1. A NEW LOOK AT RA Interactive Hot Topics Series
Understanding the Pharmacokinetics of Methotrexate: It’s Not So Simple
I’m going to spend the next 15 minutes discussing the pharmacokinetics of methotrexate, which is an important topic regarding a drug that the vast majority of rheumatologists use in the treatment of their patients with rheumatoid arthritis.
MP-RA-0285

2. Pharmacokinetics: What Does the Body Do to the Drug?
Absorption from the subcutaneous site into the circulation
Distribution to tissue compartments or binding to plasma proteins (albumin)
Metabolism and degradation after absorption into circulation
Excretion via both renal and hepatic mechanisms
I’m sure that all you remember this from your first or second years in medical school, but just to be sure: pharmacokinetics is simply a description of how the body handles a drug and it is typically divided into absorption, distribution, metabolism, and excretion or elimination. A second topic that is usually considered along with pharmacokinetics is pharmacodynamics. This is a description of what the drug does to the body.
Pharmacodynamics: What Does the Drug Do to the Body?

3. Absorption

4. Methotrexate (MTX) Pharmacokinetics: Absorption
Several parameters describe the two key aspects of absorption:
How much of the drug dose is absorbed:
Maximum plasma concentration (Cmax)
Area under the curve (AUC)
Bioavailability (F):
100% with an intravenous (IV) dose
For other routes of administration: AUC for dose administered for the route in question divided by the AUC for IV delivery
How fast is the drug absorbed:
Time to maximum plasma concentration (Tmax)
Several parameters describe the two key aspects of absorption. The amount of drug absorbed is described by
maximum plasma concentration (abbreviated Cmax),
area under the time versus plasma concentration curve or AUC, and;
bioavailability (abbreviated as F). Bioavailability is (by definition) 100% when a drug is given intravenously (IV). For other routes of administration, it is the AUC for dose administered for the route in question divided by the AUC for IV delivery. Consideration of absorption also includes how fast is the drug absorbed, which is reflected by time to maximum plasma concentration or Tmax.

5. MTX Time vs Plasma Concentration Curve after Oral Administration
600
Cmax
500
400
MTX Plasma Concentration (µg/L)
300
200
100
This slide shows a time versus plasma concentration curve for orally administered methotrexate. The study included 15 patients with rheumatoid arthritis. The slide highlights the high Cmax and the rapid decline in plasma drug concentration after oral administration. Tmax is the time that Cmax occurs and the mean value for patients in this study was 1.2 hours. 4 8 12 16 20 24
Time in hours
Adapted from Hoekstra M, et al. J Rheumatol. 2006;33:

6. MTX Time vs Plasma Concentration Curve after Oral Administration
600
500
400
AUC
MTX Plasma Concentration (µg/L)
300
200
100
This is the same curve shaded to make clear the AUC which provides a good reflection of total exposure to the drug. 4 8 12 16 20 24
Time in hours
Adapted from Hoekstra M, et al. J Rheumatol. 2006;33:

7. Bioavailability (F) of Oral MTX Is Highly Variable
AUC for oral administration
AUC for IV administration F=
Oral Bioavailability of 10 mg/m2 MTX (Oral AUC/IV AUC)
The bioavailability of oral methotrexate varies greatly from one patient to another. This slide shows results from a study in which the pharmacokinetics and bioavailability of low-dose methotrexate (10 mg/m2) were evaluated in 41 subjects who had rheumatoid arthritis as defined by the American Rheumatism Association criteria. Subjects received methotrexate in a single oral dose and a single IV dose one week apart. The results shown are the values for AUC after oral dosing divided by that for IV dosing. This is a measure of oral bioavailability and it is evident that it varies greatly from one subject to another.
Subject Number
Adapted from Herman RA, et al. J Pharmaceutical Sci. 1989;78:

8. MTX Concentration (µM)
Time vs Plasma Concentration Curves for Oral MTX Vary Greatly from One Patient to Another
1.2
1.0
0.8
0.6
0.4
0.2
MTX Concentration (µM) 1 2 3 4 5 6
Time (hr)
Patient #1
Patient #2
This slide, from a study carried out in cancer patients over 30 years ago, is instructive for two reasons. First, it again demonstrates the differences in AUC for patients who received similar doses of oral methotrexate. Second, it shows the great variability in the overall time-related patterns of exposure. For Patient #1, there is a clear peak and decline like that shown in the previous slide. In contrast, Patient #2 has very slow rise in methotrexate concentration and exposure extending past the end of the time when plasma samples were collected.
Balis FM, et al. Cancer Res. 1983;43:

9. The Bioavailability of Oral MTX Declines with Dose80 60 40 20
Dosage (mg/m2) 70 50 30 10
F (%)
The bioavailability of oral MTX declines with higher doses1-3
Absorption may decline as much as 30% with dose increases3
In the study shown, F for an oral MTX dose of <40 mg/m2 was 41% and that for a dose of ≥40 mg/m2 was 17% (P<0.0081)
The pharmacokinetics of orally administered methotrexate are dose dependent. Most notably, the bioavailability of the drug declines with higher doses. The graph on this slide is from a small number of pediatric patients who received methotrexate both orally and IV so that bioavailability could be calculated accord to the formula shown in a previous slide. The decline in bioavailability with dose is readily apparent.
van Roon EN, van de Laar MA. Clin Exp Rheum. 2010;28:S27-S32.
Bannwarth B, et al. Clin Pharmacokinet. 1996;30:
Teresi ME, et al. J Pedriatr. 1987;110:

10. Decline in Bioavailability with Increase Also Occurs with MTX Doses Employed to Treat RA
This effect is reflected by declining bioavailability with oral dose (reflect by measuring AUC/dose)1,2
140
120
100 80 60 40 20 10 15 25
Dose (mg)
AUC0-24/Dose1 90 80 70 60 50 40 30
7.5 15
22.5
Dose (mg)
AUC0-t/Dose2 20 10
A decline in methotrexate exposure with increasing oral doses has also been observed in adult patients with rheumatoid arthritis and healthy volunteers. This slide shows results from two studies in which subjects received different doses of oral methotrexate. These two studies did not deliver methotrexate IV so bioavailability could not be determined. However, they both showed that exposure to methotrexate, as reflected by AUC, declined with increasing dose.
Adapted from Schiff MH, et al. Ann Rheum Dis. 2014;73:
Pichlmeier U, Heuer KU. Clin Exp Rheumatol. 2014;32:

11. The Limited Bioavailability for Oral MTX Results from Saturable Transport Out of the Intestinal Lumen
MTX/folate transport into intestinal cells:1-3
Reduced folate carrier (RFC)
Proton-coupled folate transporter (PCFT)
Folate receptor α and b (FRα/b) isoforms
PCFT is probably the main transporter for folic acid and MTX in the small intestine
Both RFC and PCFT are saturable: they can only transport so much MTX into the cell regardless of how much is in the intestinal lumen
RFC
PCFT
MTX
Fra/b
OP- H+
Endosome
The reason that absorption of oral methotrexate is limited is almost certainly the result of the fact that the drug is transported out of the intestinal lumen by two saturable transport molecules: the reduced folate carrier (abbreviated RFC) and the proton-coupled folate transporter (abbreviated PCFT). The term saturable simply means that no matter how much methotrexate is present in the intestine, these transporters can only carry so much into cells over a given period of time.
Qiu A, et al. Cell. 2006;127:
Blits M, et al. Arthritis & Rheumatism. 2013;65:
Murakami T, Mori N. Pharmaceuticals. 2012;5:

12. MTX Plasma Concentration (µg/L)
Bypassing Intestinal Absorption with SC Delivery of MTX Increases Bioavailability
100
200
300
400
500
600 4 8 12 16 20 24
Time in hours
Oral SC
MTX Plasma Concentration (µg/L)
It has been known for quite some time that bypassing intestinal absorption can increase the bioavailability of methotrexate. This slide shows the time versus plasma concentration curve for oral methotrexate (in purple) that you have seen in prior slides. It also shows a corresponding curve for the same methotrexate dose delivered by subcutaneous (abbreviated SC, in green) injection.
Hoekstra M, et al. J Rheumatol. 2006;33:

13. MTX Plasma Concentration (µg/L)
Bypassing Intestinal Absorption with SC Delivery of MTX Increases Bioavailability
600
Lower Cmax with SC MTX and small reduction in AUC
500
Oral SC
400
MTX Plasma Concentration (µg/L)
Large gain in AUC with SC MTX
300
200
100
The shaded are on this slide shows the increased exposure; that is, greater AUC for SC versus oral methotrexate. Results from this study showed that the bioavailability of oral methotrexate was only 64% of that for SC administration. 4 8 12 16 20 24
Time in hours
Hoekstra M, et al. J Rheumatol. 2006;33:

14. Oral MTX Exposure Plateaus Above 15 mg/week
3000
MTX Dose (mg)
AUC (ngh/mL)
2600
2200
1800
1400
1000 10 15 20 25
Oral MTX (n=47)
SC MTX Auto Injector (n=47, thigh)
This slide shows results from a 12-week, randomized, open-label 3-way crossover study of 49 adult patients with rheumatoid arthritis, all of whom were undergoing treatment with methotrexate for ≥3 months prior to randomization. The results showed that AUC for oral methotrexate plateaued with doses >15 mg/week while that for SC methotrexate increased progressively with dose.
Schiff MH, et al. Reproduced from Ann Rheum Dis. 2014;73: © 2014 with permission from BMJ Publishing Group Ltd.

15. Splitting High Dose Oral MTX to Improve Bioavailability
Crossover study in 10 adult RA patients comparing a single oral dose of MTX with the same dose split and separated by 8 hours
Median MTX dose = 30 mg weekly (range mg)
Bioavailability of split dose 28% higher than single dose (p=0.007)
While the bioavailability of the high dose oral MTX was enhanced by splitting the dose, it remained ~10% lower than SC administration of the same dose
Given that absorption of oral methotrexate is limited by saturable transport out of the gut, it is reasonable to suggest that splitting high-dose oral methotrexate should improve bioavailability. To examine this hypothesis, a crossover study was done in 10 patients with rheumatoid arthritis and compared a single oral mg/week dose of methotrexate with the same dose split and separated by 8 hours. The bioavailability of the split dose versus the single dose increased by 28%; however, this was ~10% lower than SC administration of the same dose of methotrexate.
Adapted from Hoekstra M, et al. J Rheumatol. 2006;33:

16. SC Administration Lowers Interpatient Variability in the AUC for MTX
SC administration of methotrexate has another important advantage over oral dosing: it results in much lower inter-patient variability in bioavailability. Inter-subject variability in the response to a given treatment can be determined by calculation of the coefficient of variation for the treatment group. This measure is simply the mean value divided by the standard deviation. The results on this slide are from one of several studies that provided information about bioavailability of SC methotrexate and they show that inter-patient variability in AUC for a given methotrexate dose is lower with SC versus oral administration.
SC, subcutaneous; AUC, area under the curve
Adapted from Pichlmeier U, Heuer KU. Clin Exp Rheumatol. 2014;32:

17. Distribution

18. Metabolism

19. MTX Pharmacokinetics: Metabolism
Three metabolic pathways
Intestinal bacteria convert MTX to 4-amino-deoxy N10- methylpteroic acid; metabolite accounts for less than 5% of dose
MTX converted in the liver to 7-hydroxy-MTX:
May contribute to nephrotoxicity
Hepatic first pass effect of MTX is low
7-hydroxy-MTX is a 10-fold less potent inhibitor of dihydrofolate reductase (DHFR), one of the intracellular target enzymes for MTX
Intracellular conversion of MTX to polyglutamates (PGs) is the most important pathway related to efficacy
Methotrexate has three metabolic pathways. The first is the metabolism by intestinal bacteria to form 4-amino-deoxy-N10-methylpteroic acid. This metabolite accounts for <5% of the administered dose and is rarely detectable in plasma urine. The second is metabolism of methotrexate in the liver to 7-hydroxy-methotrexate, which may contribute to acute nephrotoxicity because of its precipitation in acidic urine. The most important metabolic pathway with respect to clinical efficacy is the intracellular conversion of methotrexate to polyglutamates (PG) and this is considered in the following slides.
Grim J, et al. Clin Pharmacokinet. 2003;42:

20. Polyglutamation Is a Determinant of MTX Response
MTX is a prodrug that is activated to MTX polyglutamates (MTX-PGs) by the addition of new glutamyl groups1
The accumulation of MTX-PGs appears to be an important determinant of response to MTX treatment2
Polyglutamation leads to longer retention of MTX within cells, increasing with the number of glutamate moieties3
MTX
Glutamate Moieties
Methotrexate is a prodrug activated to methotrexate polyglutamates by a folylpolyglutamate (FPGS)-mediated sequential addition of glutamic acid residues to the parent drug. This process selectively modifies the properties of methotrexate and enhances its retention in cells.
Dervieux T, et al. Rheumatol. 2010;49:
Dervieux T, et al. Pharmacogenet Genomics. 2009;19:
Stamp LK, et al. J Rheumatol. 2011;38:

21. Pharmacodynamics

22. Pathway From MTX-PG to Decreased Inflammation
MTX-PG inhibits AICAR transformylase (AICAR-T)
TNF-α (inflammatory)
IL-6 (inflammatory)
Adenosine 2A Receptor
IL-10 (anti-inflammatory)
This results in accumulation of AICAR
Leading to accumulation and release of adenosine
Adenosine acts at adenosine type 2A receptors to decrease synthesis of inflammatory cytokines and increase levels of anti-inflammatory cytokines
Methotrexate may have up to 5 glutamate moieties added by folylpolyglutamate synthetase; and the number added; that is, the length of the polyglutamate chain; is important. A greater number of glutamate moieties added to methotrexate results in an increased likelihood that methotrexate polyglutamate will remain inside the cell. In addition, longer chain methotrexate polyglutamates are more potent inhibitors of 5-aminoimidazole-4-carbox-amide ribonucleotide transformylase than shorter chain molecules. Methotrexate polyglutamates inhibits 5-aminoimidazole-4-carboxamide ribonucleotide transformylase, causing accumulation of 5-aminoimidazole-4-carbox-amide ribonucleotide, which results in increased secretion of adenosine and an increase of intracellular and extracellular levels of adenosine. Adenosine is a strong anti-inflammatory mediator. Adenosine acts at specific receptors on inflammatory cells to decrease levels of tumor necrosis factor-α and interleukin-6 and increase interleukin-10.
AICAR-T, 5-aminoimidazole-4-carboxamide ribonucleotide – transformylase
Romão VC, et al. BMC Medicine. 2013;11:17.  
Chan ESL, Cronstein BN. Arthritis Res. 2002;4: 18

23. Levels of MTX PG Are Correlated with Clinical Response
71 patients with RA diagnosed according to 2010 American College of Rheumatology/European League Arthritis Rheumatism criteria, who had never been treated with MTX or biologic agents
MTX was started with a dose of 8 mg/week and increased by 4 mg by every 4 weeks until reached 16 mg/week unless adverse events occurred
Blood samples were taken at baseline, week 4, 8, and 12, and after that every 12 weeks
Clinical responses were correlated with MTX PG1-5 levels
180
160
140
120
100 80 60 40
-15
-10 -5 5 10 15 20 25 30
ΔSDAI (12w SDAI- 0w SDAI)
Total MTX-PG (nmol/l)
R=0.433, P=0.044
Several studies have provided evidence that methotrexate levels in red blood cells, a surrogate for levels in white blood cells, are correlated with clinical responses in patients with rheumatoid arthritis. In one recent study, 71 patients with rheumatoid arthritis who had never been treated with methotrexate or biologic agents were treated with methotrexate at a starting dose of 8 mg/week that was increased by 4 mg by every 4 weeks until reached 16 mg/week unless adverse events occurred. Clinical responses measured by the Simplified Disease Activity Index were correlated with methotrexate polyglutamate levels.
Greater Improvement
SDAI, Simplified Disease Activity Index
Takahashi C, et al. Ann Rheum Dis. 2014;73:

24. Elimination

25. MTX Pharmacokinetics: Excretion
Renal excretion is the primary route of MTX elimination1
Renal excretion occurs by glomerular filtration and active tubular secretion
Impaired renal function, as well as concurrent use of drugs such as weak organic acids that also undergo tubular secretion, can markedly increase MTX serum levels1,2
There is limited biliary excretion amounting to 10% or less of the administered dose1
Category2-4
Examples
NSAIDs
Ibuprofen, aspirin, naproxen, diclofenac, etoricoxib, ketoprofen, nimesulide
Antibiotics
Penicillin, amoxicillin, piperacillin, cephalosporins, ciprofloxacin, doxycycline, and vancomycin
Proton pump inhibitors
Omeprazole, lansoprazole, rabeprazole, esomeprazole, pantoprazole
Antifungals
Amphotericin
Antivirals
Acyclovir
The primary route of methotrexate elimination is through renal excretion which occurs by glomerular filtration and active tubular secretion. Impaired renal function and concurrent use of drugs that also undergo tubular excretion can increase methotrexate serum levels. The table on this slide lists some of the most commonly used drug classes and individual agents that reduce the clearance of methotrexate.
Methotrexate PI. Roxane Laboratories, Inc., Columbus, OH 2012.
Bourré-Tessier, J, et al. J Rheumatol. 2010;37:
Leveque D, et al. Expert Rev Clin Pharmacol. 2011;4:
Batagini F, et al. Rev Bras Reumatol. 2011;51:20-39.

26. Summary

27. Summary: MTX Pharmacokinetics (cont.)
Oral route of administration represents a limiting factor when attempting to optimize MTX dosing1
Given that absorption varies greatly between oral and parenteral routes, switching from oral to parenteral may be an important option to improve patient outcomes2
The oral route of administration represents a limiting factor when attempting to optimize methotrexate dosing, particularly in patients who require doses >15 mg/week. Given that absorption varies greatly between oral and parenteral routes, switching from oral to parenteral delivery may be an important option to improve patient outcomes.
van Roon EN, van de Laar MA. Clin Exp Rheum. 2010;28:S27-S32.
Braun J, et al. Arthritis Rheum. 2008;58:73-81.