DRUG DISPOSITION

~in. Pharmacokinel. 24 (6): 472-495, 1993 0312-5963/ 93/0006-0472/$12.00/0 © Adis International Limited. All rights reserved.

CPK1320

Cyclosporin Clinical Pharmacokinetics Alfred Fahr Sandoz Pharma Ltd, Basel, Switzerland

Contents 473 474 474 475

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Summary I. Analytical Methods 1. 1 High Performance Liquid Chromatography 1.2 Radioimmunoassay 1.3 Cyclosporin Monitoring 2, Absorption of Cyclosporin 2.1 General Principles 2.2 Influence of Bile 2.3 Food Interaction 2.4 Other Effects 2.5 Bioavailability 3. Metabolism 3.1 Amount and Number of Metabolites 3.2 Metabolites in Patient Groups 4. Distribution 4. 1 Cyclophilin : A Special Protein 4.2 Distribution of Cyclosporin in Blood 4.3 Distribution of Cyclosporin in the Body 4.4 Pharmacokinetic Models 4.5 Distribution and Pharmacokinetics of Metabolites 5. Elimination 5. 1 Variability of Data 5,2 Factors Influencing the Elimination of Cyclosporin 5.3 Clearance Depends on Age 5.4 Other Factors 6. Pharmacokinetic-Therapeutic Relationships 6. 1 Pharmacokinetics and Pharmacodynamics 6.2 Pharmacokinetics and Renal Adverse Events 6.3 Do Metabolites Cause Renal Side Effects? 7. Dosage and Administration 7.1 Administration 7,2 Dosage 8. Clinical Implications 8. 1 Activity of Metabolites 8.2 Future Research 8.3 Place of Cyclosporin in Therapy

Cyclosporin Clinical Pharmacokinetics

Summary

473

Cyclosporin is a powerful immunosuppressive drug used in transplantation medicine and to treat autoimmune diseases. It is a lipophilic molecule, with its bioavailability dependent on food, bile and other interacting factors. Cyclosporin is extensively metabolised in the liver by the cytochrome P450 3A system, which is subject to considerable interindividual variation. Distribution of cyclosporin depends not only on physicochemical characteristics, but also on biological carriers such as lipoproteins and erythrocytes in blood. Cyclophilin, a binding protein for cyclosporin, influences distribution of cyclosporin in the body. Despite its lipophilicity, cyclosporin does not appear in the brain. The distribution of metabolites in the body can differ from that of cyclosporin itself. Elimination of the drug is mainly via the bile as metabolites, other routes not being very important. Pharmacokinetic parameters of cyclosporin are highly variable and depend on factors such as age, the physical condition of the patient, type of organ transplant or comedication. Renal side effects of cyclosporin are dose-related, but the influence of the dosage regimen has not been thoroughly investigated. An important factor in the reported variability is the different analytical methods used. Following the recommendations of recent consensus documents to monitor blood concentrations, this source of variability may diminish in the future. Several metabolites are reported as having less immunosuppressive activity than the parent drug. Metabolites with renal side effects have been reported. These and other effects of metabolites have not been clearly defined in the literature, presumably because of the highly variable activity of cyclosporin-metabolising liver enzymes and the paucity of data available on metabolite pharmacokinetics. The therapeutic range and dosage of cyclosporin are therefore highly dependent on many individual parameters in patients. Dosages of less than 5 mg/kg/day, however, rarely cause renal side effects. Further studies to correlate the clinical pharmacokinetics of metabolites with their activity and adverse effects are needed.

Cyclosporin (CyA, Sandimmun®), a cyclic endecapeptide (fig. 1) produced by the fungus Tolypocladium injlatum Cams, is a powerful immunosuppressant drug (Borel et al. 1976). In human medicine it is widely used in transplantation to prevent rejection of the transplanted organ and for the treatment of autoimmune disorders (see Bach 1989). Cyclosporin selectively inhibits the interleukin2 (IL-2) driven proliferation of activated Tlymphocytes. By impairing IL-2 production by Thelper cells, the drug suppresses proliferation and generation of T-cytotoxic lymphocytes, while sparing T-suppressor cell subpopulations. This feature distinguishes cyclosporin from traditional immunosuppressive drugs, which often suppress the complete immune response. The molecular mechanism of action of cyclosporin, however, is still unclear (Schumacher & Nordheim 1992). The discovery that cyclosporin

apparently binds to and inhibits the peptidyl-prolyl cis-trans isomerase (e.g. Fischer et aI. 1989) and that this cyclosporin-isomerase complex can further inhibit other key enzymes (Liu et al. 1992) promises some leads, which may answer basic questions such as the sequence of lymphocyte activation or the significance of such isomerases in signal transduction. Cyclosporin also binds to P-glycoproteins and reduces multidrug resistance in malignant cancer cell models by a mechanism involving this binding (Foxwell et al. 1989). Originally suspected of being an antibiotic substance, cyclosporin has, in fact, some fungicidal and antiparasitic effects (Bolas-Fernandez et al. 1988). It is not clear why Tolypocladium injlatum Cams produces cyclosporin, but a teleological reason may be that mycophilic mosquito larvae die if they are fed with the drug (Weiser & Matha 1988). Cyclosporin is only one of several similar substances

474

Clin. Pharmacokinet. 24 (6) 1993

Fig. 1. Structure of cyclosporin as established by Riiegger et al. (1976) and Petcher et al. (1976).

produced by the fungus. Some of the others also exhibit immunosuppressive activity (von Wartburg & Traber 1986). Pharmacokinetic parameters for cyclosporin in humans show large ranges (table I). High variability [up to 2-fold intraindividual and more than 3fold interindividual variations in area under the plasma or blood concentration-time curve (AUC)] are present even in healthy volunteers under standardised conditions (Lindholm et al. 1988).

drug but also different metabolites (for a recent review see Rosenthaler & Keller 1990). Difficulties in using this method are related to the high molecular weight, lipophilicity and lack of a chromophore for cyclosporin. Because of the expense, HPLC is mostly reserved for investigational use and to study the metabolism of cyclosporin. The sensitivity of the test is high (20 to 40 ILg/L), but large sample volumes (about 1ml) are needed. Table I. Pharmacokinetic characteristics of cyclosporin sum-

1. Analytical Methods Comparative pharmacokinetic studies can only be done with valid data obtained by similar methods. The low concentrations of cyclosporin (10 to 500 ILg/L) observed in therapy represent a challenge in the development of the various analytical methods available (for recent reviews see Lindholm 1991 and Kivisto 1992). 1.1 High Performance Liquid Chromatography

marised by Scott and Higenbottam (1988). The range for peak plasma concentrations is about 150 to 1000 I'g/L, the cyclosporin dosage is about 5 to 12 mg/kg/day Parameter

Value

Bioavailability (%) First-pass metabolism (%) Enterohepatic recirculation Hepatic metabolism (%) Clearance (L/h/kg) Vss (L/kg)

8-60 10-27 Metabolites only 99 0.15-0.7 1.8-13.8 0.1-1.7 2.9-15.8 >90

tV,a (h) tv,~ (h)

Biliary excretion (%)

If done appropriately, high performance liquid chromatography (HPLC) is a very reliable assay method. It measures not only the amount of parent

Abbreviations: Vss = volume of distribution at steady-state;

tv,a

= absorption

phase half-life;

tv,# = elimination half-life.

475

CycIosporin Clinical Pharmacokinetics

1.2 Radioimmunoassay

In clinical monitoring, radioimmunoassay (RIA) methods are used because of their easy handling and small sample volumes needed ("" IO~I). The antiserum of the first commercial test kit (no longer available) was polyclonal and measured several metabolites besides cyclosporin. The monoclonal antibody of the RIA-kit produced by Sandoz ('Sandimmun®-Kit'; 3H-based, also containing a nonspecific monoclonal antibody for metabolites) is specific for the parent drug and agrees quite well with HPLC methods (Copeland & Yatscoff 1988). Small deviations from agreement at low concentrations « 100 ~g/L) were observed (Vine & Bowers 1988), presumably indicating cross-reaction of the antibody with metabolites. Additionally, a 125I-based RIA with the same specific monoclonal antibody is available from Incstar Corp. (Stillwater, USA). Another sensitive test method is the fluorescence polarisation immunoassay (FPIA), which was available first with a polyclonal antiserum and then with a specific monoclonal antibody. This test is described as a rapid and simple procedure (for a recent article see Wang et al. 1991). 1.3 Cyclosporin Monitoring

Measurements of cyclosporin are done in different biological fluids (blood, plasma, serum), which add another source of variability to published pharmacokinetic data. A 'physiological' reason for measuring cyclosporin in plasma was that plasma transports cyclosporin to tissues and is therefore responsible for distribution of drug in the body. Cyclosporin binds rapidly ("" 10 min) to red and white blood cells in a temperature-dependent manner. When serum or plasma is separated from blood at temperatures other than body temperature, the measured concentrations do not reflect actual values in blood. An additional argument for measuring cyclosporin in whole blood comes from the finding (Rosano et al. 1986), that the metabolite pattern of

cyclosporin in tissues resembles more the pattern in whole blood than in plasma. All this is in line with the recommendations of consensus documents, which strongly recommend cyclosporin monitoring in whole blood (Consensus Document 1990; Kahan et al. 1990). The bioanalytical method used has a direct and significant influence on the calculated pharmacokinetic parameters. If an assay does not differentiate between metabolite and parent drug, pharmacokinetic parameters based on such data will be of limited value. This was shown, for example, by Reynolds et al. (1988), who found a range of 36 to 43% for the average bioavailability and 8.4 to 13 mlfmin/kg for the mean clearance (CL) with different assay methods. The differences in pharmacokinetic parameters using specific RIA and HPLC are much smaller. Speck et al. (1989) found that, except for CL, no significant differences occurred in the pharmacokinetic parameters volume of distribution at steadystate (V ss), mean residence time or bioavailability measured in kidney transplant recipients. For these reasons and because of ongoing methodological problems encountered using even the most up-to-date assay facilities (Frey et al. 1987; Johnston & Holt 1988), this review will identify the assay methods used in the cited publications, whenever appropriate. Not only the analytical methods, but also the collection of blood samples can cause severe problems. Cyclosporin binds reversibly to silastic and silicone catheters and to some extent even to polyurethane catheters (Lorenz et al. 1991). Drawing blood samples from the same line as used for cyclosporin administration therefore leads to unreliable results.

2. Absorption of Cyclosporin 2.1 General Principles

Cyclosporin is slowly and incompletely absorbed from the gastrointestinal tract, absorption taking place predominantly from the small intestine (Drewe et al. 1992). Absorption is highly variable (even in otherwise comparable studies) and is

476

influenced by many factors, e.g. food, comedication, type of transplant or amount of bile in the gut. One reason for this variability might be the lipophilicity of cyclosporin (partition coefficient 4000) [unpublished data on file, Fahr et al.]. Therefore, the concentrate for intravenous infusion contains cyclosporin 50 gIL in Cremophor-EL® (polyoxy-ethylated castor oil base) and alcohol and is diluted 1 : 20 to 1 : 100 with normal saline or 5% glucose. Cyclosporin is administered orally in an oily solution (corn oil, polyoxyethylated corn oil and ethanol as excipients). This solution contains cyclosporin 100 gIL, which partitions into alcohol and the oil. The oily solution is dispersed in a drink (milk, fruit juice, etc.) of the patient's choice. The process of mixing cyclosporin and the drink contributes to the observed variability of cyclosporin absorption. Grevel (1988) noticed that lack of dispersion of the drug (e.g. improper mixing) is one cause of incomplete absorption. Tarr and Yalkowsky (1989) showed that a reduction of emulsion droplet size by homogenisation yields 1. 7-fold increase in cyclosporin blood concentration in rats. The kind of drink itself seems to have a minor influence on the variability (Johnston et al. 1986). Quite recently, however, Edwards et al. (1993) observed an increase (32%) in cyclosporin blood concentrations in renal transplant patients when they dispersed the drug in grapefruit juice. The investigators suggested that grapefruit juice components might cause inhibition of cyclosporin metabolism in the gut wall (see section 3). Experiments with lymphocytes and liposomes showed that diffusion is sufficient to explain how cyclosporin passes through cell membranes (LeGrue et al. 1983), supporting the general rule that lipophilic drugs can traverse cell membranes easily. Veda et al. (1983a) showed in rats that increasing the cyclosporin dose (given in an olive oil solution) from 6 to 22 mglkg did not decrease bioavailability, which can be explained by a nonsaturable diffusional absorption process. This group showed, also in rats, that little of an oral cyclosporin dose appears in the lymph.

Clin. Pharmacokinet. 24 (6) 1993

Absorption of cyclosporin in humans is highly variable. Cyclosporin appears in the blood after 0 to 0.9h, the absorption half-life (t'ha) ranging from 0.5 to 2h (Lindberg et al. 1986), measured in whole blood and plasma by HPLC at body temperature. This is in line with findings of Grevel et al. (1986), who calculated an absorption window of about 2.8h. A study in which blood samples were taken every hour for 12h and assayed by HPLC in whole blood (Phillips et al. 1988) revealed a variety of possible absorption profiles, including biphasic behaviour or slow absorption profiles which cause cyclosporin concentrations to rise for up to 12h. 2.2 Influence of Bile The influence of bile on cyclosporin absorption was shown in a study using a whole blood HPLC assay (Mehta et al. 1988), where bile flow in patients with orthotopic liver transplantation was regulated by a T-tube. The bioavailability after T-tube clamping - allowing the bile to flow into the gut showed a considerable increase of AVC in comparison with that with an open T-tube (increase of AVCo_ oo for 8 patients was in the range of 36 to 1561% with a median of 192%). This increase is not related to enterohepatic recycling of cyclosporin, because only 1% of the drug administered is excreted in the bile (and less than 6% into urine). Bile seems to serve as an emulsifier, which allows cyclosporin to be transferred from the lipophilic formulation into a more hydrophilic milieu allowing better absorption. Lindholm et al. (1990) showed in healthy volunteers a slight increase (1.25-fold, p < 0.05) in cyclosporin absorption, when 500mg of bile acids and a light breakfast were given (compared with the fasted state). The assay was by HPLC in whole blood. Difficulties in bile duct reconstruction in liver recipients may hinder the free flow of bile and can diminish the absorption of cyclosporin (Busuttil et al. 1986). 2.3 Food Interaction The influence offood on cyclosporin absorption is not clearly defined. All 3 possibilities have been reported: food diminishes absorption (Keown et al.

Cyclosporin Clinical Pharmacokinetics

1982}, has no significant effect (Keown et al. 1983) and enhances absorption of cyclosporin (Ptachcinski et al. 1985). Comparisons between these studies are not easy. A major influence of the variable absorption may be food composition (e.g. fat, lipids) or the physiological status of the gastrointestinal tract (e.g. bile production, motility). In a recent publication, Gupta et al. (1990) described the influence of a 'fat-loaded breakfast' on bioavailability in healthy volunteers. When they were fasting, the bioavailability of cyclosporin 10 mg/kg was 21 %; when the drug was administered with fatty food, the bioavailability increased to 79%. Food may also playa role in the appearance of a second serum cyclosporin peak (measured by HPLC at body temperature) after oral administration followed by a standardised meal (Lindholm et al. 1988). The investigators explain this peak by the outflow of bile after the meal thereby enhancing absorption (especially after the usual fasting conditions in studies in volunteers). 2.4 Other Effects Drugs influencing gastric and intestinal motility may change the absorption of cyclosporin. Metoclopramide for example, which increases gastric emptying, enhances the cyclosporin absorption by about 30% (measured by HPLC in whole blood) [Wadhwa et al. 1987]. Drug interactions affecting cyclosporin absorption have been discussed in several studies. Erythromycin (Gupta et al. 1988), is thought to increase absorption; phenytoin (Rowland & Gupta 1987) to decrease cyclosporin absorption. Another explanation is inhibition of cyclosporin metabolism by erythromycin (Kessler et al. 1986) or the induction of metabolism by phenytoin (Freeman et al. 1984). Dysfunction and severe disease of the gastrointestinal tract diminish the absorption of cyclosporin. Atkinson et al. (l984), using a polyclonal RIA assay in serum, found that the amount of absorbed cyclosporin decreases during diarrhoea to less than half of normal values. The tlha for cyclosporin 600mg ranges from 0.6

477

to 1.8h in patients (HPLC assay in plasma and serum at less than body temperature) [Beveridge et al. 1981] and from 0.4 to 4h in volunteers receiving 300mg cyclosporin (polyclonal RIA and HPLC assay of whole blood) [Grevel et al. 1986]. The peak concentration (C max ) of cyclosporin is observed 3 to 4h after administration (Beveridge et al. 1981). 2.5 Bioavailability In one of the first published pharmacokinetic studies on cyclosporin in transplant patients (Beveridge et al. 1981), using HPLC assay of plasma and serum at <37°C, the absolute bioavailability of cyclosporin was estimated at 30% (range 10 to 60%). This and larger ranges are observed in various populations of transplant patients. Mean values of bioavailability data collected from different patient groups and analysed with different methods in the literature (for a survey see Grevel 1986; Shaw et al. 1987) are graphically summarised in figure 2. Noteworthy is the broad range of mean bioavailability values. Lower and more closely clustered values ofbioavailability have been measured in plasma separated at room temperature and can be seen in figure 2. This can be explained by the partitioning of cyclosporin into erythrocytes at low temperature, giving rise to lower values of bioavailability. Another influence may come from variations in haematocrit. Patients often have a subnormal haematocrit and there is an inverse correlation between haematocrit and plasma concentration in vitro (Rosano 1985). A 10% decrease in haematocrit, for example, causes an increase in plasma cyclosporin concentrations of 12%. The variability in the relative bioavailability (assayed by HPLC in plasma at body temperature) is quite high (2-fold intraindividual and 3-fold interindividual) even in healthy volunteers (Lindholm et al. 1988). Wideman (1983) used a polyclonal RIA assay of plasma at <37"C to show a clear time dependency of bioavailability during the transplant period. Before renal transplantation, bioavailability is 10.4%. This increases to 20% in the first post-.

Clin. Pharmacokinel. 24 (6) 1993

478

50

45

40

~

35

~

~

o 30

'OJ >

o

<0

o

iii

25

20

15

o 10~------------------------------

Whole blood

Plasma separated at 37°C

Plasma separated at < 37°C

Fig. 2. Boxplot of bioavailability data from the studies of Grevel (1986) and Shaw et al. (1987). and some newer studies (e.g. Gupta & Benet 1990).

operative week, 29% after 2 weeks and to 57% for longer periods in this patient population. The highest mean values for bioavailability were found for patients after renal transplantation [RIA of serum, bioavailability 49% (Kahan & Grevel 1988); HPLC assay of serum at body temperature, bioavailability 47% (Odlind et al. 1986)). Findings of in vitro studies by Ziegler et al. (1988) suggest cholestasis by inhibition of the uptake of bile acids and bilirubin of liver cells caused by cyclosporin. An increase in bioavailability to 47% in patients treated for 3 months (Odlind et al. 1986) or a 50% increase in AUC (assayed by polyclonal RIA in whole blood) [Wilms et al. 1988) is therefore difficult to explain on the basis of liver and bile recovery. Kahan et al. (1983) using a polyclonal RIA assay of serum and plasma at < 37"C, tried to ex-

plain the increase of bioavailability after longer treatment by induction of cyclosporin receptors in the gut. Kasiske et al. (1988) used an HPLC assay of whole blood and correlated increased AUC after a 3-month treatment period with increase in serum lipoprotein and haematocrit, concluding that higher levels of cyclosporin-binding material increase the AUC of cyclosporin in blood. Frey et al. (1988) estimated bioavailability with different methods (30% using HPLC assay of whole blood and 41% using polyclonal RIA in whole blood) in kidney recipients. They explain these differences by presystemic metabolism. The metabolites entering the systemic circulation would be detected by polyclonal RIA and a higher bioavailability was therefore calculated. Most bioavailability studies were performed with renal transplant patients. Few studies in bone marrow and nephrotic syndrome patients are published. It may be that patients with these indications are generally in a worse condition. Little is known about bioavailability in the immediate postoperative period in transplant patients. Morse et al. (1988) performed a pharmacokinetic study using HPLC of whole blood with 8 renal transplant patients covering this time period. They report a range of bioavailability values from II to 47% after conversion from intravenous to oral therapy with unpredictable results in the first month postoperatively (bioavailability was 30 ± 25%). Grevel and Kahan (1991 a) used a monoclonal specific RIA in whole blood to find a rapid increase in cyclosporin absorption from day 3 to day 5 (from 30% to 65% of 'adequate' absorption), which then increased in I month to adequate absorption. Not only the variable absorption of cyclosporin may account for the highly variable bioavailability. Because cyclosporin is mainly eliminated by the liver, the extraction capability of this organ can be another source of variation. This elimination also depends on drug interaction. Ketoconazole, for example, inhibits elimination of cyclosporin to such a degree that some investigators recommend its use for lowering cyclosporin dosages (First et al. 1989). These and other factors influencing elimination are presented below.

479

Cyclosporin Clinical Pharmacokinetics

3. Metabolism Ordinarily, metabolism starts at the first contact of a drug with the body. Being a peptide, it would be expected that cyclosporin would be extensively metabolised in the gastrointestinal tract by enzymes and intestinal flora. However, 7 of the amino acids of cyclosporin are N-methylated and may therefore delay but do not completely prevent degradation of the drug in the gastrointestinal tract. Recent reports confirm this assumption for humans and show non-negligible amounts of metabolites in gastrointestinal mucosa (Tjia et al. 1991) and in the intestinal membrane of anhepatic liver transplant patients when assayed in whole blood by HPLC (Kolars et al. 1991). The main site of metabolism for cyclosporin is the cytochrome P450-dependent mono-oxygenase (CYP3A) system in the liver and in intestinal membranes (Combalbert et al. 1989; Kronbach et al. 1988). All other drugs metabolised by this system may potentially interfere with cyclosporin metabolism. The main metabolic pathways of cyclosporin in humans are mono- and dihydroxylation, and N-demethylation. 3.1 Amount and Number of Metabolites Classification of cyclosporin metabolites was in the past mainly according to the nomenclature of Maurer (1985), who counted 21 different peaks in his HPLC analysis. Claims for the number of metabolites vary. Thus, 18 metabolites (Christians et al. 1988) or up to 27 (Wallemacq et al. 1989) have been found in humans. Wenger (1990) predicted more than 60 metabolites from chemical considerations. Because of the large array of possible metabolites, a new nomenclature was suggested (Kahan et al. 1990) to classify the metabolites by site and type of derivatisation. About 12 of the metabolites have been identified structurally in the last 8 years (Hashem et al. 1988; Maurer et al. 1984; Maurer & Lemaire 1986; Wenger 1990). All metabolites seem to retain their cyclic oligopeptide structure, conjugation of cyclo-

sporin or of the formed metabolites not being important in humans. An overview of the pathway and the structure of the prominent metabolites is shown in figure 3. The most prominent metabolites found in human blood are M I (new nomenclature AM9) and M 17 (AM 1), which also show the highest crossreactivity (M 1 15%, M 17 55%) with the polyclonal RIA kit. A major biliary metabolite is the acid metabolite (AM 1A) identified by Hartman et al. (1985). Henricsson (1990) reported a sulphate conjugate of cyclosporin. The structure of 12 'new' mostly hydroxylated metabolites have been reported recently (Christians et al. 1991 a). Other metabolites are present in human volunteers in only small amounts. Of the total amount of cyclosporin and metabolites in blood, M8 (AM 19) represents 8%, while M I 0 (AM49), M 13 (AM4N9) and M18 (AMlc) each account for less than 2% (Maurer & Lemaire 1986). 3.2 Metabolites in Patient Groups The picture is different in patients, where the metabolic capacity of the body changes with time after transplantation. Shortly after liver transplantation the relative amount of metabolites is high and decreases over time, correlating with lower serum bilirubin (Wallemacq et al. 1987) [assayed using HPLC and polyclonal RIA in serum at <37°C]. 01dhafer et al. (1988) found a polyclonal RIA: HPLC ratio 4 for serum bilirubin concentrations larger than 75 j.Lmol/L. This can be explained by the fact that shortly after transplantation the liver may not be able to eliminate metabolites into bile. This ability seems to recover 2 or 3 months posttransplantation. Wang et al. (1988) investigated the metabolism in liver transplant patients and reported high amounts of metabolites M 17 and M I in an HPLC assay of whole blood, as seen in figure 4. Concentrations of metabolites are variable in different patient populations. Wang et al. (1988) investigated cyclosporin concentrations by HPLC in liver, heart, kidney and bone marrow transplant patients. M 17 in blood is about 1.8 times higher

Clin. Pharmacokinet. 24 (6) 1993

480

7.

, o..y

4; NO - - - - - - ' - - - -... M21

Fig. 3. Schematic metabolic pathway of cyclosporin for the most important metabolites in humans. Amino acids numbered on the structure are those that are not subject to major oxidative metabolism. Steps in metabolism are shown by a number and letter combination alongside arrows denoting the step, where numbers refer to the amino acid metabolised and letters describe the metabolic reaction. Old (new) nomenclature: M I (AM9), M8 (AM 19), M9 (AM4N69), M I 0 (AM49), M 16 (AM69), M17 (AMI), MI8 (AMlc), M21 (AM4N), M25 (AMI4N), M26 (AMlc9), acid metabolite (AMIA). AA = amino acid; OH = hydroxylation; ND = N-demethylation; CB = carboxylation; 3',6'-C = 3',6' cyclisation.

than cyclosporin for liver transplant patients. The lowest M 17 : cyclosporin ratio was found in bone marrow transplant patients and is still about 1 (about 0.4 after intravenous administration, when measured in whole blood by HPLC) [Schwinghammer et al. 1991]. This 'low' value may indicate a normal excretory function of the liver. Subbarao et al. (1989) used HPLC, polyclonal and monoclonal specific RIA assays to investigate trough whole blood samples in heart and renal transplant patients. The blood samples of renal transplant patients show a markedly elevated polyclonal RIA: HPLC ratio for cyclosporin concentrations (ratio ,,=,4.8). In heart transplant recipients

the polyclonal RIA: HPLC ratio varies considerably and unpredictably (factor 2 to 12),

4. Distribution A schematic overview of the distribution of cyclosporin in the body is depicted in figure 5, which also takes metabolite distribution into account. Because of its lipophilicity, cyclosporin distributes across most of the biological membranes and partitions readily into many cell membranes and tissues. Kahan et al. (1983) published cyclosporin tissue concentrations (measured by polyclonal RIA)

Cyclosporin Clinical Pharmacokinetics

from organs obtained during postmortem examination of a single patient. They reported remarkably low concentrations of cyclosporin in fat compared with other tissues. The same group (Ried et al. 1983) found preferential distribution of cyclosporin in fat (10 times more than in serum) and pancreas and a low distribution in the brain and spinal cord using a polyclonal RIA assay. A recent publication (Lensmeyer et al. 1991) reports accumulation of cyclosporin and metabolites in pancreas, spleen, liver, fat and kidney, as assayed by HPLC in whole blood. De Groen (1988) not only sees low-density lipoprotein (LDL) as a carrier vehicle for cyclosporin, but also postulates that LDL may facilitate also the entrance of cyclosporin into cells via the LDL receptor. 4.1 Cyclophilin: A Special Protein Inside the cells cyclosporin binds to a cytosolic protein named cyclophilin. In human erythrocytes, cyclosporin seems to bind in a 1 : 1 fashion to cyclophilin (Foxwell et al. 1988) at concentrations of 1000 ILg/L. In therapeutic concentrations 50 to 90% of cyclosporin is bound to erythrocytes; the total binding capacity of cyclosporin being 2.56 mg/L in packed erythrocytes (Legg & Rowland 1988). Its common presence in many eukaryotic cells (Koletsky et al. 1986) and conservation of structure suggests that cyclophilin may play an important role in cell physiology, maybe even in the early events of T cell activation. Recent findings suggest (McDonald et al. 1992) that cyclosporin increases the amount of cyclophilin in tissue. One hint for the importance of cyclophilin distribution is given by Ryffel et al. (1991). This group investigated tissues from 13 different organs of human origin. They found cyclophilin in all samples with a mean concentration of "" 1 ILg/mg protein (range 0.8 to 2.8). The distribution of cyclophilin inside an organ, however, can differ significantly. 4.2 Distribution of Cyclosporin in Blood Lemaire and Tillement (1982) studied the distribution of cyclosporin in blood components in

481

vitro. At room temperature they found 58% of added cyclosporin (concentration 500 ILg/L) in erythrocytes, 9% in leucocytes, 4% in plasma water, 21 % bound to lipoproteins (mostly to high density lipoprotein cholesterol, HDL) [Urien et al. 1990] and 8% bound to other plasma proteins. The distribution depends on the cyclosporin concentration (fig. 6). The distribution of cyclosporin between erythrocytes and plasma is also temperature-dependent (Wenk et al. 1983). A reduction of cyclosporin concentrations in plasma by a factor of 0.5 to 0.68 by cooling from 37"C to 20·C was found.

Intravenous

2500

o M17 DM1 • M18

::r 2000

i~ Iii

o

8

eCyA

• M21

1500 1000 500

Ol~~~~~~~~~~ 4

6

Time (h)

2000

i

Oral

1500

.~

1

1000

8

500

o

2

468

10

12

Time (h)

Fig. 4. Typical blood concentrations of cyclosporin (CyA) and metabolites MI, M17, MI8 and M21 in c1inicaIly stable liver transplant recipients (redrawn trom data of Wang et al. 1988) after intravenous and oral administration. Mean cyclosporin doses were 2.7 mg/kg (intravenous) and 4.5 mg/kg (oral). The intravenous dose was administered a day after normal oral administration.

Clin. Pharmacokinel. 24 (6) 1993

482

Tissue Urine

~ CyA metabolites t1(P Memblanes

CP

Blood Liver

RBC~~, r

L::::...-=:t1r+ e V, "A• . ~,

Llpo-

proteins

I

Metabolite,

;:==!~ eyA

<

Cytochrome

~I---+-+ C y A _ Metabolites I

Metabolites

P4S0 III

GIT Metabolites eyA

Fig. 5. Schematic overview of the distribution and the fate of cyclosporin (CyA) and its metabolites in the body. RBC = red blood cells; CP = cyclophilin; GIT = gastrointestinal tract.

The unbound fraction of cyciosporin in plasma of renal transplant patients is in the range of 0.04 to 0.12 (Legg & Rowland 1987). Sgoutas et al. (1986) used a po1ycional RIA assay in plasma at < 37T and found differing cyciosporin distribution profiles among the lipoproteins in fasted, non-fasted and Iipaemic non-fasted persons. An inverse correlation between plasma AVC and serum HDL-

100

4.3 Distribution of Cyciosporin in the Body

• Plasma A Erythrocytes • Leucocytes

50

Co cO"-

o-.:U) t.l (/)

tUtU

rl:E

O~---------r----------~--------~

100

cholesterol in healthy volunteers was found by Lindholm et al. (1988) using an HPLC assay at body temperature. Cyciosporin itself may change the lipoprotein and lipid composition in blood. Long term treatment with cyciosporin causes higher LDL: HDL ratios and higher triglyceride levels, as reported by Schorn et al. (1988), but this is a common feature for immunosuppressive therapy (Irish et al. 1992).

1000

10000

Cyclosporin concentration &tg/L)

Fig. 6. Distribution of 3H-cyclosporin in human blood: fraction of total mass in plasma (circle), erythrocytes (triangle) and leucocytes (square). Each point is the mean (± SD) of 3 determinations in 3 different blood samples (figure kindly provided by M. Lemaire).

Because of its action on lymphocytes, the distribution of cyciosporin into the lymphatic fluid is of interest. About 40 to 60% of blood cyciosporin concentration values are observed in the lymph of rats (Veda et al. 1983b). The low passage of cyciosporin through the blood-brain barrier (Cefalu & Pardridge 1985) is surprising because of the lipophilicity of the drug and the long durations of treatment. The low concentrations in the brain make the reported central nervous system side effects after cyciosporin treatment (Adams et al. 1987; De Groen et al. 1987) difficult to explain. Recent publications (Begley et

Cyciosporin Clinical Pharmacokinetics

al. 1990; Pardridge et al. 1990) demonstrate that a large fraction of any cyclosporin that crosses the blood-brain barrier is sequestered within the microvasculature. Because of the lipophilicity of cyclosporin and its distribution in fat (measured in whole blood by HPLC) [Lensmeyer et al. 1991], it would be expected that the volume of distribution (Vd) is larger for obese than for non-obese patients. However, pharmacokinetic studies (Flechner et al. 1989; Yee et al. 1988a,b), using whole blood assays by polyclonal RIA and HPLC, showed that this is not the case. Yee et al. suggest that the distribution of cyclosporin is restricted to lean body mass. Cyclosporin is present in the fetal circulation at similar concentrations to those found in the mother's blood. Cyclosporin was also present in maternal breastmilk and in the placenta of a pregnant woman (measured by polyclonal RIA in serum at <37"q [Flechner et al. 1985]. 4.4 Pharmacokinetic Models Grevel et al. (1986) consider the absorption phase as the rate limiting step (300mg oral dose) and used a zero-order model to describe absorption data, whereas Beveridge et al. (1981) applied a firstorder pharmacokinetic model to describe data in a study with a higher dosage (600mg). Kutz et al. (1985) used a Michaelis-Menten type absorption model, which can be considered here as a mixture of zero- and first-order absorption. Reymond et al. (1988) suggested that limited solubility of cyclosporin in the gastrointestinal tract may account for some of the observed phenomena. It is often sufficient to describe the concentration-time curves of cyclosporin in oral and intravenous pharmacokinetic studies by a 2-compartment model and most studies published rely on this model. However, a 2-compartment model does not cover the rapid distribution of cyclosporin between erythrocytes and plasma, which occurs in about 10 min (Vine & Bowers 1987). This rapid distribution finds its equivalence in only few pharmacokinetic publications. Description of the time course with a 3-com-

483

partment model is rare because of the regimen to take blood samples at early times after or during infusion. Follath et al. (1983) used polyclonal RIA and HPLC of whole blood samples to estimate in 4 renal failure patients values of the rapid distribution half-life (t'12.-) of 0.1 ± 0.03h, distribution half-life (t'12,,) of 1.08 ± 0.25h and terminal elimination half-life (t'l2/l) of 15.8 ± 8.4h. These were estimated with HPLC in whole blood. Values from analyses with polyclonal RIA are slightly higher, but not significantly different. Lindberg et al. (1986) reported a t'12.- of 0.33 ± 0.28h, t'll" of 2.84 ± 1.11 h and t'1211 of 26.9 ± 10.8h in 20 uraemic patients using a whole blood HPLC assay. Recently, Karlsson and Lindberg-Freijs (1990) recommended a simultaneous fitting of oral and intravenous data with 3-compartment models to estimate pharmacokinetic parameters rather than noncompartmental analysis, but this may be difficult to handle in all transplantation facilities. A physiological pharmacokinetic model for cyclosporin in rats was recently developed (Bernareggi & Rowland 1991) involving 14 tissue and 2 blood compartments. On scaling up to humans, the animal data seem to suggest that partition of cyclosporin into fat is an important parameter in the pharmacokinetics of the drug. Because of high variability in pharmacokinetic parameters with model calculations, Clardy et al. (1988) performed noncompartmental analysis using HPLC assay of whole blood from different patient groups. Vss after oral administration was about the same in all transplant types [7.9 ± 5.1 L/kg] with high individual variation. However, the mean residence time values, stated by the investigators as the most useful parameter, differed between patients with different transplant types [e.g. kidney 5.8 ± I.2h, liver 10.7 ± 4.4h]. 4.5 Distribution and Pharmacokinetics of Metabolites The distribution of the cyclosporin metabolites is different from that of cyclosporin because of their usually higher hydrophilicity. Another reason might be the different binding of metabolites to cyclo-

484

philin (except M 17, see Fahr et al. 1990). The binding of metabolites to blood components is a complex interplay of metabolite type, haematocrit, temperature and concentration (Lensmeyer et al. 1989). Generally, metabolites M I, M8, M9, MIO, M16, MI7 are associated mainly with cells, whereas M 18, M21, M25 are mainly associated with plasma. A decrease in haematocrit, increase in temperature or metabolite concentration causes a nonlinear increase in plasma association. There are few data regarding the pharmacokinetics of metabolites in humans. The Cmax of M 17 after oral administration occurs at ,.,6h. Cyclosporin and MI7 have about the same elimination profile (Keown et al. 1986), measured by HPLC, polyclonal RIA and monoclonal specific RIA in whole blood, indicating that metabolite formation is the rate-limiting process. Awni et al. (1987, 1988), using HPLC assays of whole blood, investigated the time dependency of pharmacokinetic parameters for cyclosporin, M I 7, MI and M21 in renal transplant patients over a 12-week period. The apparent clearance, V55 and t'12j3 were not altered significantly in this period, whereas cyclosporin AUC and trough concentrations increased (possibly due to better absorption). Some metabolites partition into the placenta and umbilical cord in pregnant women (measured in whole blood by HPLC) [Venkataramanan et al. 1988b]. Only M 17 was present in the umbilical cord in similar concentrations (97 to 137 ~g/L) to those in the maternal blood (132 to 134 ~gfL). Cyclosporin was present in the maternal blood in comparable concentrations (,.,100 ~g/L), but in the umbilical cord concentrations were highly variable.

5. Elimination 5.1 Variability of Data Clearance of cyclosporin was investigated in a number of publications. In clinical studies, clearance is often calculated by taking the intravenous dose divided by the AUC. Clearance, terminal elimination rate constant (ke) and Vd are related by the equation: Vd = CL/ke

Clin. Pharmacokinet. 24 (6) 1993

Table II. Drugs altering the blood concentrations of cyclosporin (from Yee & McGuire 1990) Drugs increasing cyclosporin concentration

Drugs reducing cyclosporin concentration

Erythromycin Verapamil Nicardipine Diltiazem Steroids Ketoconazole

Phenytoin Rifampicin (rifampin) Phenobarbital Carbamazepine

Studies using long infusion periods (up to equilibrium of cyclosporin in blood) allow a quite reliable estimation of CL. The accuracy of the Vd values is dependent on the precision of AUC and ke. Calculating the t'1211 depends critically on the number of data points in the terminal phase. It is difficult to compare literature values obtained from studies which measure cyclosporin blood concentrations for 12h with those with measurements over 24h. The dosage regimen (once or twice a day) is often the limiting factor in such studies. Nevertheless, the interpatient variability can be quite high. In . I study, for example, the reported range of t,;~ In 41 patients was 4.3 to 53.4h. Generally, otherwise-healthy individuals have smaller values for t'12j3, CL and V55 than transplant pat~ents (Ptachcinski et al. 1987). The lower CL, whIch would cause a higher t'12j3, is compensated by a significant reduction of V55. High variability of V55 and CL even in a restricted population such as kidney recipients led Kahan et al. (1992) to perform a strategy with intravenous and oral doses using a monoclonal specific RIA assay in whole blood to tailor cyclosporin dosage better to individual patients and to avoid early adverse events. 5.2 Factors Influencing the Elimination of Cyclosporin Not only manifold factors described below but also drugs which are metabolised by the cytochrome P450-dependent mono-oxygenase system in the liver are suspected of interacting with cyclo-

Cyclosporin Clinical Pharmacokinetics

485

sporin metabolism and its subsequent elimination. Drugs (especially calcium antagonists) seem also to reduce the Vd of cyclosporin. Some excellent reviews about drugs interacting with cyclosporin have been published (Lake 1991; Scott & Higenbottam 1988; Yee & McGuire 1990). Table II gives an overview of drugs altering the blood concentration of cyclosporin. Cyclosporin is a drug with a low to intermediate extraction ratio. It does not undergo excessive first pass metabolism and the extraction of the drug does not exceed 50% of the dose (Kahan 1985). CL is therefore dependent on its unbound fraction in blood. This is shown by several reports. Lithell et at. (1986) used an HPLC assay of plasma at 37°C and reported an inverse linear relationship between CL and lipoprotein concentration in the blood of uraemic patients. Lipoprotein concentrations, however, are quite variable in renal and liver transplant patients (Ptachcinski et at. 1986a), which adds to the variability in CL. Kahan et at. (1986) used a polyclonal RIA assay in serum at <37"C to investigate demographic factors affecting CL and Vd. Figures 7 and 8 show the

30

-

25

20

,.... r 10

,-

5

o

In 0

4

8

12

Innnnnn 16

20

24

28

32

n 36

Volume of distribution (L/kg)

Fig. 7. Frequency distribution of cyclosporin clearance among 212 renal transplant patients (redrawn from Kahan et al. 1986).

30

20

10

o

0

8

16

24

32

n 40

48

56

64

n

72

Clearance (mlfmin/kg) Fig. 8. Frequency distribution of cyclosporin volume of distribution among 212 renal transplant patients (redrawn from Kahan et al. 1986).

frequency distribution of cyclosporin CL and Vd among 212 renal transplant patients. An exhaustive collection of pharmacokinetic studies was published by several groups (Ptachcinski et at. 1986a; Shaw et at. 1987; Vine & Bowers 1987). All pharmacokinetic parameters in these and newer publications show a large range. All my attempts to find correlations between pharmacokinetic parameters in these studies failed, with I exception. There is a clear trend for Vss to increase and a less pronounced trend for ke to decrease if the cyclosporin dose is increased (fig. 9). A decrease in ke and increase in Vss by increasing the intravenous dose (cyclosporin 5 to 20 mg/ kg) has also been found in animal studies using HPLC assays of whole blood (Awni & Sawchuk 1985), which was interpreted as nonlinear pharmacokinetic behaviour of the drug. In healthy human volunteers, Reymond et at. (1988) used an HPLC assay of whole blood to find a significant increase of the tl/2{j from 8.9 to 11.9h when the dose was increased from 350 to 1400mg. On the other hand, in a case report, Kruger et at. (1988) suggest linear pharmacokinetics exist (but a tl/2{j ",,30h), as-

486

Clin. Pharmacokinet. 24 (6) 1993

a 25% reduced Vss compared with younger patients (II to 40 years). CL is 1.7 times higher in paediatric liver recipients (Burckart et al. 1986) and is doubled in paediatric kidney recipients (Ptachcinski et al. 1986b) compared with adults. One of the reasons for this age-dependency may be the change of lipoprotein concentration (Schaefer & Levy 1985; Vee et al. 1986a). Similar results were found in the serum of renal transplant patients aged over 45 years (Kahan et al. 1986), in whom CL and Vd are decreased by 30%. The tlh/l of the drug is in many cases shortened in children because of higher CL (and a smaller increase in Vd). In paediatric patients with renal transplants the tlh/l is 7.3h in contrast to 10.7h in the comparative group (assayed by HPLC in whole blood) [Ptachcinski et al. 1986b].

5

4

-::'" 3

2

0.15 ke 0.10 0.05 0.00

5.4 Other Factors Fig. 9. Three-dimensional graph from different publications showing the relationship between volume of distribution at steady-state Vss (L/kg), elimination constant kc (h- I ) and dosage (mg/kg/day).

sayed using HPLC of plasma after extremely high oral dosages of cyciosporin (150 mg/kg). The higher Vss values at higher doses (fig. 9) can be explained by this scenario: at high concentrations proportionally less cyciosporin is bound to erythrocytes because of saturation of binding sites, therefore cyclosporin migrates to tissues. The lower values of ke in figure 9 could also be explained by analytical artefacts: the higher the dose, the longer postdose that detectable cyclosporin concentrations could be detected, yielding longer t lh~ values (i.e. smaller ke values). 5.3 Clearance Depends on Age Correlations between age and CL, Vss and tlh~ have been discovered. In paediatric bone marrow transplant patients « II years) CL and Vss are doubled in comparison with older (11 to 40 years) patients (Yee et al. 1986a, 1987). Bone marrow recipients over 40 years display 50% lower CL and

As discussed above, it would be expected that lipophilic cyciosporin drug distributes readily into body fat. But Flechner et al. (1989) report that obese uraemic patients receiving dosages scaled according to bodyweight show trough concentrations twice those of nonobese patients. When the dosage given to the patients was normalised to their ideal bodyweight, no differences in bioavailability, t'h{i' CL or Vss were found. CL and Vd are increased with high fat meals (Gupta et al. 1990). Plasma CL rises in healthy volunteers from 0.47 L/h/kg (no high fat meal) to 0.7 L/h/kg (high fat meal). The investigators suggest that lipids from high fat meals serve as a carrier of cyciosporin to the body and to liver. In female patients CL and Vd is higher than in males (Kahan et at. 1986). The t'h/l values are, however, not significantly different between the sexes. Grevel et al. (1989) found a negative correlation between serum alanine transaminase activity (indicator of liver cell integrity) and CL in adult uraemic patients. Cyciosporin binding parameters such as cholesterol, triglyceride, haemoglobin or haematocrit did not correlate with CL. There are some indications that cyciosporin CL

Cyclosporin Clinical Pharmacokinetics

shows diurnal variation. In I study the reported CL is 1.45-fold higher at night (Venkataramanan et al. 1986) in patients. But findings from Bowers et al. (1986) and Cipolle et al. (1988) using the same assay methods suggest rather a smaller CL at night. Lower night-time metabolite concentrations relative to those of cyclosporin (Canafax et al. 1988) point to a decreased metabolic rate at night. CL of cyclosporin by haemodialysis is negligible «1% of a cyclosporin dose in 4h) [Venkataramanan et al. 1984]. Elimination parameters of metabolites in humans are not easy to measure. The similar t'1211 values obtained by RIA and HPLC (Robinson et al. 1983) suggest that the rate-limiting step in metabolite pharmacokinetics is the formation of metabolites and not their elimination.

6. Pharmacokinetic-Therapeutic Relationships 6.1 Pharmacokinetics and Pharmacodynamics A clear relationship exists between dosage, blood concentrations and immunosuppressive action, which is outlined in section 7. Pharmacokinetic studies are therefore useful for therapeutic monitoring. The balance of perfecting pharmacokinetic studies and drawing the minimum number of blood samples from the patient is not easy to achieve. Attempts such as that of Grevel and Kahan (1991 b) are therefore quite helpful for finding the optimum both for monitoring and for patient comfort. The site of action of cyclosporin is not only in the blood. Many investigators speculate that lymph and lymph nodes are important sites for the drug to exert its action on T cells, which are its main target. Increasing the transport of cyclosporin into lymph seems a difficult task. Only about 0.4% of a cyclosporin dose is recovered in the thoracic duct of rats (Ueda et al. 1983a). If other pharmaceutical formulations are developed which direct more cyclosporin into this presumed target fluid (Takada et al. 1988), blood concentrations or AUC values will not alone be an indicator of cyclosporin efficacy. Peak to trough differences of a factor of 5 or

487

more are observed when cyclosporin is administered only once daily. The inhibitory action of cyclosporin on lymphocytes may need a low steady concentration of cyclosporin, whereas the transient high cyclosporin concentrations might be associated with renal side effects. 6.2 Pharmacokinetics and Renal Adverse Events Actions of cyclosporin on the kidney have been widely discussed since cyclosporin was first used clinically. Other reported side effects of cyclosporin (except hypertension, see Thompson et al. 1986) are of less importance (Caine et al. 1978; Ferguson et al. 1982; Starzl et al. 1980). Cyclosporin was administered in higher dosages in the early days of its clinical use, no proper assay methods being available at that time. Clinical complications were noticed and dosages adjusted. Monitoring of blood concentrations of cyclosporin coupled with concomitant use of other immunosuppressive drugs allowed a reduction in the cyclosporin dosage and a consequent decrease in the incidence of side effects. Holt et al. (1986), for example, report that adjusting the cyclosporin concentration to between 400 and 800 J.Lg/L (measured by polyclonal RIA of whole blood) keeps the incidence of renal adverse events low and the immunosuppressive activity high. A complete discussion of renal side effects is beyond the scope of this review, but some aspects relating renal side effects to pharmacokinetic parameters of cyclosporin and its metabolites can be described (for recent work on kidney pathology after cyclosporin treatment see Mason 1990). In clinical terms, 2 forms of kidney impairment caused by cyclosporin are known. First, reduced renal function is a dose-dependent reduction of renal plasma flow and glomerular filtration rate which is largely reversible by dosage reduction and completely reversible by drug withdrawal. Secondly, structural changes of the smallest arteries, afferent arterioles and the proximal tubular epithelium may occur. The mechanism of cyclosporin action on. the

488

kidney is not completely understood. Perhaps the identity of cyclophilin with an enzyme and the presence of cyclophilin in many cell types may give some clues on the issue of morphological changes. Ryffel et al. (1991) found cyclophilin in kidney predominantly located at the proximal tubular epithelium, where cyclosporin side effects are presumed to occur. Another attempt at finding a mechanism for renal side effects of cyclosporin comes from Burke and Whiting (1986), who speculate that the high molecular weight of cyclosporin causes it to accumulate in the proximal tubule cells and this 'might be a contributory factor to CsA tubular toxicity'. Functional changes in the kidney are, for many groups working on this problem, seen as a consequence of the renal vasoconstrictive effects of cyclosporin (e.g. Perico et al. 1986). Are trough or peak concentrations responsible for the kidney impairment observed? Dieperink et al. (1988) studied the influence of once-daily doses (high peak: trough ratio) or divided daily doses on immunosuppression and renal side effects in rats (using a polyclonal RIA assay of whole blood). Both dosage forms produced similar immunosuppressive activity, but kidney function was significantly impaired in rats which received cyclosporin once a day. Williams et al. (1986) report an increase (from 2 to 8%) in adverse effects after intravenous vs oral administration. Several investigators suggest that part of these side effects may stem from the intravenous vehicle CREMOPHOR-EL@, which causes renal side effects in animals (Luke et al. 1987; Thiel et al. 1986). Other studies could not confirm this result (Albrechtsen et al. 1986). 6.3 Do Metabolites Cause Renal Side Effects? A similar controversy to that just described concerns the question of whether metabolites cause renal side effects. Part of this discussion parallels the controversy about the activity of the cyclosporin metabolites (see section 8.1). As described, renal side effects of cyclosporin were found to be

Clin. Pharmacokinet. 24 (6) 1993

related to the dosage of cyclosporin. This allows speculation about the cause: I. Cyclosporin in high concentrations causes renal side effects. 2. Cyclosporin is metabolised in the body, the higher concentrations of metabolites are toxic. 3. At higher concentrations of cyclosporin other metabolites may be formed because of saturation of the normal metabolic pathway, the others being toxic. 4. Any combination of I, 2 and 3 is possible. Renal side effects of metabolites is therefore a controversial subject much discussed in the literature. High metabolite concentrations occur - as outlined - in many clinical cases. Direct observations of metabolite action in humans are of course not available. It might be evident from the review so far that many factors interacting unpredictably with each other will cloud reported observations of the renal side effects of metabolites. Vee et al. (l986b) reported that in bone marrow transplant patients the occurrence of renal dysfunction correlated better with RIA than with HPLC measurements of cyclosporin. They suggest that 'cyclosporin metabolites playa role in the development of renal dysfunction'. Rosano et al. (1988) investigated blood from a single renal transplant patient. During a 'nephrotoxic period', high amounts of metabolites were measured (using polyclonal RIA). Values were about 8-fold higher than after the 'nephrotoxic period', but cyclosporin concentrations as measured by HPLC stayed about the same. The metabolites appearing during the 'nephrotoxic period' were more polar than M I or M 17. The Cmax values for these polar metabolites accounted for 53% of the nonspecific RIA activity. These polar metabolites were not structurally identified. Burke and Whiting (1986) speculate, in their work on induced nephrotoxicity of cyclosporin in rats, that different forms of P450 may produce different metabolites which may be toxic. Kronbach et al. (1988) report high variations of P450 system activity (up to 25-fold) in the liver of kidney transplant recipients. This may shed light on the me-

489

Cyclosporin Clinical Pharmacokinetics

tabolism found in case studies, where special metabolites are reported. Lucey et al. (1990) report renal failure and neurological disturbances in a liver recipient when cyclosporin concentrations (measured by HPLC in whole blood) were in the therapeutic range. They attribute this to a genetic defect in CYP3A metabolism, which seemed to produce toxic metabolites. Harfmann et al. (1990) found that ratios of monoclonal nonspecific RIA and specific RIA in whole blood of > 3.2 during the first week posttransplant are related to side effects. They suggest that metabolites rather than cyclosporin are responsible for side effects. Recent reports (e.g. Christians et al. 1991 b) attribute renal adverse events to specific metabolites (in this case M26 and AMIA) in patient groups.

7. Dosage and Administration 7.1 Administration The normal route of administration of cyclosporin in solid organ transplantation therapy is either oral or intravenous. Cyclosporin is inadequately absorbed after intramuscular administration (Keown et al. 1981). Routes of administration have been investigated for systemic delivery following subcutaneous (Wassef et al. 1985) and topical ocular (Gregory et al. 1989) administration in animals, but have not been tested for clinical use. 7.2 Dosage Renal side effects are related to dosage. A few selected papers can demonstrate how these side effects were controlled by clinicians during the last decade. CaIne et al. (1978) reported both renal and hepatic side effects in renal transplant patients. The cyclosporin starting dosage in the first 7 patients was 25 mgjkgjday and either 10 or 15 mgjkgjday in later patients. Empirically, 17 mgjkgjday was found to be best and, when this dosage was reduced about 10 mgjkgjday in the maintenance phase, this group noticed an improvement of renal function (CaIne et al. 1979). The concomitant use of smaller dosages of

cyclosporin (7.4 to 17.5 mgjkg) with steroids (Starzl et al. 1980) diminishes the risk of renal side effects. Adjusting the cyclosporin dosage (during prednisone coadministration) to 6 to 12 mgjkgjday by monitoring serum creatinine, Ferguson et al. (1982) could 'manage' the renal side effects. Loertscher (1987) advocates cyclosporin dosages of 3 mgjkg when it is given together with other drugs. At that dosage he found no differences in renal function compared with control groups. Moyer et al. (1988) demonstrated that minimising the trough concentration of cyclosporin (measured by HPLC in whole blood) to 150 to 250 J.LgjL during the first 4 months of therapy and 80 to 200 J.LgjL afterwards prevents renal side effects of the drug. Mihatsch et al. (1988) recommend for use in autoimmune diseases an initial dosage of less than 5 mgjkgjday. During the last decade new regimens combining cyclosporin with other immunosuppressive agents have evolved in transplantation. This means that the initial dosage of cyclosporin has been greatly reduced. As a result, renal side effects of cyclosporin can be reduced or even avoided.

8. Clinical Implications The pharmacokinetic studies with cyclosporin discussed in this review show high variability in bioavailability, CL, Vd, etc. Several factors such as age and liver function have some influence on this variability. Nevertheless, individual values in patients with comparable demographic characteristics vary considerably. Generally, the investigated factors do not explain satisfactorily the observed variability. In addition, there are also too many uncertainties remaining (bioanalytical methods, different patient and transplant groups, etc.) to allow a correlation of all studied factors quantitatively. It has therefore become routine clinical practice in some centres to individualise cyclosporin dosage in patients by pre-estimating the pharmacokinetics of the patient. Drug-induced renal side effects are a frequent phenomenon in drug treatment. In the case of

490

cyclosporin it seems that renal side effects caused by this drug can now be managed by careful monitoring of cyclosporin in patients and adjustment of the dosage accordingly. The condition of the patient and their liver function play important roles in the absorption, metabolism and excretion of drug and metabolites. Increased bile production and excretion can enhance absorption considerably. Since the first use of cyclosporin in transplantation, much information has been gained on how to use cyclosporin safely for the transplant patient. The transplantation community has accepted that monitoring cyclosporin regularly in whole blood with a specific analytical method is extremely helpful. Although the concentration of metabolites cannot be measured precisely by means of specific monoclonal RIA in patients, this method is the best choice in clinical practice. 8.1 Activity of Metabolites The possible activity and renal adverse effects of the metabolites of cyclosporin formed in humans after cyclosporin administration is a controversial topic. There are many publications with contradictory results concerning activity, nephrotoxicity, concentrations of metabolites and the correlations of these parameters with relevant clinical effects. Analysis of metabolite data in humans gives a cloudy impression. Occurrence and concentrations of metabolites depend on correlated factors, notably the status of the liver. The picture concerning metabolites is unclear: which are immunosuppressive, which affect renal function? Animal models are of value, but the large differences in metabolism between humans and animal species may lead to divergent conclusions. Additionally, conclusions from published in vitro experiments so far do not add consistent information about the relationship of immunosuppressive activity vs renal side effect potency of metabolites. The immunological activity of metabolites is a highly controversial and much discussed issue.

Clin. Pharmacokinet. 24 (6) 1993

Measurement of activity is restricted to in vitro and animal tests (for a review see Fahr et al. 1990). It is difficult to extrapolate in vitro activities of metabolites to those in patients, where many metabolites in different time-dependent concentrations may synergistically act on their target. Different tissue distribution of different metabolites has to be taken into account as well. Animal studies might also be misleading because metabolism may be very different in amount and composition (Fahr et al. 1990; Venkataramanan et al. 1988a). The biological action of cyclosporin and some metabolites might be caused by binding to cyclophilin. Binding of M 17 (the metabolite with the highest activity) to cyclophilin is not very different from the binding of cyclosporin (Fahr et al. 1990). The finding of different membrane permeability for M 17 in lymphocytes and erythrocytes supports the notion that drug delivery of cyclosporin and metabolites into the target cells may be responsible for different results in vitro and in vivo. 8.2 Future Research Progress toward optimising cyclosporin therapy might come from 2 alternative approaches: I. Knowledge of cyclosporin function at the basic level may help to optimise dosage schemes and to evaluate the reasons for the variability of the pharmacokinetic/pharmacodynamic parameters. Eventually, these studies may lead to a better understanding of the immune system. 2. Synergistic immunosuppressive and side effects of metabolites and cyclosporin in blood and tissues might have caused dissent in the scientific discussion. This picture is even more complicated by different distribution and amounts of metabolites produced in patient groups. This calls for further studies of metabolites, in an attempt to correlate metabolite pharmacokinetics, activity and adverse effects. Of course, the main concern was and has to be the patient. More clinical studies are emerging where the investigators are aware of the problems discussed above, and try to solve them in a multidisciplinary approach (Faulds et al. 1993).

Cyclosporin Clinical Pharmacokinetics

8.3 Place of Cyclosporin in Therapy Cyclosporin is used today in almost all immunosuppressive regimens. Despite the long experience with cyclosporin and the number of publications about its therapeutic use (",,4500 references in MEDLINE as of 1992), there is still no agreement about the optimal use of the drug. However, the common objectives of all clinicians are to get the greatest efficacy from cyclosporin and to avoid possible side effects. To fulfil this objective, cyclosporin is used alone, together with steroids, or in triple and quadruple therapy. During the recovery of the patient, the need for additional therapy besides cyclosporin often diminishes. This has to be accompanied by pharmacokinetic monitoring, which helps the clinician to navigate through the sometimes difficult waters of immunosuppressive therapy.

Acknowledgements I would like to thank Drs T. Beveridge, P. Hiestand, M. Lemaire, G. Maurer and B. RyfTel for helpful discussion; T. Beveridge and M. Lemaire for critical reading of the manuscript and H. Eckert for his interest in this work.

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Correspondence and reprints: Dr A. Fahr. Sandoz Pharma Ltd. Research & Development, Building 145/1062. CH-4002 Basel. Switzerland.