Combined oral cyclosporin and methotrexate therapy in patients with rheumatoid arthritis elevates methotrexate levels and reduces 7-hydroxymethotrexate levels when compared with methotrexate alone

R. I. Fox, S. L. Morgan1, H. T. Smith2, B. A. Robbins3, M. G. Choc4 and J. E. Baggott1,

Division of Rheumatology, Scripps Memorial Hospital and Research Foundation, La Jolla, California,
1 Departments of Nutrition Sciences and Medicine, University of Alabama at Birmingham, Birmingham, Alabama,
2 Novartis Pharmaceutical Corporation, East, Hanover,
3 Bayer Consumer Care Division, Morristown and
4 Aventis, Bridgewater, New Jersey, USA


    Abstract
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 Abstract
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 Methods
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 Discussion
 References
 
Objective. To study the pharmacokinetics of methotrexate (MTX) plus cyclosporin A (CSA) in patients with rheumatoid arthritis (RA).

Methods. On day 1 of the study, patients with RA receiving stable doses of MTX had blood and urine levels of MTX and its metabolite 7-hydroxymethotrexate (7-OH-MTX) measured post oral dosing of the drug. MTX was then discontinued and CSA therapy was started on day 8. On day 20, blood levels of CSA and CSA metabolites were measured post drug dosing. On day 23, MTX therapy was restarted and levels of MTX, CSA and their metabolites were again measured as described above.

Results. In the 30 patients, coadministration of CSA and MTX led to a 26% increase in mean peak plasma MTX concentration (P < 0.01), an 18% increase in the mean plasma MTX concentration area under the curve (AUC, P=0.01) and an 80% decrease in plasma 7-OH-MTX AUC (P < 0.01). In 13 patients receiving a 10 mg MTX dose, CSA reduced urinary 7-OH-MTX excretion by 87% (P < 0.01) without altering MTX excretion. MTX did not alter the pharmacokinetics of CSA or its metabolites.

Conclusion. CSA may block oxidation of MTX to its relatively inactive metabolite, 7-OH-MTX, thereby potentiating MTX efficacy.

KEY WORDS: Methotrexate, 7-Hydroxymethotrexate, Cyclosporin A, Pharmacokinetics.


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
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Rheumatoid arthritis (RA) is a chronic inflammatory disease. Treatment options for patients with progressive disease include disease-modifying anti-rheumatic drugs (DMARDs) such as methotrexate (MTX), D-penicillamine, antimalarials, leflunomide, gold compounds, sulphasalazine and cyclosporin A (CSA). The need for early aggressive treatment and dissatisfaction with the long-term disease outcome in RA using single agents has resulted in an interest in using combinations of these agents, MTX being the cornerstone drug [1, 2].

On the basis of modest benefit in RA patients treated with CSA alone, Tugwell et al. [3] evaluated the safety and efficacy of CSA plus MTX in 148 RA patients in a 6-month double-blind trial. They were randomized to receive either (i) cyclosporin (2.5 or 5 mg/kg/day) plus MTX (7.5–10 mg/week) or (ii) placebo plus MTX (7.5–10 mg/week). Patients in the CSA plus MTX group, when compared with patients given MTX alone, showed significant improvement in six of seven clinical assessments and the degree of disability measured by the Health Assessment Questionnaire. The CSA plus MTX therapy did not substantially increase the adverse effects of MTX in this study [3]. Stein and Pincus [4] completed an open-label extension of the previous study. Patients who had previously received CSA and MTX continued for an additional 24 weeks (group 1), while the MTX-only group had CSA added to their regimen for 24 weeks (group 2). Clinical improvements were maintained in group 1, while group 2 had significant improvements in four of seven clinical assessments. The type and number of adverse effects were reported to be comparable with those in studies using CSA alone in RA. Thus, it appears that CSA plus MTX is a more efficacious therapy than MTX alone and is no more toxic.

Oral low-dose MTX achieves peak concentrations in plasma in 1–2 h [5]. MTX elimination varies with renal function and concurrent use of NSAIDs, and prolonged use of MTX itself may also cause deterioration in renal function that increases the elimination half-life of MTX [5]. Previous studies in RA patients receiving MTX showed that the drug is metabolized to 7-hydroxymethotrexate (7-OH-MTX) [6]. The enzyme responsible for this metabolism is aldehyde oxidase [7]. This enzyme is found in liver and kidney and there appears to be substantial individual variation in the level of this enzyme in the human liver [8]. Also, 7-OH-MTX is the major drug metabolite found in the bone marrow of children 24 h after an oral MTX dose (30 mg/m2) and it is two- to five-fold higher than the concentration of MTX in this tissue [9]. Importantly, 7-OH-MTX, in the rat adjuvant arthritis model, is less efficacious than MTX [10].

The bioavailability of CSA, in the oral formulation as Sandimmun®, has relatively high inter- and intra-patient variability in transplant patients [11, 12]. Bile flow, food composition and gastrointestinal motility influence the absorption of CSA in the Sandimmun formulation. A microemulsion of CSA (Neoral®) was developed to increase the bioavailability of CSA and produce more consistent pharmacokinetics [13]. In patients with autoimmune disease, the bioavailability of Neoral is 20–30% higher than that of Sandimmun [14].

CSA is reported to decrease renal blood flow, resulting in decreased creatinine clearance [15]. Thus, changes in cellular MTX levels in patients receiving MTX plus CSA could result from decreased MTX clearance. Thus, one hypothesis to explain the increased clinical efficacy of MTX plus CSA in RA would be a simple increase in cellular MTX levels due to its decreased renal clearance. Indeed, it was this hypothesis that led to the pharmacokinetic studies reported here. Another possibility is that MTX could lead to the formation of certain CSA metabolites that are nephrotoxic and thus reduce the clearance of both drugs [16]. Alterations in either MTX or CSA bioavailability and pharmacokinetics could contribute to the therapeutic benefit noted when MTX plus CSA is used in RA patients, and studies were performed to answer these questions.


    Methods
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Subjects and protocol
Subjects who satisfied the selection criteria and fulfilled the revised ARA criteria for defined RA [17] were enrolled as in-patients at the General Clinical Research Center of Scripps Clinic and the Halifax Clinical Research Institute on study days 1–3 and 20–26. Institutional Review Board approval was obtained at both sites. Vital signs, physical examination, electrocardiogram and laboratory testing (such as complete blood count with differential counts), quantitative excretion of sodium, and magnesium were evaluated. Patients with a history of uncontrolled hypertension, irritable bowel syndrome, cholecystitis, major gastrointestinal tract surgery, abnormal liver function tests, pancreatitis or impaired renal function were excluded. Patients were chosen for this study without bias of age or gender. Thirty-three patients (seven male, 26 female) were enrolled. The age of subjects ranged from 26 to 74 yr, with a mean of 55±12.8 yr. Mean weight was 72.3±14 kg and height 166.5±10.4 cm.

Each RA patient was receiving a stable weekly dose of MTX (7.5–22.5 mg/week) for 2 months prior to being enrolled. Coadministration of certain non-steroidal anti-inflammatory drugs (NSAIDs) was allowed during the study. Starting on day 1 of the protocol, plasma samples for MTX and 7-OH-MTX levels were drawn through an indwelling catheter up to 24 h after the dose of MTX. Plasma samples were also obtained at 36, 48 and 72 h, and 72-h urine was collected. The patients then discontinued their MTX. On day 8, daily dosing of CSA (Neoral; 1.5 mg/kg every 12 h or total 3 mg/kg/day) was initiated and continued until day 20, when whole blood samples were obtained at the times stated above. Whole blood was obtained for measurement of CSA levels; however, inadvertently only plasma was obtained from some patients, thus CSA blood levels are reported for only a subset of the patients (n=17). CSA dosing was continued and on day 23 each patient received their weekly MTX dose with CSA. Whole blood, plasma and urine samples were again obtained as described above. Clearance measurements were obtained with patients on a fixed diet. The clearance was calculated as follows: (urinary concentration x urine volume/time)/plasma concentration.

Thirty patients (12 from Scripps Clinic and 18 from Halifax Clinical Research Institute) completed all 3 periods of the study. A complete set of whole blood, plasma, and urine samples for MTX, 7-OH-MTX, CSA and CSA metabolites analysis were obtained from patients at Scripps. At Halifax, a complete set of whole blood, plasma and urine samples was available for five of 18 patients because of the error in collection described above.

All patients received folic acid (1 mg/day) throughout the study. The dose of CSA for pharmacokinetic measurements was administered with 300 ml of water after a fast of at least 2 h, and subjects fasted at least 2 h after dosing. Meals were served at intervals to allow for this schedule. Intake of xanthine-containing food or beverages (i.e. coffee, tea, sodas and chocolate) was prohibited for 2 h before or after any drug dose. Grapefruit and grapefruit juice, alcohol consumption or strenuous exercise were not permitted during the pharmacokinetic studies. During the study period, patients received stable doses of ketoprofen (Orudis®, Aventis Pharmaceuticals, Inc, Bridgewater, NJ, USA; 33 mg/day), piroxicam (Feldene®, Pfizer, Providence, RI, USA; 10 mg/day) or flurbiprofen (Ansaid®, Pharmacia & Upjohn Co., Kalamazoo, MI, USA; 100 mg twice daily). Patients were receiving a wide array of NSAIDs prior to the study, and to minimize the potential effects of NSAIDs during MTX and CSA pharmacokinetic studies all patients received ketoprofen, piroxicam or flurbiprofen during the study period [5]. No other drugs or over-the-counter medications (including vitamins and analgesics) that might influence drug metabolism were allowed during the 4 weeks prior to the study or during the study.

Drug and metabolite analyses
Plasma and urine levels of MTX and 7-OH-MTX were assayed by Pharmaco International (Richmond, VA) using an HPLC (high-performance liquid chromatography) method with ultraviolet detection. Daily standardization of the assay for both MTX and 7-OH-MTX was conducted using seven calibration standards over the range of 5–500 ng/ml. A linear weighted (1/concentration2) least-squares regression algorithm was used to plot peak height ratio (MTX or 7-OH-MTX/internal standard) vs concentration. Unknown and quality control concentrations for both components were calculated by inserting the observed peak height into the appropriate regression equation. Concentrations were reported in ng/ml. Whole blood CSA and four metabolites (AM1, AM9, AM1c and AM4N) were determined at the Immunosuppressive Drug Research Laboratory (University of Alberta, Canada) by HPLC with ultraviolet detection.

Statistics
The comparisons of the pharmacokinetic parameters were made using the Estimate statement of the SAS Proc GLM statistics program (SAS Institute, Inc, Cary NC, USA). Renal function data and mean urinary excretion of MTX and 7-OH-MTX were compared by the paired t test. Data are reported as means ± standard deviations.


    Results
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 Methods
 Results
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Laboratory values
Mean baseline values for serum creatinine, blood urea nitrogen, uric acid and potassium were all within normal limits. None of these parameters exhibited any clinically relevant or statistically significant mean change during the study period. Although a small change in creatinine clearance was noted during administration of MTX with CSA (Table 1Go), it did not reach statistical significance. Mean MTX clearance was not significantly altered by CSA therapy (P > 0.05); however, there was a decrease (Table 1Go). Mean renal clearance of 7-OH-MTX was significantly decreased from 14.0 to 8.4 ml/min when CSA was given with MTX. There was little change in the clearance of sodium and a significant increase in clearance of magnesium (Table 1Go). Mean white blood counts (7.6±2.9 109/l) were within normal limits at baseline. One episode of leukopenia occurred on MTX alone, which continued during combination MTX and CSA therapy. Mean baseline liver function tests [SGOT (serum glutamic-oxaloacetic transaminase), SGPT (serum glutamic-pyruvic transaminase), alkaline phosphatase and GGT ({gamma}-glutamyl transferase)] were all within normal range at baseline.


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TABLE 1. Change in renal function data (±S.D.) in patients receiving MTX and MTX plus CSA

 

Pharmacokinetics
As shown in Fig. 1Go, coadministration of CSA led to a 26% increase in the mean peak plasma MTX concentration (Cmax, P < 0.01), a 60% increase in the time to peak plasma concentration (tmax, P<0.01) and an 18% increase in mean plasma MTX concentration area under the curve (AUC, P=0.01). The data in Fig. 2Go show that CSA with MTX lowered the mean plasma 7-OH-MTX concentration AUC to 20% of that observed after administration of MTX alone (P < 0.01). The plasma Cmax for 7-OH-MTX was also reduced, to 26% (P < 0.01) of that observed with MTX alone. The mean MTX volume of distribution for the two treatments, using the mean clearance and half-life values, was unchanged and was estimated to be 41.7 l for MTX alone and 33.7 l for the combination of CSA and MTX.



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FIG. 1. Plasma concentrations (mean±S.D.) of MTX after the administration of MTX with CSA (Neoral) and MTX alone in patients with RA. The line with closed circles represents mean MTX concentrations in patients (n=30) who received MTX with CSA (Neoral) and that with open circles represents those in patients (n=30) who received MTX alone.

 


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FIG. 2. Plasma concentrations (mean±S.D.) of 7-OH-MTX after administration of MTX with CSA (Neoral) and MTX alone in patients with RA. The line with closed circles represents mean 7-OH-MTX concentrations in patients (n=30) who received MTX and CSA (Neoral) and that with open circles represents those in patients (n=30) who received MTX alone.

 
An analysis of the 72-h urinary excretion of MTX and 7-OH-MTX was consistent with the plasma data above. In a subset of 13 patients receiving a 10 mg MTX dose, the mean excretion of MTX with and without CSA was 7.89±1.66 and 8.37±2.47 mg respectively (P > 0.1). In contrast, mean 7-OH-MTX excretion was 0.09±0.11 mg and 0.69±0.45 mg with and without CSA respectively (P < 0.01). Thus, CSA plus MTX lowered 7-OH-MTX excretion by 87% but reduced MTX excretion by only 6%. In all of these patients, 7-OH-MTX excretion decreased. In three patients, no urinary 7-OH-MTX was detected when MTX plus CSA was given. The CSA dose was not significantly correlated with a reduction in MTX metabolism to 7-OH-MTX, and this finding was probably the result of adjusting the CSA dose to body weight.

In contrast to the alteration of the pharmacokinetics of MTX by CSA, the combination of CSA plus MTX produced less than a 7% change in CSA mean plasma concentration AUC and Cmax (P>0.05) (Fig. 3Go). Similar results were observed for AM1 and AM9, the major metabolites of CSA, as well as the minor metabolites AM1c and AM4N (data not shown).



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FIG. 3. Blood CSA concentrations (mean±S.D.) after the administration of CSA (Neoral) alone and CSA with MTX in patients with rheumatoid arthritis. The line with closed circles represents mean CSA concentrations in patients (n=17) who received CSA (Neoral) with MTX and that with open circles represents those in patients (n=17) who received CSA alone.

 

Adverse events
One serious study-related adverse event was noted. On the third day of treatment with CSA only, a patient (66-yr-old white female with a history of hypertension for 28 yr) experienced severe hypertension and chest pain of gastrointestinal origin requiring an emergency room visit. Of importance, her primary physician had discontinued her blood pressure medication (diltiazem) 2 weeks prior to starting CSA. No electrocardiogram or laboratory abnormalities were noted in the emergency room. The patient was discontinued from the protocol, and her diltiazem treatment was reinstated.

On day 8, one patient had elevations of liver function tests (less than 2-fold elevation) that normalized during the protocol. Eight subjects reported adverse events with MTX alone, 17 with CSA alone and nine with the combination therapy. The most common adverse events were gastrointestinal disturbances (e.g. nausea) in 13 subjects and nervous system disorders (e.g. headache and dizziness) in 14 subjects.


    Discussion
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 Methods
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Although CSA has been used as the sole agent for the treatment for RA, the results are modest in comparison to those with other DMARDs [13, 18]. However, CSA plus MTX is more efficacious than MTX alone [3, 4], and thus CSA is most frequently used in combination with MTX in RA patients who have had an inadequate response to MTX alone. Because of its efficacy in controlling organ allograft transplant rejection, there is no question that CSA is an effective immunosuppressive agent [11]. Thus, the beneficial effects in RA of CSA in combination with MTX could be entirely due to CSA providing additional suppression of the immune system. In addition to this mechanism, CSA could also be beneficial by altering the pharmacokinetics of MTX.

In the absence of CSA therapy, plasma curves for MTX and 7-OH-MTX (Figs 1 and 2GoGo) are similar to those reported previously in patients receiving low oral doses of MTX [6, 9]. The plasma MTX AUC was elevated by 18% when CSA was coadministered. This increase in plasma MTX AUC may be partially explained by a decrease in MTX plasma clearance level. However, the difference in the plasma AUC for MTX (Fig. 1Go) was essentially equal to the differences in the plasma AUC for 7-OH-MTX (Fig. 2Go) for the 0–24 h time period. This suggests that CSA increases the plasma AUC of MTX by the same amount that it decreases the plasma AUC of 7-OH-MTX, and further suggests that the inhibition of 7-OH-MTX formation by CSA may explain a substantial part of the differences in MTX pharmacokinetics shown in Figs 1 and 2GoGo. On the other hand, the greatly decreased plasma 7-OH-MTX levels in RA patients receiving MTX plus CSA could be a result of increased rates of renal secretion and excretion of 7-OH-MTX, leading to decreased blood 7-OH-MTX levels. However, decreased (not increased) 7-OH-MTX urinary excretion was observed in a subset of patients receiving 10 mg of MTX plus CSA, and is not consistent with this explanation.

The oxidation of MTX to 7-OH-MTX is catalysed by aldehyde oxidase [7], an enzyme that exhibits a high degree of variability in human liver [8]. Although changes in aldehyde oxidase activity have not been reported in RA patients receiving CSA, reduced oxidation of MTX to 7-OH-MTX in the rabbit has been reported when MTX is coadministered with 4'-(9-acridinylamino)methanesulfon-m-anisilide, which is known to be a potent inhibitor of aldehyde oxidase [19]. Thus, it is possible that CSA and/or its metabolites inhibit aldehyde oxidase. Also, in a recent report on five ulcerative colitis patients receiving MTX followed by MTX plus CSA, decreased blood 7-OH-MTX levels were reported with MTX plus CSA therapy, which is consistent with the results presented here [20].

The preponderance of 7-OH-MTX (over MTX) in bone marrow in patients receiving low-dose MTX suggests that this metabolism is an important factor governing the efficacy of MTX [9]. In human cell culture experiments, MTX is 4- to 17-fold more cytotoxic than 7-OH-MTX [21]. The efficacy of 7-OH-MTX in rat adjuvant arthritis has been shown to be highly variable but, in general, is approximately one-eighth that of MTX [10]. If these substantial differences in cytotoxicity/efficacy of MTX and 7-OH-MTX can be translated to RA, blockage of the metabolism of MTX to 7-OH-MTX may improve the efficacy of MTX. Therefore, independently of its own efficacy in treating RA, CSA could potentiate the action of MTX by blocking its metabolism to 7-OH-MTX, a less efficacious compound. It is important to note that our findings do not rule out an additional beneficial effect of CSA by suppressing the immune system in RA.

CSA potentiation of MTX efficacy may translate into using a lower MTX dose when this combination therapy is started. In addition, it may be desirable to reduce the amount of 7-OH-MTX formed as this metabolite is less water-soluble than MTX and is formed in the kidney and accumulates there [22]. Although not convincingly established, it is likely that 7-OH-MTX is responsible, at least in part, for the reduced renal function seen in some RA patients treated with low-dose MTX [23]. CSA plus MTX therapy may reduce 7-OH-MTX-induced renal toxicity. To our knowledge, this is the first report of combination therapy with MTX which alters its metabolism to 7-OH-MTX, and raises the possibility that other drugs may act similarly. We speculate that CSA may not be unique in its ability to reduce 7-OH-MTX formation; other drugs frequently used in combination with MTX should be investigated.


    Acknowledgments
 
This study was in part supported by the General Clinic Research Center of Scripps Clinic (NIH-M0I-RR000833) and grants from the Novartis Pharmaceutical Corporation and the National Institutes of Arthritis and Musculoskeletal and Skin Diseases and the Office of Dietary Supplements (1R29 AR42674).


    Notes
 
Correspondence to: J. E. Baggott, 326 Webb, Department of Nutrition Sciences, University of Alabama at Birmingham, Birmingham, AL 35294–3360, USA. E-mail: jwrainey{at}uab.edu Back


    References
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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Submitted 27 September 2002; Accepted 9 January 2003