SINGLE-DOSE AND STEADY STATE PHARMACOKINETICS OF CSA AND TWO MAIN PRIMARY METABOLITES , AM 1 AND AM 4 N IN PATIENTS WITH RHEUMATIC / AUTOIMMUNE DISEASES

a Department of Clinical Pharmacology, Faculty Hospital Plzen, Czech Republic b Clinical Pharmacokinetic Research Laboratory, Department of Biomedical and Pharmaceutical Sciences, College of Pharmacy, University of Rhode Island, Kingston, 02881, RI, USA c Department of Clinical Pharmacology, Faculty Hospital Ostrava d Center for Metabolic Bone Disease, 2 Department of Internal Medicine, Faculty Hospital Plzen E-mail: suchyd@fnplzen.cz


INTRODUCTION
Cyclosporine A (CsA), a calcineurin inhibitor with potent immunosuppressive activity, is used in standard immunosuppressive regimens in solid organ transplantations as well as in the treatment of autoimmune diseases.Cyclosporine A, is effective in the treatment of rheumatoid arthritis (RA) as a single drug, as well as in combination with methotrexate or sulfasalazine.Several consensus conferences have provided guidelines for the use of CsA in rheumatology.Clinical researchers generally agree that starting low-doses of CsA should range from 2.5 to 3.5 per day [1][2][3] .However, data comparing its efficacy and safety over the long-term and in a large number of patients are limited.
CsA is also effective in the treatment of articular and cutaneous manifestations of psoriasis.A clearing effect on the dermatitis has been demonstrated in a number of studies; however, no data are available for the different forms of psoriatic arthritis 4 .In one controlled study from 1995, CsA was equally clinically effective as methotrexate.In another, from 2001, it was shown to be more effective than sulfasalazine or symptomatic treatment 4,5 .Apropos the use of CsA in other autoimmune/ rheumatic diseases, less data are available.In uncontrolled, small studies of systemic lupus erythematosus (SLE), a significant decrease in overall disease activity was described [6][7][8] with best results in controlling proteinuria, leukopenia and thrombocytopenia but with little effect on arthritis 6,7 .D. Suchy, M. Dostalek, I. Perinova, H. Brozmanova, M. Grundmann, V. Vyskocil, O. Mayer In a subgroup of patients with lupus nephritis, CsA treatment was reported favourably in almost all studies and some data showed that it might have beneficial effects at a histological level [9][10][11][12] .
There are also case reports and retrospective case series supporting the efficacy of CsA in the treatment of inflammatory myopathies [12][13][14] .A randomised controlled clinical trial in patients with poly(dermato)myositis comparing a combination of glucocorticoids and methotrexate vs. glucocorticoids and CsA alone showed comparable efficacy for the two treatment arms 15 .Cyclosporine A acts more rapidly than azathioprine and may be a useful second-line drug.There are limited data on the use of CsA in patients with seronegative spondylarthropathies.No data are available for other spondyloarthropathies, including ankylosing spondylitis.
After administration of cyclosporine, there is an initial absorption phase, during which blood concentrations reach a peak level (C max ).Typically, C max occurs within the first 2-3 h CsA is strongly lipophilic, insoluble in water and has limited solubility in other solvents (olive oil, ethanol).Therefore, CsA is absorbed incompletely from the gastrointestinal tract, predominantly from the small intestine, and resulting bioavailability ranges widely between 5-80% (ref. 15,16).The original oil-based preparation of CsA showed wide inter-and intra-patient variability.Absorption is significantly affected by solubilisation of CsA in bile and a number of other factors, e.g diet, gastrointestinal transit time, concurrent medications and others [16][17][18][19][20][21] .The currently used CsA formulation, Neoral ® has greater and more balanced gastrointestinal absorption and improved bioavailability than the older formulations.Neoral ® is a microemulsion pre-concentrate of particle size <100 nm 20,21 compared with Sandimmun ® , similar C 0 levels, but higher C max as well as AUC were achieved during the steady state in patients who received Neoral ® .Pharmacokinetic data in patients with RA and psoriasis, as well as patients with solid organ transplantations, showed better bioavailability for Neoral ® than Sandimmun ® .0][21] ).
The pharmacokinetics of CsA have been the subject of a large number of articles and reviews, mainly in the area of solid organ transplantation 21 .Data describing the pharmacokinetics of CsA in autoimmune diseases however are relatively rare.Few data are available for patients with rheumatoid arthritis [30][31][32] , but more consistent data were found for psoriasis treatment [33][34][35][36][37] .None of these stud-ies however described the pharmacokinetics of the main primary CsA metabolites AM1 and AM4N.
The aim of our study was to evaluate basic pharmacokinetic parameters of parent drug and its primary metabolites AM1 and AM4N after single and repeated administration of cyclosporine A in patients with the autoimmune/rheumatic diseases listed.

Subjects
The study was performed in the Rheumatology Department, Faculty Hospital Plzen, Czech Republic.After informed consent and with the approval of the local Ethics Committee, 38 subjects average age (± SD) 46.82 (±11.67) years with rheumatoid arthritis, systemic lupus erythematosus, psoriatic arthritis, ankylosing spondylitis and undifferentiated spondylartropathy (off-label use in the last two indications, CsAwas administered to patients with recurrent ocular complications of these diseases and failure of previous treatment).were included in an observational open study.Patient data are are summarized in (Table 1).Inclusion criteria were if they had normal renal and hepatic function, were normotensive, and had stable concomitant medication one month prior to entry.Exclusion criteria were if they had severe hypertension, clinically relevant cardiovascular disease, active infection, malignancy or other unstable medical conditions, had received any drug with the potential to interact with cyclosporine metabolism or were pregnant or lactating.

Study design
This was an open, observational, single centre study.In total 38 CsA naive patients were recruited.Pharmacokinetic data were obtained after administration of a single dose of CsA as well as during steady state conditions after the 3-month period of continual stable CsA therapy.
Single dose pharmacokinetics over a 24 h interval was determined in 38 patients by giving them 1.3 mg/kg oral CsA.In 12 patients who were be willing to participate in follow-up, a second series of blood samples was taken in order to calculate area under the concentration -time curve of CsA and its metabolites (AUC) and other PK parameters in steady state conditions.

Pharmacokinetic and Statistical Analysis
During each pharmacokinetics part of the study, 10 venous blood samples were collected (pre-dose and at times 0.5 h; 1.h; 1.5 h; 2 h; 4.h; 6.h; 8.h; 12.h; and 24 h after the morning dose of CsA).Venous blood samples (5 ml) were collected into tubes containing ethylenediaminetetraacetic acid (EDTA) for determination of CsA and its main metabolites (AM1 and AM4N) concentrations.
The following pharmacokinetic parameters were individually analysed by model-independent analysis: • Absorption constant (Ka) • Elimination constant (Ke) Single-dose and steady state of CsA and two main primary metabolites, AM1 and AM4N in patients with rheumatic/autoimmune diseases Area under the blood concentration-time curve over 2 h during absorption phase (for CsA) and 24 h interval (for CsA and metabolites AM1, AM4N) was determined using the trapezoidal rule.
The data were evaluated as means and standard deviation (SD).The significance of the intraindividual pharmacokinetic differences during the study period (single dose, steady state) was not tested statistically, due to the small number of patients who participated in the follow-up.
Analyses: Concentrations of CsA in whole blood were determined using high performance liquid chromatography (HPLC).Whole blood was precipitated with zinc sulphate, extracted with diethyl ether, evaporated, dissolved in aqueous methanol and partitioned twice with n-hexane.Chromatography was carried out using a microbore RPcolumn under isocratic elution with acetonitrile-methanolwater (200:80:140, v/v/v) at 70 °C and a detector set at 205 nm, as described previously (Brozmanova et al. 38 ).

RESULTS
The average dose of CsA was 1.28±0.34mg/kg/day.In individual patients, the same dose was maintained during the study, divided into equal doses, taken at 12 h intervals.Basic pharmacokinetic parameters for single and repeated dose administration expressed as means ± SED are summarized in (Table 2).

DISCUSSION
The aim of our study was to evaluate the basic pharmacokinetic parameters of low dose CsA and its metabolites, AM1 and AM4N.
Cyclosporine A is a drug with a narrow therapeutic window and large interindividual differences in pharmacokinetic parameters.The pharmacokinetic data showed that the absorption phase during the first four hours after oral administration of CsA is an area of the greatest intraand interpatient variability.The interpatient variability in pharmacokinetic profiles is smaller for microemulsion forms of CsA than conventional oil-based formulations, indicating more consistent and more predictable absorption of CsA from microemulsion formulations.Due to the need to balance the efficacy and toxicity for this narrow therapeutic range drug, considerable effort has been made to define pharmacokinetic relationship for CsA therapies, mainly in transplant medicine.In autoimmune disease, clinical pharmacokinetic data are available for adult patients with psoriasis and those with rheumatoid arthritis and lupus nephritis (Table 3).When we compare these data with those of transplant patients, it is clear that the doses used in autoimmune diseases are generally lower.In the case of our patients, the average CsA dose was very low (average dose 1,28 mg/kg/day), only half recommended dose.Consequently, the daily exposure (AUC) was lower and thus the risk of toxicity.In these patients, therapy is typically controlled through targeting the initial and maximal dose levels.Monitoring of blood CsA concentrations is usually not performed for the common adjustment of therapy.Routine measurements of serum creatinine and blood pressure can alert the clinician to the onset of toxic manifestations.
There are limited pharmacokinetic data autoimmune diseases.These data were obtained mainly from pharmacokinetic conversion studies using two different CsA formulations -usually conventional oil in water emulsion (Sandimmun ® ) and microemulsion preconcentrate (Neoral ® ).None of these studies described the pharmacokinetics of the main cyclosporine primary metabolites (AM1 and AM4N).
The patients in our observational study were treated with two CsA formulations-Neoral and the Czech alternative Consupren ® , which was developed as an emulsion albeit with a different type of emulsifier, resulting in dif-ferent physical character of the emulsion (water/oil emulsion).
The pharmacokinetic data (CsA AUC ) over the whole 24 h interval were very similar to the findings in other published sources, mainly during steady state conditions.According to the manufacturer's information, after oral administration, patients with RA achieved maximum concentrations (C max ) between 1.5-2 h after administration C max (±SD) was 728 ± 263 ng/ml, through concentration 94 ± 37.7 ng/ml at a dose of 2.37 ±0.36 mg/kg/day, average AUC (± SD) 2641 ± 877 ng/ml.h.
The available results from pharmacokinetic conversion studies are summarized in (Table 3).
The proportion of AM1 metabolite (AUC 0-24 ) after a single dose of CsA corresponded to 40% of the parent compound and approximately 35% of the parent compound in steady state conditions.The proportion of metabolite AM4N was low in both conditions and represented only 3 or 4.5% after a single dose and steady state, respectively.We were unable to compare our data with findings from other studies due to the lack of relevant data on rheumatic diseases.During the 3 month follow-up, changes in some pharmacokinetic parameters were ob- In conclusion, this observation study provides basic pharmacokinetic data on the behaviour of cyclosporine and its metabolites in patients with different rheumatic diseases after single administration and under steady state conditions.In comparison with transplant medicine, this issue is not very well documented.Data on the behaviour of metabolites in this group of patients are still not well documented.ABBREVIATIONS AUC, Area under the curve; C0, Concentration before drug administration; Cl, Clearance; Cmax, Peak concentration; CsA, Cyclosporine A; DMARDs, Disease-modifying anti-rheumatic drugs; EDTA, Ethylenediaminetetraacetic acid; HPLC, High performance liquid chromatography; Ka, Absorption constant; Ke, Elimination constant; MRT, Mean residence time; NSAIDs, Nonsteroidal anti-inflammatory drugs; RA, Rheumatoid arthritis; SD, standard deviation; tmax, Time to peak concentration; t 1/2 el , Half-life of drug elimination; Vd, Volume of distribution, • Half -life of drug elimination (t 1/2el ) • Maximum blood concentrations (C max ) in the dosing interval and corresponding t max.• Area under the blood cyclosporine concentration/time curve over a 2 and 24 h interval (AUC 0-2, AUC 0-24 )

Table 2 .
Pharmacokinetic parameters of CsA and its AM1 and AM4N metabolites after single dose and repeated administration.

Table 3 .
Pharmacokinetics of Neoral ® and Sandimmun ® in Autoimmune Patients.Single-dose and steady state of CsA and two main primary metabolites, AM1 and AM4N in patients with rheumatic/autoimmune diseases served, such as reduction of AUC during the absorption phase, lower C max , increase in Cl and Vd.Unfortunately, due the small number of patients with steady state pharmacokinetics, it was not possible to obtainrelevant statistics evaluation.