in 2 ml of n-hexane and added to 200/xl of mobile phase, by vortexing for 1 min. The sample was finally centrifuged (8 min, 1000g) and 130 /zl of the lower mobile phase layer transferred into the inserts. The tubes were then tightly capped and 50-/zl aliquots were injected onto the HPLC column for analysis.

Calibration graphs. CsA (in solution in methanol 10 mg 1-1) was added to drug-free pooled plasma to provide concentrations of 50, 100, 200, 400, 750, 1000, 2000 and 5000/~g 1-1, in the presence of internal standard CsD (500 /xg 1-~). Each plasma standard was then taken through the sample preparation procedure described above.

Quantitation was done by determination of peak- area ratio of CsA/CsD against the drug concentrations. The concentrations of unknown samples were determined by using the linear regression line (unweighted) of the concentration of the calibration standard versus peak-area ratios.

Statistical evaluation. Linear regression, analysis of covariance (ANCOVA), two-way analysis of variance (ANOVA), nonlinear regression analysis and multiple comparison of the parameters were used. The assumptions underlying these statistical

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analyses (normality and homoscedasticity) were checked up. A probability level of a < 0.05 was considered significant.

Pharmacokinetic study. A three-compartment open model was fitted to the plasma concentration data. Parameters were estimated assum- ing linear pharmacokinetics and elimination oc- curring from the central compartment only. The plasma concentration-time curves were described by the following equation:

3

C(t) = ~ A i exp(-Ait )

I

Half-lives t~/2, area under the adjusted plasma c o n c e n t r a t i o n - t i m e graph A U C a, clearance CL, and apparent volume of distribution V a were calculated by

Perier, 1982).

A U C A =

standard formulae (Gibaldi and

o e

f C(t) dt = l~i

o

+ 7

1

^{CsA CsD}

On the basis of non-compartmental methods, statistical moments were also determined. The zero-order statistical m o m e n t (AUCT) and the area under the first m o m e n t curve (AUMCT) were calculated using the trapezoidal rule. Values were determined from zero time to infinity.

f-1 ( C i .q_ Ci+l )

A U C T = E i=0 2 (ti+l - ti) + "~3 (f: last point) Ce

The m e a n residence time M R T was derived from the quotient A U M C r / A U C T , the clearance C L = D o / A U C T and the apparent volume of distribution at stead~,-state Vss = C L . M R T were determined.

3. R e s u l t s a n d d i s c u s s i o n

Analytical variables

Chromatography. Fig. 1 represents chromatograms of extracts from plasma (a) spiked with CsA (1000 /xg 1-1) and (b) sample from a rat (CsA 5 mg kg-1). Cyclosporins A and D elute at 24 --. 1 and 32 --- 2 min, respectively. No interfer- ing substances have been encountered. The limit

a b

Fig. 1. Chromatograms of extracts from plasma (a) spiked with CsA (1000 ~g 1 -~) and (b) sample (336 /lg 1-1 ) from a rat (CsA 5 mg/kg) [internal standard CsD (500 ~g 1-~)].

of detection defined as signal-to-noise ratio (3:1) was 25 /z g 1-1.

Linearity. The standard curve was linear to 5000 /~g 1-1 (R 2 = 0.98). Linear regression analysis of the relative peak area (y) vs. cyclosporine concentration (x) describes the line y = a + bx with CL95 % (a) = - 0.100-+ 0.147 and CL95 % (b) = 0.00342 ___ 0.00037 (/zg -t I).

Repeatability, precision and accuracy. T h e between-day variation was assessed by analyzing three standard curves obtained on three non- consecutive days. The parallelism of slopes of y on x and the homogeneity of the y-intercepts was demonstrated by the A N C O V A . The precision of the method was expressed as the within-day and between-day coefficient of variation ( C V = 1 2 - 7%). The accuracy was shown as the m e a n CV of all concentrations from the theoretical value ( C V = 14.7 _+ 8.6%).

Efficiency o f extraction procedure. CsA and CsD were added to methanol to provide the same

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concentrations as for the calibration graph and the relative peak areas were determined. The recovery of CsA after extraction in supplemented plasma (500/zg 1-1) was about 75%.

Analysis of temporal dosing-stage influence on plasma CsA concentrations

A two-way A N O V A of the plasma concentration data was designed to study the effects of the two fixed factors: the temporal dosing-stage (02, 08, 14 and 20 H A L O ) and the sampling time (t = 0.25 to 72 h). Highly significant differences (a < 0.0001) in the pharmacokinetics of cyclosporine A when administered at different times of the day were demonstrated. The presence of a significant interaction (a < 0 . 0 2 5 ) between the two fixed factors clearly indicated that the effect of sampling time on plasma concentrations de- pended on dosing time. This could be due to different dosing time-dependent effects on the distributive and postdistributive phases of the kinetics.

A n o t h e r point arising from the A N O V A is concerned with the nested design of the within- subgroups error, divided into "rats" and "dupli- cate" sources of variation. There was evidence of a significant component among rats (a < 0.0001).

The components of both "among rats" and

" a m o n g duplicates" expressed as percentages of the unexplained variance were about 70% and 30%, respectively. Therefore, the interindividual variation exceeded the experimental error which was mainly due to the extraction procedure.

Kinetic study

The plasma concentration-time profiles of the four different dosing-stage kinetics were described by a triexponential disposition model (Fig. 2). Some discrepancies between the observed [C(t)] and expected [(~(t)] concentrations were apparent during the first 10 h after dosing.

The differences d(t) = C(t) - ~(t) were submitted to statistical analysis to determine the effects of the two fixed factors: sampling time t (from 2 to 10 h after dosing) and dosing time. In this case, there is not evidence for a significant effect of the time of administration, whereas the effects of sampling time were significant (a < 0.0001).

M e a n values A m ( ± s . e . ) determined from the pooled d values from the four kinetics are presented in Fig. 3 as a function of t. The observed fluctuations, reaching about 10% of the plasma concentration level, can be described as damped

oscillations with a period of about 3 h. W h e t h e r an experimental bias leads to these discrepancies must be considered: this eventuality seems doubt- ful, since the rats (i) were randomly sampled into the different groups or subgroups, and (ii) were never punctured twice during the first 10 h after dosing. The presence of these secondary peaks could be related to a reabsorption process.

Biliary excretion of CsA and its metabolites is the major route of drug elimination. Substances in bile can be reabsorbed and recirculated. Roman- ski and Dabrowski (1989) reported an ultradian pattern of bile flow in the rat. In this study, spectral analysis was used to detect oscillations in the bile flow rate. A significant periodic component of bile flow (2 -+ 0.5 h period) was found.

The observed periodic oscillations of plasma CsA levels may perhaps reflect this ultradian pattern of bile flow. Future studies should accurately quantitate each of the two processes in our own experimental conditions.

The results of the pharmacokinetic compartmental analyses are summarized in Table 1. The statistical analysis (2-way A N O V A and AN- COVA) of the terminal portion of the kinetics - - i.e. from 20 h to 72 h after dosing - - showed that the temporal dosing-stage effects on plasma levels were significant (a <0.001) and that no interaction was present between sampling and dosing times. However, there was no hetero- geneity between the four log-linear regression slopes so it can be inferred that the post-distributive kinetics were parallel but not confounded.

The mean terminal half-life was about 22.5 ± 2 h.

The data concerned with the distributive portions could not be submitted to such an analysis. The mean half-lives for the initial and subsequent distributive phases were about 13---3 min and 4 _ 1.5 h, respectively. These estimations are in good agreement with previously reported values derived from experiments using tritiated cyclosporine (Wagner et al., 1987). These kinetic findings indicate that cyclosporine is rapidly dis- tributed into extravascular tissues. The apparent first-order elimination rate constant k~o (0.22 + 0.03 h -1, C V = 4%) does not exhibit important variations in relation to dosing time. The intercompartmental rate constants kl~ (0.19--+0.07 h -1 C V = 11.6%) and k31 (0.08 ± 0.02 h -1 CV = 7.8%) and furthermore k~2 ( 1 . 6 8 ± 0 . 9 6 h -~

C V = 1 8 % ) and k2~ ( 1 . 0 ± 0 . 0 5 h -~ C V = 1 5 % ) are more dependent on the temporal dosing time.

A n examination of the evolution of the ratio

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~O00G

10000.

t000

t00

10

CsA (gg/I)

14 HALO t (h)

0 10000

24 48 72

tO00

100

10 20 HALO t (h)

~ CsA (p.g/1)

tO00

t00

10

02 HALO t (h)

24 a8 72

C ~ (~.@) iO000

tO00

t00

08 H A L O t (h)

0 24 48 72 0 24 48 72

Fig. 2. Plasma c o n c e n t r a t i o n - t i m e profiles of cyclosporine A (mean - s.e., n = 4 to 6) following four different dosing times (02 and 08 H A L O , resting span; 14 and 20 H A L O , active span).

300 ' /~m (/J'g 1"I )

2 0 0 •

,oo I

t ( h i o

-100

Fig. 3. Time variations ( f r o m t = 2 to 10 h) of the differences d(t) between observed C(t) and expected &d'(t) plasma concentration levels (mean -- s.e., n = 16 to 20).

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Table 1

Pharmacokinetic parameters from the four different dosing- time profiles

02 H A L O 08 H A L O 14 H A L O 20 H A L O

1 / 2

t 2 (h) 0.18 0.24 0.22 0.25

t2 ~'2 (h) 3.65 -5.46 2.95 3.71

t~/2 (h) 18.2-+ 3.4 24.8-+ 4.4 21.0-+ 6.4 21.0-+ 2.0

klo (h - l ) 0.22 0.20 0.22 0.24

k12 (h -1) 2.27 1.91 1.68 0,85

k21 (h -1) 1.26 0.72 1.23 0.70

k13 (h -1) 0.18 0.15 0.25 0.17

k31 (h -1) 0.10 0.07 . 0.09 0.07

Half-lives of distributive (1 and 2) and elimination (3) (t~/2+ - C195%) phases. A p p a r e n t first-order elimination rate constant klo and intercompartmental rate constants (k~2 to k3~ ) from an open three-compartment model (1: central compartment).

k~3/k3~ and k~2/k2~ as a function of the dosing time (Fig. 4) shows that exchanges between the central (1) and the peripheral (2 and 3) compartments seem to be dosing-stage-dependent. The drug is more strongly retained in compartment 2 when administered in the middle of the light

0 2 HALO 0 8 HALO 14 HALO 2 0 HALO

Fig. 4. Variations of the intercompartmental rate-constants ratio k~3/ka~ and kt2/k2~ as a function of dosing time (expressed as hours after light on, HALO). Black and white zones (horizontal axis) correspond to dark (activity) and light (rest) spans, respectively.

period (08 H A L O ) and in compartment 3 when administered during the dark period (14 and 20 H A L O ) . This could be interesting in view of the known dosing time-dependent toxic effects of the drug. In a recent study carried out in the Wistar rat (CsA, oral dose 20 mg kg -~ per day for 21 days), we found that renal constriction, assessed by rises in creatinine and B U N levels and the decrease in creatinine clearance, was maximal when the drug was administered at 07 or 19 H A L O . The tubular injury, demonstrated by the increase of G G T urinary excretion, seemed to be more serious after 19-HALO than 0 7 - H A L O dosing (Batalla et al., 1994). Thus the temporal changes in CsA-induced renal toxicity might result in part from chronokinetic changes, not only due to the known temporal variations in absorption processes (Malmary et al., 1992).

Cyclosporine concentration-time data were analyzed using the theory of statistical moments, to determine area under the curve (AUCa-), mean residence time (MRT), total clearance (CL) and apparent steady state volume of distribution (Vss). The results are presented in Table 2, together with values derived from the compartmental approach, i.e. via analytical ways ( A U C A and Va). The analytically determined area A U C A underestimates the more realistic values A U C x obtained through the trapezoidal rule. These discrepancies - - about 6.4% (20 H A L O ) to 11.6% (08 H A L O ) - - are partially explained by the fact that the small secondary peaks were not taken into account when the three-compartment model was established. Whatever the method of calculation, the higher A U C was obtained when the drug was administered in the middle of the resting period (08 H A L O ) and the lower A U C corresponds to the 20-HALO dosing. According- ly, the total clearance CL varied as a function of the temporal dosing-stage from 0.142---0.003 1 h -1 kg -1 (08 H A L O ) to 0.172 - 0.004 1 h -1 kg -1

Table 2

Pharmacokinetic parameters from the four different dosing-time profiles

02 H A L O 08 H A L O 14 H A L O 20 H A L O

A U C A (/xg 1- h -1) 30 009 34 929 28 762 22 850

V~ (1 kg - I ) 4.1 5.1 4.6 5.2

A U C T (/xg 1 -~ h -1) 32 045 __- 684 35 275 - 1185 32 737 -+ 902 29 087 - 752

M R T (h) 22.3 29.5 23.8 21.0

CL (1 h -~ kg -~) 0.156 +-- 0.003 0.142 -+ 0.005 0.153 --- 0.004 0.172 +-- 0.004

V~s (1 kg -1) 3.5 4.2 3.6 3.6

Analytical determinations: A U C A and apparent volume of distribution V a. Non-compartmental determination: AUCT(+-S.E.), mean residence time M R T , total clearance CL(---S.E.) and apparent steady-state volume of distribution Vss.

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(20 H A L O ) . The mean residence time MRT lay between 21 h (20 H A L O ) and 29.5 h (08 H A L O ) . The apparent volume of distribution was about V a = 4 . 8 1 kg -1 or Vss=3.8 1 kg -1 depending on the method of calculation. Com- parable values were early reported (Sangalli et al., 1988). The daytime to nighttime differences in the blood level of CsA following oral dosing were studied in recipients of pancreas allografts (Canafax et al., 1988). The authors reported substantial dosing time-dependence in drug exposure to CsA; the A U C and the MRT were greater during the resting cycle than the activity cycle of the patients. The same group of authors (Bowers et al., 1986) noticed contradictory re- suits in the same category of patients, receiving two equal - - unspecified - - doses of CsA. Morn- ing through levels, corresponding to the dose given during the active period, were lower than those for the resting period. In rats injected intraperitonally with a toxic dose of 60 m g / k g of CsA the mean blood levels seemed to be greater

- - standard deviations were not given - - when

the drug was administered during the active period (Bowers et al., 1986). In previous experiments, non-specific R I A assay was used to quan- tify plasma CsA in Wistar rats after oral dosing (40 mg/kg) (Malmary et al., 1992). The lowest A U C corresponded, in those conditions, to a 08 H A L O administration, the highest A U C corres- p o n d e d to 20 and 02 H A L O administration. The differences from the present experiment, where much lower CsA doses were administered, could be explained by specific dosing time-dependent variations in absorption a n d / o r metabolism of the drug. When large doses of CsA are given, the metabolic enzymes are saturated and this can mask any real difference.

The circadian variations in the i.v. kinetics could be due to parallel changes in the physiolog- ical functions and variables involved in the distribution, metabolism and excretion of the drug.

The factors influencing drug distribution in a specified species are blood flow to various organs, binding of the drug to plasma proteins and membrane permeability to the drug. Studies carried out in rats indicated that blood flow to the kidneys, the liver and the intestine was maximal during the activity period and minimal when the animals were asleep (Labrecque et al., 1988).

This fact could partially explain the observed variations of the exchanges between the central and the third compartments: the ratio k ~ 3 / k 3 ~ was

maximal when the drug was administered during the night. Temporal variations in the binding of some drugs to plasma proteins were described in rodents. The lowest free drug levels occurred during the night, when the albumin concentration was high (Bruguerolle, 1987). The time-dependent variations in drug binding to plasma proteins are likely to have important consequences for drugs with high protein binding. It was estimated that 30-40% of the systemic content of CsA is found in the plasma and that almost all of this was bound to the proteins (Lemaire and Tille- ment, 1982). The time dependency of drug binding to erythrocytes has only been reported in but a few cases (Bruguerolle, 1992). Further studies are n e e d e d to assess temporal changes in the passage of CsA through biological membranes, using red blood cells as a model. Cyclosporine A is extensively metabolized in the liver. These biotransformations depend on the enzymatic activity of the hepatocytes which undergoes temporal changes too (Radzialowsky and Bousquet, 1968).

In conclusion, the present study has shown that there are significant differences in the pharmacokinetics of cyclosporine A when i.v.-administered at different times of the day (to our knowledge, previous chronokinetic studies were essentially devoted to oral dosing). These modi- fications specifically concern the distribution phases of the drug since the terminal half-life is dosing time-independent. Dosing time-dependent variations of the intercompartmental rate constants could explain, at least in part, similar variations in the toxic effects of the drug, already mostly dependent on fluctuations in the amount of absorption when cyclosporine is administered orally. The measurement of A U C was introduced as an alternative to trough level monitoring of CsA therapy (Grevel et al., 1990). Time-sensitive therapeutic dosing regimens based on individual patient a.m.-to-p.m, differences in A U C a n d / o r MRT were proposed in pancreas transplant patients (Cipolle et al., 1988). Moreover, as chronokinetic changes induce some non-linearity into the pharmacokinetic concepts (Hecquet, 1990), A U C appears to be the best candidate for chronopharmacological evaluation.

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