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Methotrexate pharmacokinetics after intraarticular injection in patients with rheumatoid arthritis.

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Cell proliferation of synovial cells, monocytes,
lymphocytes, and fibroblasts, is thought to be fundamental to the pathophysiology of rheumatoid arthritis
(RA). Methotrexate (MTX) readily blocks the proliferation of a wide variety of human tissues. Nevertheless,
when injected intraarticularly in small single doses of
2.5-5 mg, MTX does not adequately control the synovitis of RA (1-3). This lack of clinical efficacy may be
due to the cell cycle phase specific action of MTX
whose toxicity is limited to cells in S-phase (4). MTX
can produce a clinical effect only when present in inhibitory concentrations for periods long enough that a significant proportion of the target cells enter S-phase (5).
Thus a dose schedule is required that maintains cytotoxic concentrations of the drug in the synovial tissues
for prolonged periods of time, ideally several times the
cell cycle generation time for cells of the rheumatoid
pannus (6). This can be achieved only if other sensitive
tissues, primarily bone marrow and gastrointestinal epithelium, can be protected from MTX reaching the systemic circulation.
Determination of the pharmacokinetic profile of
intraarticular MTX allowed us to design a dose schedule involving concurrent administration of intraarticular MTX and systemic Leucovorin, a competitive
antagonist of MTX (7,8), that permitted cytotoxic concentrations of MTX to be maintained in the knee joint
for up to 48 hours while completely avoiding systemic
toxicity. These preliminary observations suggest the feaFrom the Department of Medicine, University of California,
San Diego, La Jolla, California 92093.
Supported in part by USPHS grants CA-23334, CA-28100,
AM-07062, and by the UCSD General Clinical Research Center, National Institutes of Health/Division of Research Resources, grant RR00827.
Address reprint requests to Stephen B. Howell, M.D., Department of Medicine T006, University of California, San Diego, La
Jolla, California 92093.
Submitted for publication July 23, 1979, accepted in revised
form October 10, 1979.
Arthritis and Rheumatism, Vol. 23, No. 1 (January 1W)
sibility of developing a methotrexate-based treatment
program using even longer durations of exposure.
Materials and Methods. Three patients with
seropositive RA and one patient with RA variant disease, all with persistent knee effusions uncontrolled by
conventional agents, received either one (1 patient) or
two (3 patients) kjections of 5 mg MTX 24 hours apart
in the same knee joint. Leucovorin 10 mg/mm was administered orally beginning with the first MTX injection and repeated every 6 hours until the serum methotrexate concentration was <10-'M. (MTX and
Leucovorin were kindly supplied by Lederle Laboratories, Pearl River, New York.) MTX concentrations were
measured by radioimmunoassay (9) by using antibody
kindly provided by Diagnostic Biochemistry, Incorporated, San Diego, California. Incorporation of (3H) deoxyuridine into DNA was used to quantitate the degree
of MTX effect on normal human marrow cells in vitro
as described elsewhere (10). In vivo MTX effect on marrow was quantitated by flow cytometric analysis of serially aspirated marrow using a Phywe ICP22 flow cytometer (10). Methotrexate levels in serum and synovial
fluid were arranged by computer into a two compartment model of the form C = A e P
Be-@ using
least mean square approximation.
Results. Pharmacokinetics of intraarticular methotrexate. Four patients were given a total of seven intraarticular injections of 5 mg MTX into the knee. The
geometric mean MTX concentrations in synovial fluid
and serum during the 24 hours following injection are
shown in Figure 1, and the pharmacokinetic parameters
derived from fitting these data to a two compartment
model are presented in Table 1. The elimination of
MTX from the joint was biphasic over the 24 hours following injection, with half lives of 0.54 and 2.90 hours.
Initial synovial fluid MTX concentrations averaged 7 X
lO-"M and had fallen four orders of magnitude to the
range of 8 X 10-*M by 24 hours. Sustained MTX concentrations of 2 x 10-'M or greater are toxic to normal
Figure 1. Synovial fluid (0)and blood (0)
MTX concentrations following intraarticular injection of 5 mg MTX at time zero into the
knee joint. Each point is the geometric mean value (k SD) obtained
on seven courses of treatment in 4 patients. MTX levels were determined by radioimmunoassay.
human cells (10); thus on the average, a cytotoxic concentration of drug was present in synovial fluid for at
least 24 hours after the injection of 5 mg of MTX. Clinical estimates of knee effusion volumes in these patients
ranged from 30-70 ml; the calculated mean volume of
distribution (V,) based on the derived pharmacokinetic
parameters was 69 ml. The mean clearance of MTX
from the joint was 0.28 ml/min. The highest mean concentration of MTX found in serum was 1.9 X 10-7M,
and this occurred 2 hours after MTX injection.
Throughout the whole 24 hour period, the mean joint
MTX concentration exceeded the serum concentration
by a minimum of 10-fold, and at the time of the peak
serum MTX, the ratio was more than 250.
MTX/Leucovorin ratio for protection of normal
human marrow. The MTX to Leucovorin ratio required
to completely protect normal human bone marrow cells
from MTX was determined for MTX concentrations of
1.0 X 10-6Mand 3.0 X lO-’M. These two concentrations
were selected as representative of the range of peak
serum MTX levels achievable following intraarticular
injection of 5 mg of MTX. Methotrexate-induced inhibition of dihydrofolate reductase as measured by (3H)
deoxyuridine incorporation into DNA was used to
quantitate the degree of Leucovorin protection. The re-
sults shown in Figure 2 demonstrate a steep dose-response relation between the MTX/Leucovorin ratio and
the degree of inhibition of dihydrofolate reductase. For
an MTX concentration of 3 x lO-’M, a ratio < 1:3 was
required for full protection. A ratio of 1: 10 was required
for a level of 1 X lO-‘M MTX. These results suggest
that if a sufficient concentration of Leucovorh is provided to the systemic circulation to block the effect of
MTX reaching the blood, and if the joint methotrexate
concentration exceeds that of blood by a factor of 10 or
greater, then the MTX/Leucovorin ratio in the joint
will be high enough to permit the full cytotoxicity of
MTX to be expressed against the synovial tissues.
I n vivo protection of marrow during intraarticular
MTX therapy. To block the cytotoxicity of MTX reaching the systemic circulation, all patients were given Leucovorin 10 mg/mm by mouth every 6 hours for 24 hours
following the intraarticular injection. Methotrexate
causes a characteristic change in the cell cycle phase distribution of bone marrow cells, with an accumulation of
cells in S-phase that is detectable by flow cytometry
(10). Marrow aspirates were performed at time zero and
again in 48 hours in 2 patients receiving 2 intraarticular
injections of MTX 24 hours apart. In one patient 16.8%
of cells were in S-phase before treatment, and this fraction declined to 10.7%at 48 hours. In the other patient
13.6% of cells were in S-phase before treatment, and
13.7% at 48 hours. These results demonstrate no accumulation of cells in S-phase, indicating protection by
Leucovorin. In addition there were no clinical or laboratory signs or symptoms of local or systemic toxicity.
Specifically there was no exacerbation of inflammation
in the knee joints, and no patient developed nausea,
vomiting, stomatitis, diarrhea, alopecia, myelosuppression, or any change in renal or liver function tests.
Discussion. This analysis of the pharmacokinetics of intraarticular MTX provides a reasonable
explanation for the failure of MTX to produce remission of inflammation in RA joints when used in single
small doses (1-3). The joint fluid elimination half-life of
Table 1. Pharmacokinetic parameters describing the disappearance
of MTX from the rheumatoid knee joint
a (h-’)
P 0-7
T1/2a (h)
T 1/2P (h)
A (X 10-4M/liter)
B (X 10-4M/liter)
Vd (ml)
Clearance (ml/min)
Calculated value f SE
0.24 f 0.012
0.54 & 0.046
2.90 f 0.15
6.14 f 0.47
0.29 f 0.58
0 1:l
Methotrexate/Leucovorin ratio
Figure 2. Inhibition of (3H) deoxyuridine incorporation into human
marrow mononuclear cells as a function of MTX/Leucovorin ratio;
0 = 3 x 10-7M MTX 0 = 1 x 10-6M MTX. Marrow cells were incubated with MTX and varying concentrations of Leucovorin for 1
hour at 37°C. (3H) deoxyuridine was added and the incubation continued for an additional hour, and then the amount of label incorporated into nucleic acids was determined by scintillation counting. The
data are plotted as a percent of the (3H) deoxyuridine incorporation
into cells not exposed to MTX.
2.9 hours is short relative to the cell cycle generation
time of even rapidly dividing tissues such as marrow
(1 l), and it is very unlikely that the exposure to MTX
resulting from a single injection of drug is of adequate
duration to permit even a partial chemical synovectomy. The MTX disappearance curve is biphasic and
can be adequately described by a two compartment
model. The initial half-life of 0.54 hours probably represented distribution of drug throughout the joint space.
The mean volume of distribution calculated from the
pharmacokinetic parameters was 69 ml which correlates
well with clinically estimated effusion volumes. The second half-life of 2.9 hours reflects a relatively low clearance of MTX from the joint of 0.28 ml/min. This articular clearance rate is several orders of magnitude less
than that of blood, which exceeds 100 ml/minute (12),
and probably accounts for the observation that a greater
than 10-fold concentration gradient was maintained be-
tween synovial fluid and blood throughout the initial 24
hours after injection.
In a preliminary attempt to achieve longer duration exposures to MTX, we found that it was feasible to
maintain cytotoxic levels of MTX in knee joints for up
to 48 hours without causing systemic toxicity by using
repeated intraarticular injections of MTX and oral Leucovorin. Peak serum MTX concentrations ranged between 1.3 x lO-’M and 2.7 x 10-7M. Our experiments
suggested that a MTX/Leucovorin ratio of less than 1:3
was required to protect human marrow cells from MTX
concentrations in this range, which is in close agreement
with previous studies of mouse marrow (13) and human
lymphoid cells (14). Although measurements of serum
reduced folate were not made in these patients, Mehta
(15) reported steady-state levels of approximately 5 x
10-7M using a similar dose schedule of Leucovorin.
Even if Leucovorin equilibrated into the synovial fluid,
little protection would be expected in this compartment
where the MTX concentration ranged from 10 to
>1000-fold higher than the corresponding serum level.
Even a 48 hour exposure to MTX failed to produce a favorable therapeutic response in the knee joints
of this small group of patients, and further study is required to determine the efficacy of longer exposures.
Application of the pharmacokinetic information derived from this study should permit this to be done with
safety. MTX may not prove to be the cytotoxic drug of
choice for the treatment of RA. However, the same
pharmacologic principles are relevant to other antimetabolites for which antagonists are available.
Acknowledgments. Our thanks to Ms Deborah
Gamelin and Ms Rita Tamerius, RNP for their secretarial and skilled nursing services, and to Drs. John
Rice and Richard Olshen for mathematical assistance.
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pharmacokinetics, patients, injections, arthritis, methotrexate, rheumatoid, intraarticular
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