25 Clinical pliarmacology in inflammatory bowel disease: optimizing current medical therapy
LAURENCE J. EGAN AND WILLIAM J. SANDBORN
Introduction
Effective therapy of inflammatory bowel disease (IBD) often requires the implementation of diverse pharmacologic strategies (Fig. 1). Unlike, for exam- ple, the treatment of an acid-peptic disease where blockade of the proton pump, a specific target enzyme, is highly effective in inhibiting the patho- genic process, investigators have been unable to identify a specific unifying etiologic or pathogenic factor in IBD to pharmacologically target. For this reason, current therapy of ulcerative colitis (UC), Crohn's disease (CD) and pouchitis often requires the use of more than one drug that belong to different therapeutic classes, and that are non-specific in action.
In this chapter the clinical pharmacology of drugs that are effective in IBD is reviewed. The pharmaco- kinetics and mechanisms of action of these agents are discussed, with particular reference to their use in IBD patients. Although the list of medicines avail- able to treat IBD is expanding, none is universally effective or safe. Therefore, we also explore various strategies to individualize and optimize drug therapy in IBD, including therapeutic drug monitoring and the potential use of pharmacogenetic data to guide
Systemic inhibitors of inflammation
Glucocorticoids
I mmunosuppressants
Azathioprine, methotrexate cyclosporine
Cytokine modulators
Anti-TNFa, IL-10
Topical inhibitors of inflammation
Aminosalicylates
Figure 1. Effective therapy of IBD often requires a combination of diverse pliarmacological approaches.
therapeutic decision-making. Several agents recently introduced for use in IBD, such as mycophenolate mofetil, interleukin (IL)-10 and thalidomide are not discussed because of a paucity of information on their use.
Aminosalicylates
Chemistry
Drugs that contain 5-aminosalicylic acid (5-ASA) have been used successfully in the treatment of IBD for decades. The aminosalicylates have evolved to become a cornerstone of modern IBD therapy since the observation that the 5-ASA-containing agent sulfasalazine, designed to provide both anti-inflam- matory and antibacterial activities for rheumatoid arthritis therapy, produced symptomatic improve- ment of bowel disease in patients with coexisting colitis [1]. Sulfasalazine consists of one molecule of 5-ASA azo-bound to a molecule of the sulfa anti- biotic sulfapyridine. A number of studies have sug- gested that the therapeutic action of sulfasalazine in IBD resides with the 5-ASA moiety, rather than with sulfapyridine [2-4]. Olsalazine is an azo-conjugated dimer of 5-ASA, and balsalazide consists of a mole- cule of 5-ASA azo-bound to 4-aminobenzoylalanine, an inert carrier (Fig. 2).
Mechanisms of action
5-ASA differs from salicylic acid only by the addition of an amino group at the 5 (meta) position. However, this change produces a chemical entity with pharmacologic properties different from conven- tional salicylates, and appears to be necessary for activity in IBD. The primary anti-inflammatory, antipyretic and analgesic actions of salicylates such as aspirin are due to blockade of prostaglandin
Stephan R. Targan, Fergus Shanahan and Lor en C. Karp (eds.), Inflammatory Bowel Disease: From Bench to Bedside, 2nd Edition, 495-521.
© 2003 Kluwer Academic Publishers. Printed in Great Britain
COOH COOH
^ ^ - • ^ S _ < ^ _ N = N - - < Q
OHS-Aminosalicylic acid Sulfasalazine
HO2CH2CH2HNC
COOH HOOC COOH
H O ^ N = . - H 0 - O H HO_^H=H-HP^OH
Balsalazide Olsalazine
Figure 2. The aminosalicylate drugs that are used in IBD all consist of 5-ASA in free form as mesalamine, or azo-conjugated to a carrier molecule. In sulfasalazine, 5-ASA is conjugated to sulfapyridine, in olsalazine to another molecule of 5-ASA, and in balsalazide to 4-aminobenzoylalanine.
synthesis by inhibition of the cyclo-oxygenase 1 and 2 enzymes. However, aminosalicylates such as 5- ASA and sulfasalazine have highly variable effects on prostaglandin production. Depending on the model system studied, and the doses of drug tested, higher concentrations of aminosalicylates inhibited production of prostaglandins [5, 6], while lower concentrations could promote prostaglandin pro- duction [7, 8]. Thus, while modulation of prostaglan- din metabolism in the inflamed gut may be a relevant anti-inflammatory action of aminosalicylates, it is unlikely to be the sole therapeutic action since more potent inhibitors of cyclo-oxygenase, such as indo- methacin, have no effect or even worsen IBD.
Cytokines such as tumor necrosis factor-oc (TNF- oc), IL-1 and interferon (IFN)-y are recognized to have important proinflammatory roles in IBD [9, 10]. Because of the potent inflammatory activities of these substances, attention has also focused on the factors that regulate the production of proinflamma- tory cytokines and other peptides that contribute to intestinal inflammation, such as adhesion molecules and inducible nitric oxide synthase. In this context the inducible transcription factor nuclear factor kappa B
( N F - K B )has emerged as a dominant reg- ulator of the expression of genes that are important for innate immune responses in the gut [11-14] and in other tissues [15]. Conventional salicylates, such as aspirin, potently inhibit the activity of N F - K B in response to various stimuli [16]. Azo-bound amino-
salicylates such as sulfasalazine inhibit the activity of N F - K B by blocking inducible degradation of its cytosolic inhibitor,
IKBOC[17]. The free aminosalicy- late 5-ASA also inhibits the activity of
N F - K B[18,
19], possibly a unique mechanism: inhibition of phosphorylation of the
N F - K Bprotein RelA [18].
Interestingly, sulfasalazine is considerably more potent than 5-ASA as an inhibitor of
N F - K B ,sug- gesting that the parent drug may in fact possess pharmacologic activity that is independent of its 5- ASA moiety. The azo-conjugated aminosalicylates sulfasalazine [20] and olsalazine [21] have been demonstrated to dose-dependently block cytokine receptor binding in vitro.
The therapeutic actions of aminosalicylates such
as 5-ASA have also been evaluated with respect to
other pathways of intestinal inflammation and
injury. 5-ASA has been found to inhibit colonic
production of chemoattractant leukotrienes in UC
[22]. The importance of this process is challenged by
the lack of clinical efficacy of a specific leukotriene
inhibitor [23]. Aminosalicylates have also been
shown to inhibit intestinal epithelial cell injury and
apoptosis stimulated by oxidant stress [24, 25], and
to augment the intestinal epithelial cell heat-shock
protein response [26]. It remains to be determined if
any of these actions, or modulation of other inflam-
matory pathways, are the true basis for the beneficial
effect of aminosalicylates in patients with IBD.
Pharmaceutics and pharmacokinetics
Aminosalicylates act topically on the diseased bowel in IBD. Standard, non-delayed-release preparations of 5-ASA are efficiently absorbed in the proximal small bowel [27]. Thus, the drug is not available at the site of disease unless given rectally, or in an oral delayed-release formulation. Two strategies have been developed to improve delivery of oral 5-ASA to the site of disease in IBD: coating the free 5-ASA with polymers that dissolve slowly and release the drug gradually; and formulation of the drug as an azo-conjugate that is poorly bioavailable until bac- terial azo-reductases in the colon split the azo bond and release the active 5-ASA. The systemic and intestinal pharmacokinetics of the delayed-release and azo-bound preparations of 5-ASA have been extensively studied and are summarized in Table 1.
Table 1. 5-ASA preparations
Delayed-release formulations of 5-ASA consist of the drug in powder form that is either coated with a pH-dependent resin, or is encapsulated in semi- permeable ethylcellulose microgranules. A number of different pH-dependent resins have been used to protect 5-ASA from release in the acidic proximal small bowel. In these formulations the active drug is not released until the intestinal pH rises above a certain threshold (Table 1), theoretically allowing predictable delivery of 5-ASA to certain intestinal sites based on their acidity. Eudragit S-coated 5-ASA (Asacol) is designed to be released above pH 7, corresponding to the distal ileum, while Eudragit L- coated 5-ASA (Salofalk, Mesasal, Claversal) should be released above pH 5-6 in the more proximal small bowel. The Pentasa preparation provides delivery of drug to the entire small bowel and colon by permit- ting gradual timed release of 5-ASA from micro- granules. The semipermeable ethylcellulose coating
Generic name Mesalamine
Mesalamine Mesalamine
Mesalamine
Mesalamine
Sulfasalazine
Olsalazine
Balsalazide
Proprietary name Rowasa, Salofalk
Canasa Asacol
Salofalk, Mesasal, Claversal
Pentasa
Azulfidine
Dipentum
Colazide, Colazal
5-ASA delivery mechanism Enema suspension
Suppository
Eudragit-S-coated tablets (release at p H > 7
Mesalamine in sodium/
glycerine buffer coated with Eudragit-L (release at p H > 6 )
Ethylcellulose-coated microgranules (time and pH dependant release)
5-ASA azo bound to sulfapyridine
5-ASA dimer, linked by azo-bond
5-ASA azo-bound to inert carrier
Sites of delivery
Left colon and rectum Rectum
Terminal ileum, colon
Distal jejunum, proximal ileum
Entire small bowel, colon
Colon
Colon
Colon
Luminal 5-ASA concentration^
[Ref.]
Colon 23 mM [30]
Colon 15 mM [30], small bowel 0.3 mM [33]
Small bowel 0.3 mM [35], colon 13 mM [30]
Colonic lumen 7 mM [37]
Colonic lumen 1 4 - 2 3 mM [37], colon 24 mM [30]
Absorption^
[Ref.]
13% [28]
24% [29]
23% [31 ], 24% [32], 31% [30]
40% [34], 54% [30]
30% [36], 36% [30]
11% [31]
10% [31], 17% [32], 22% [30]
Daily dose range
1-4 g
0.5-1.5 g 1.6-4.8 g
1.5-4 g
2 - 4 g
1-4 g
1-3 g
2-6.75 g
^Luminal 5-ASA concentrations achieved in these studies of the various drug were administered in each study.
^Absorption is estimated from cumulative urinary excretion of 5-ASA +
formulations should not be directly contrasted because different doses of acetyl-5-ASA, expressed as a percentage of total administered dose.
498
of Pentasa allows 5-ASA to dissolve and diffuse into the gut lumen over several hours.
Three azo-conjugated preparations of 5-ASA are available: sulfasalazine, balsalazide and olsalazine.
Compared to free 5-ASA, oral administration of azo- conjugated 5-ASA results in greatly reduced sys- temic absorption of the active drug, and therefore greater delivery of drug to the site of presumed pharmacologic activity [30-38], Intestinal bacteria contain azo-reductase activity, and are most abun- dant in the terminal ileum and colon. Azo-conju- gated aminosalicylates reach the distal bowel largely unabsorbed, and there are effectively metabolized to yield free 5-ASA and the carrier molecule. In the case of olsalazine, two molecules of 5-ASA are released.
These preparations thus deliver high concentrations of active drug to the terminal ileum and colon.
Compared to the delayed-release preparations, azo- conjugated aminosalicylates are less subject to absorption in the small bowel and consequently, less drug is delivered systemically (Table 1).
A number of factors may affect the delivery of orally administered 5-ASA to the site of disease in IBD patients. For example, rapid intestinal transit may decrease exposure of the small intestine to delayed-release preparations of 5-ASA, and altera- tions in bacterial flora due to antibiotic use or small bowel bacterial overgrowth could affect liberation of 5-ASA from the azo-bound aminosalicylates. Clini- cally significant differences between the available oral 5-ASA preparations have been diflftcult to detect and, compared to sulfasalazine, the newer products have not been shown to have superior clinical efficacy. However, aminosalicylates lacking sulfa- pyridine are tolerated better than sulfasalazine [39].
Comparisons between doses of azo-bound amino- salicylates and free aminosalicylates should be made on the basis of molar content of 5-ASA; thus, 1 g of sulfasalazine contains approximately 0.4 g of 5- ASA.
Rectal administration of 5-ASA as a suppository or enema provides a high concentration of drug at the site of disease in distal UC. Suppositories deliver 5-ASA to the rectum only. Enemas contain an aqueous suspension of 5-ASA that gradually dissolves into the luminal free water to deliver active drug to the rectum and left colon [40]. The effective- ness and absorption of rectally administered 5-ASA depend on the patient's ability to retain the drug, which can be difficult for individuals with active proctitis. The absorption of rectal 5-ASA averages around 20% of the administered dose.
Adverse effects
Aminosalicylates are well tolerated, especially the newer drugs lacking the sulfapyridine moiety of sulfasalazine [39, 41]. Sulfasalazine, but not 5-ASA, has been associated with oligospermia [42] and folate deficiency [43]. Adverse reactions to 5-ASA are generally mild and reversible. In most of the efficacy trials the frequency of drug reactions sufficient to warrant discontinuation was not significantly greater in the active treatment group (3-4%) than in the placebo group. The most frequently reported side effects of 5-ASA include dizziness, fever, headache, abdominal pain, nausea and rash [44-46].
Certain rare and potentially serious adverse events are occasionally associated with 5-ASA use. These include pneumonitis and pericarditis [47, 48], aplas- tic anemia [49], pancreatitis [50] and rarely, exacer- bation of existing colitis [51].
High doses of aminosalicylates that are associated with higher salicylate blood levels have generated concern about potential nephrotoxicity [52, 53].
Careful studies of renal function and urinary sedi- ment have documented subtle changes indicative of tubular epithelial cell damage in IBD patients receiv- ing high doses of 5-ASA drugs [54]. However, despite this concern, clinically significant renal dysfunction appears to occur very infrequently in IBD patients treated with 5-ASA drugs.
Optimizing therapy
The effectiveness of aminosalicylate therapy of IBD
depends on getting a high concentration of the drug
to the site of disease. The different preparations of 5-
ASA listed in Table 1 offer unique characteristics that
can theoretically be exploited to deliver the greatest
amount of drug to the site of disease in individual
patients. Thus, many clinicians use 5-ASA supposi-
tories and enemas in patients with proctitis or
proctosigmoiditis. Indeed, even in the presence of
pancolitis, the use of rectal 5-ASA may be very useful
to control distal disease, the source of the most
troublesome symptoms. In more extensive UC, both
the azo-conjugated and delayed-release formula-
tions of oral 5-ASA are suitable. No individual
preparation has been conclusively shown to have an
efficacy advantage over another in UC. For the
treatment of small bowel CD, the timed-release
Pentasa preparation has the theoretical, but not
clinically proven, advantage over the other oral
aminosalicylates.
Table 2. Comparison of the potency, bioavailability and common doses of oral and rectal glucocorticoid preparations for use in IBD
Drug Hydrocortisone Prednisone Prednisolone
Budesonide,
controlled-ileal-release
Anti-inflammatory potency
1 4 4
200
Bioavailability Oral: > 50%; rectal: 16-30%
Oral: > 50%
Oral: > 50%; rectal, as metasulfobenzoate: < 10%
Oral: 10%; rectal: 16%
Usual maximum dose, mg/day 100-200 4 0 - 8 0 4 0 - 8 0
9 - 1 5
A consistent theme to emerge from clinical trials of aminosalicylates in IBD is that more is better. For active UC, doses of 5-ASA in the 2.4-4.8 g per range were more efficacious than 1.6 g or placebo [44, 46].
Similarly, in the treatment of active CD, 5-ASA doses of 3-4 g per day [55] are more effective than 1-2 g per day [56, 57]. For maintenance of medically or surgi- cally induced remission in CD, 5-ASA doses of 2.4 g or greater are effective, but lower doses are not [58- 60]. Furthermore, in studies which have compared oral and rectal 5-ASA for distal UC, combination therapy seems to be superior to either alone. Higher doses of 5-ASA produce higher mucosal drug levels [61], and the occurrence of ileal relapse after resec- tion in CD patients on prophylactic 5-ASA, 2.4 g per day, was found to be associated with lower ileal mucosa drug levels [62]. Taken together, these data support the concept that the optimum use of amino- salicylates in active IBD demands the highest toler- ated dose. In quiescent disease, lower doses may be more tolerable to the patient and are less costly.
5-ASA is primarily metabolized by A^-acetylation to a product that does not appear to possess significant anti-inflammatory activity. Of the two A^- acetyltransferase isoenzymes, A^-acetyltransferase-1 has much greater affinity for aminosalicylates than A^-acetyltransferase-2 [63]. A^-acetyltransferase-1 is highly expressed in the epithelium of the small bowel and colon [64]. The NATl gene is polymorphic, and inactivating mutations are associated with a pheno- type of decreased enzymatic activity [65]. Conver- sion of 5-ASA to acetyl-5-ASA by A^-acetyltransfer- ase-1 takes place in the gut epithelium [66, 67] and in the liver, where phase II conjugation with glucuronic acid also takes place. After oral or rectal administra- tion of an aminosalicylate, acetyl-5-ASA is the chief
species present in the serum and urine [32, 33, 68]
and in the intestine [36].
One study has examined the potential role of NATl polymorphisms and A^-acetyltransferase-1 activity in colonic mucosa as a determinant of clinical response to 5-ASA [69]. In this study IBD patients classified on clinical grounds as normal or poor responders to 5-ASA were compared with respect to NATl polymorphisms and colonic N- acetyltransferase-1 activity. Interestingly, poor responders had a lower rate of 5-ASA acetylation than normal responders, and three out of five poor responders, but none of the normal responders, were heterozygous for NATl polymorphisms. These intri- guing but preliminary data run contrary to the notion that acetylation inactivates 5-ASA, but require confirmation.
Glucocorticoids
Chemistry
The glucocorticoids that are useful in the therapy of
IBD are synthetic molecules that bear a number of
modifications or substitutions of the naturally occur-
ring steroid, Cortisol (hydrocortisone). These altera-
tions to the steroid molecule result in dramatic
differences in its absorption, transport, metabolism
and molecular actions. The synthetic analogs of
Cortisol have been developed with the primary goal
of maximizing glucocorticoid activity while mini-
mizing mineralocorticoid effects, and decreasing the
protean adverse effects of high quantities of systemi-
cally active glucocorticoid. Prednisone and predni-
solone are the steroids most commonly used to treat
IBD, and have an intermediate duration of action
500
that allows once-daily dosing. A variety of high glucocorticoid potency, low-bioavailability synthetic molecules have been developed. Delayed-release oral preparations of these molecules, for example controlled-ileal-release budesonide, allow topical delivery of the drug to the site of disease in IBD (Table 2).
Mechanisms of action
Glucocorticoids exert a diverse range of effects on most tissues of the body. When present at supraphy- siological amounts after exogenous administration, the glucocorticoids have pronounced anti-inflamma- tory and immunosuppressive actions. The majority of the physiologic and pharmacologic etfects of glucocorticoids are dependent on diffusion of the steroid across the cell membrane to the cytoplasm where they bind to the glucocorticoid receptor, a member of the superfamily of steroid hormone receptors (Fig. 3). The glucocorticoid receptor exists in the cytosol bound to the 90 kDa heat-shock proteins that act as chaperone molecules and prevent nuclear translocation. After steroid binding, the glucocorticoid-glucocorticoid receptor-heat-shock protein complex undergoes conformational changes that allow dissociation of the steroid and its receptor from the heat-shock proteins. The steroid-receptor complex is then transported to the nucleus where it can interact via its zinc finger domains with gluco- corticoid response elements that are found in the 5' promotor regions of up to 100 genes. Binding of the steroid-receptor complex to glucocorticoid response elements changes the rate of transcription of target genes. In some situations this favors the assembly of the transcription initiation complex and up-regulates the rate of gene transcription. In contrast, when the steroid-receptor complex binds to negatively acting glucocorticoid response elements, repression of gene transcription results. In addition to these direct effects on the rate of gene transcription, activated glucocorticoid receptors also physically associate with other transcription factors in the nucleus via leucine zipper interactions, and reciprocally modu- late each others' activities. One important example of this mechanism is the inhibition of
N F - K Band activator protein-1 binding to DNA by activated glucocorticoid receptors [70, 71]. Glucocorticoids also inhibit the activity of
N F - K Bby inducing tran- scription of its natural inhibitor, IKBOC [72, 73].
These direct effects, and numerous indirect effects resulting from the modulation of gene expression,
Figure 3. Mechanism of action of glucocorticoids (GC). After dissociation from corticosteroid-binding globulin (CBG), GC diffuse across tfie plasma membrane. In the cytoplasm the steroid binds to the glucocorticoid receptor (GCR), inducing conformational changes that cause dissociation of two chaperone molecules, heat-shock protein-90 (hsp90). The GC-GCR complex migrates to the nucleus and binds to glucocorticoid response elements (GRE) in the promoter regions of various target genes, regulating the rate of gene transcription. In addition, the GC-GCR complex interacts with important peptide transcription factors in the nucleus such as NF-KB, and can regulate their activity (not illustrated).
are the major molecular mechanisms of action of glucocorticoids. The dramatic capacity of gluco- corticoids to inhibit inflammation relates to the potent inhibitory actions that these drugs exert on molecules that promote the activity of immune and inflammatory processes (Table 3). Effects of gluco- corticoids that are well documented include inhibi- tion of the production of proinflammatory cytokines such asTNF-a and IL-1, and chemokines such as IL- 8; repression of the transcription of the genes for certain enzymes such as inducible nitric oxide synthase, phospholipase A2 and cyclo-oxygenase II;
and blockade of adhesion molecule expression.
These molecular actions of glucocorticoids result in a blockade of leukocyte migration and function, and inhibit the effects of numerous important peptide- and lipid-derived mediators of inflammation.
A number of studies have examined the anti-
inflammatory or immune-modulating effects of glu-
cocorticoids in patients with IBD. Nitric oxide is
found in elevated quantity in the inflamed mucosa in
IBD [79], where it is produced by the inducible form
of nitric oxide synthase. Glucocorticoids inhibited
in-vitro production of nitric oxide by cultured muco-
sal biopsy specimens, but glucocorticoid treatment
of severe UC was not shown to down-regulate
immunoreactive nitric oxide synthase [80]. In
Table 3. Gene targets of glucocorticoids that are important in mediating their anti-inflammatory actions Effect of
Target substance glucocorticoid Target cell Mechanism [ref.] Result
Cytokines and chemokines IL-1JNFa,IL-8 IL-2
Enzymes
Inducible nitric oxide synthase
Phospholipase A2
Cyclo-oxygenase II
Receptors
IL-1 receptor type II
Adhesion molecules E-selectin
Intracellular adhesion molecule-1
Decrease Decrease
Decrease
Decrease
Decrease
Increase
Decrease Decrease
Many Lymphocytes
Epithelial and endothelial Many
Many
Many
Endothelial
Epithelial, monocytic
Inhibition of N F - K B [15]
Inhibition of AP-1 [74]
Inhibition of N F - K B [15]
Inhibition of N F - K B , induction of lipocortin gene expression [75]
Inhibition of N F - K B [15]
Direct induction of gene expression [76]
Inhibition of N F - K B [77]
Inhibition of N F - K B [78]
Decreased inflammatory signaling
Decreased lymphocyte activation
Decreased vascular permeability
Decreased eicosanoid production
Decreased prostaglandin production
Decreased availability of IL-1 for biological activity
Decreased leukocyte extravasation Decreased leukocyte
migration
patients with active CD prednisolone, but not bude- sonide, has been demonstrated to inhibit neutrophil and peripheral blood lymphocyte expression of acti- vation markers and the proinflammatory cytokines, IL-1 and TNF-a [81]. Similarly, expression of the adhesion molecules intracellular adhesion molecule- 3 and P2-integrin by peripheral blood lymphocytes in CD and UC was lower in patients receiving predni- sone [82]. At the level of the inflamed mucosa in UC, prednisolone enemas and oral tablets have been shown to decrease the luminal concentrations of the arachidonic acid metabolites prostaglandin-E2 and leukotriene-B4 [22]. At the mechanistic level predni- solone therapy inhibits the activation of N F - K B in the gut mucosa [11, 14], supporting an important role for this transcription factor in mediating the beneficial effects of glucocorticoids in IBD. Overall, these human studies provide evidence that the effects of glucocorticoids on expression of the genes listed in Table 3 underlie the in-vivo anti-inflammatory effects of these drugs in IBD.
Pharmaceutics and pharmacokinetics
The conventional synthetic corticosteroids predni-
sone and prednisolone are the drugs of this class
most commonly used in IBD. Prednisone is a pro-
drug that requires metabolic activation in the liver to
prednisolone. There is some evidence that chronic
liver disease may impair this process [83], and for this
reason prednisolone may be preferable in patients
with advanced liver disease. Both of these drugs are
well absorbed after oral administration and bioavail-
ability is high, averaging over 70%. There is some
evidence suggesting decreased absorption of predni-
solone in patients with CD of the small bowel [84,
85], but it is not clear if this difference is cHnically
significant. After absorption, glucocorticoids circu-
late in the plasma partially bound to corticosteroid-
binding globulin and albumin, but it is only the free
form that is available for biologic activity. The
affinity of different corticosteroids for these carrier
proteins varies considerably and in part determines
anti-inflammatory potency. A l t h o u g h various
physiologic and pathologic conditions alter the
plasma concentration of the steroid-binding pro-
502
teins, the resulting changes that occur in the concen- tration of free glucocorticoid are transient and are not likely to be significant.
There has been a considerable effort to develop glucocorticoids for delivery to the site of disease in IBD, with the goal of providing high disease-tissue drug concentrations, with less systemic absorption than conventional steroids. One approach is rectal administration of the drug. Hydrocortisone enemas are used in ulcerative proctosigmoiditis to provide topical delivery of glucocorticoid directly to the diseased tissue, with good clinical results. Absorp- tion of hydrocortisone enemas is less than after oral administration; nevertheless, bioavailability ranges from about 15% to 30% [86, 87], sufficient to sup- press adrenal steroid production. A derivative of prednisolone with a metasulfobenzoate substitution is also used rectally in distal UC. Compared to prednisolone this agent is less well absorbed and should therefore provide a therapeutic advantage by acting locally and causing less systemic exposure to glucocorticoid activity [88, 89]. However, careful studies of adrenal function and bone turnover demonstrate that prednisolone metasulfobenzoate enemas do result in significant systemic glucocorti- coid activity when used to treat distal UC [90, 91].
Budesonide is a synthetic analog of prednisolone that has proved to be therapeutically effective as oral delayed-release capsules in CD of the ileum and right colon, and as an enema in distal UC. This drug has high affinity for the glucocorticoid receptor resulting in very high potency, and is subject to rapid inactiva- tion in the liver leading to low systemic bioavail- ability [92, 93]. A controlled-ileal-release formula- tion of budesonide is used orally, in which the drug is formulated as capsules containing microgranules of budesonide coated with Eudragit LI00-55, an acrylic resin that dissolves above pH 5.5 to release the drug in the distal ileum. The systemic bioavailabihty of oral budesonide capsules is approximately 10% [94], and the majority of absorption appears to occur in the distal small bowel. However, even this degree of systemic exposure is sufficient to suppress adrenal steroid production, but is less than occurs with equivalent doses of prednisolone [95-97]. For rectal delivery, budesonide is formulated as a suspension enema. The bioavailability of budesonide adminis- tered by this route, mean 16%, is greater than found with the oral controlled-ileal-release formulation, probably reflecting absorption into the distal rectal veins that drain directly into the systemic venous system [98]. Rectal administration of budesonide
produces dose-dependent suppression of adrenal function [99]. Thus, with both the oral and rectal formulations of budesonide, some degree of adrenal suppression occurs, but this appears it to be less than seen with conventional steroids.
A number of other high glucocorticoid potency, low systemic bioavailability corticosteroids have been developed and tested for use in IBD. Tixocortol pivalate is a potent, well-absorbed and rapidly meta- bolized steroid molecule that has been used as an enema for distal UC. Fluticasone is a high potency fluorinated corticosteroid that is poorly absorbed from the intestine and undergoes extensive first-pass inactivation, leading to systemic bioavailability of less than 1%. Fluticasone has been evaluated for oral use in CD and UC, but in preliminary studies was not found to efficacious [100, 101]. Beclomethasone is a highly potent glucocorticoid with low systemic bioa- vailability. Preliminary studies of beclomethasone enemas in active distal UC have revealed efficacy and little adrenal suppression, but further studies are needed [102-104].
Adverse effects
The use of glucocorticoids for prolonged periods in IBD patients is associated with serious side-effects.
These are not different from those that occur when these drugs are given chronically for other indica- tions. In the National Cooperative Crohn's Disease Study, adverse drug effects that occurred more fre- quently in prednisone-treated patients than in those receiving placebo were petechiae and bruising of skin, striae, moon face, acne, hypertension and infection [105]. About 30% of prednisone-treated patients in this study experienced a side-effect of this therapy, and the likelihood of occurrence of most of the reported adverse events increased with duration of exposure to the drug.
Physicians who care for IBD patients requiring
frequent use of glucocorticoids also recognize that
the full gamut of steroid-induced problems develop
with prolonged consumption of these drugs. Certain
adverse effects of glucocorticoids are of particular
relevance in IBD. Hyperglycemia and non-alcoholic
steatohepatitis are frequent metabolic complications
of chronic glucocorticoid therapy. Abnormal liver
chemistry test results in IBD patients can also be due
to primary sclerosing cholangitis, or short-bowel
syndrome, so one must consider steroid-induced
steatohepatitis in the differential diagnosis of abnor-
mal liver tests in IBD. Individuals with IBD are at
greatly increased risk for the development of osteo- porosis [106-108]. The use of glucocorticoids may increase this risk, as in one study 58% of steroid- treated patients had lumbar spine osteopenia, com- pared to 28% of steroid non-users [108]. In these studies the use of glucocorticoids may simply be a marker of more active disease, itself a risk factor for osteoporosis. However, in diseases other than IBD that are not strongly associated with osteoporosis, glucocorticoid use induces bone loss and fractures [109]. Aseptic necrosis of the hip is an unpredictable, devastating complication of glucocorticoid use. The newer high-potency, low-bioavailability steroids such as budesonide may be associated with less risk of inducing bone disease. Options for treating and preventing bone disease due to glucocorticoids include calcium and vitamin D, estrogen for post- menopausal women, and bisphosphonates. IBD patients who have been treated with glucocorticoids within 1 year should receive a short course of steroids perioperatively to protect against an inadequate adrenal response to the stress of surgery [110].
Optimizing ttierapy
It is paradoxical that glucocorticoids, the first ther- apy proven effective for IBD [111,112] have not been as carefully studied as most other classes of drugs.
The optimum doses of glucocorticoids to induce remission in active UC and CD are not known. Few studies have addressed the question of the optimum rate at which to taper the dose of steroids after a patient responds, so individual clinical judgement and experience, rather than data, direct practice.
Nevertheless, there are some well-established prin- ciples to guide the effective and safe use of glucocor- ticoids in IBD. Almost all IBD patients with moder- ately to severely active disease should be treated with a corticosteroid, as these drugs are highly efficacious for induction of remission and are probably respon- sible for reducing the high death rate first reported for severe UC [111]. Although oral steroids are well absorbed, most authorities recommend intravenous administration in the sickest patients, because altera- tions in gut motility and mucosal function may compromise absorption [84, 85]. However, after a patient responds, the dose should be tapered gradu- ally to minimize development of corticosteroid- related side-effects. A typical rate of withdrawal for oral prednisone is by 5 mg per day each week, but this is only a guideline. Medications other than glucocor- ticoids, such as aminosalicylates and immunosup-
pressives, should be used to maintain remission because of superior efficacy and toxicity profiles.
Rectally administered steroids have lower systemic absorption than oral, and therefore may provide a therapeutic advantage by acting locally in distal UC.
Similarly, in CD of the terminal ileum and right colon, controUed-ileal-release budesonide appears to be almost as effective as prednisolone, but has significantly less systemic effect [97].
Intravenous, oral and rectal steroids are highly efl&cacious in most patients with IBD. However, up to 20% of the IBD population do not respond clinically to oral prednisolone after 30 days [113], and on clinical grounds are resistant to steroids. The molecular basis for resistance to the anti-inflamma- tory effects of glucocorticoids is an important issue in IBD therapy. A syndrome of familial steroid resis- tance has been described not only for glucocorti- coids, but also for sex hormones, mineralocorticoids and vitamin D [114]. Familial glucocorticoid resis- tance is a rare disorder that appears to be due to a mutation in the glucocorticoid receptor. More com- mon is isolated resistance to the anti-inflammatory effects of glucocorticoids, which has been best described in asthma [115]. In asthma, steroid resis- tance is associated with defects in the suppression of peripheral blood mononuclear cell function by glu- cocorticoids [116]. This may be related to alterations in the number of glucocorticoid receptors, and to altered affinity for ligand in target cells [117-119].
The molecular basis for these abnormalities has been
linked to increased expression of the transcription
factor c-fos in the peripheral blood mononuclear
cells of glucocorticoid-resistant asthmatics [120]. In
CD, peripheral blood leukocytes have reduced sensi-
tivity to the inhibitory effects of dexamethasone on
cytokine secretion after ex-vivo stimulation, com-
pared to controls [121]. Among a group of patients
with severely active UC, a therapeutic response to
intravenous hydrocortisone was significantly more
likely in those with the greatest sensitivity of periph-
eral T cells to dexamethasone-induced inhibition of
prohferation after ex-vivo stimulation [122]. In IBD,
more study is clearly needed to delineate the mole-
cular mechanisms that underlie sensitivity and resis-
tance to glucocorticoids.
504
Azathioprine and 6- mercaptopurine
Chemistry
Azathioprine and 6-mercaptopurine are purine anti- metabolite drugs that were found to have immune- suppressing properties when used for cancer che- motherapy, leading to their investigation and ulti- mate use as immunosuppressive drugs in a variety of clinical settings. Azathioprine is a pro-drug that is rapidly converted to 6-mercaptopurine in a non- enzymatic reaction by sulfhydryl-containing sub- stances such as glutathione. These two drugs possess identical actions with respect to their use in IBD.
Considering mass (azathioprine is 55% 6-mercapto- purine by weight) and the efficiency of conversion of azathioprine to 6-mercaptopurine (88%), one must administer approximately twice as much azathiopr- ine as 6-mercaptopurine to achieve similar amounts of bioavailable active drug. 6-Mercaptopurine can enter three known metabolic pathways (Fig. 4). Two of these routes, leading to the inactive metabolites 6- methylmercaptopurine and 6-thiouric acid, are cata- lyzed by thiopurine methyltransferase and xanthine oxidase, respectively. The action of hypoxanthine phosphoribosyl transferase commits 6-mercapto- purine to conversion to the 6-thioguanine nucleo- tides, the putative active metabolites [123].
Mechanism of action
After administration of azathioprine or 6-mercapto- purine, the 6-thioguanine nucleotides accumulate intracellularly, and are believed to mediate the biolo- gic actions of these drugs. Despite over 50 years of investigation the precise molecular basis for the therapeutic effects of the purine analogs is not known. However, intracellular accumulation of 6- thioguanine nucleotides causes inhibition of the pathways of purine nucleotide metabolism and DNA synthesis and repair, resulting in inhibition of cell division and proHferation. It is plausible, but not proven, that antiproliferative or functionally inhibi- tory actions on cells of the immune system, such as lymphocytes, underlie the immunosuppressive actions of azathioprine and 6-mercaptopurine.
Pharmacolcinetics and pharmacogenetics The bioavailability of azathioprine, estimated as 6- mercaptopurine, ranges from 27%o to 83%, and of 6-
6-methyl- mercaptopurine
TPMT
HPRT Thioinosinic—^ 6-thioguanine dLc'id^^^"'^ nucleotides Azathioprine —• 6-mercaptopurine
XO
6-thiouric acid
Figure 4. Metabolic pathways of azathioprine and 6- mercaptopurlne. TPIVIT, thiopurine methyltransferase; HPRT, hypoxanthine phosphoribosyltransferase; XO, xanthine oxidase. Reproduced with permission from Chan GL et al., J Clin Pharmacol 1990; 30: 358-63.
mercaptopurine ranges from 5% to 37%. An oral preparation of azathioprine coated with Eudragit-S, designed for release above pH 7 in the distal ileum, had a mean bioavailability of 7%. Rectal administra- tion of azathioprine in hydrophilic or hydrophobic enemas consistently resulted in less than 10% sys- temic bioavailability [124]. At present there are no data on the clinical efficacy or toxicity of rectal or delayed-release oral azathioprine in IBD.
Many studies have examined the kinetics of the active metabolites of azathioprine and 6-mercapto- purine, the 6-thioguanine nucleotides. Although azathioprine and 6-mercaptopurine are rapidly cleared from plasma, 6-thioguanine nucleotides are concentrated in cells resulting in a half-life of elim- ination of many days [125]. In patients with CD and other conditions, erythrocyte 6-thioguanine nucleo- tide concentration gradually rises to a plateau after 2-4 weeks of oral azathioprine [126-128] (Fig. 5).
The rather delayed time to reach steady-state 6-
thioguanine nucleotide kinetics parallels the slow
onset of therapeutic benefit of oral azathioprine or
6-mercaptopurine therapy in IBD, which has been
estimated to average 17 weeks [129]. This observa-
tion prompted a pilot study [130], followed by a
placebo-controlled trial of an intravenous loading-
dose of azathioprine to hasten the onset of clinical
benefit in CD [126]. In this study, despite initially
higher erythrocyte 6-thioguanine nucleotide concen-
tration in those who received active intravenous
drug, a clinical response was seen no sooner than in
placebo-treated patients. However, the median time
to steady-state erythrocyte 6-thioguanine nucleotide
concentration and first clinical improvement was
only 4 weeks, suggesting that azathioprine may act
more quickly than had previously been realized.
0} E
O Placebo A IV azathioprine
0 2 4 6 8 10 12 14 16 Weeks of treatment
Figure 5. Mean erythrocyte 6-thioguanine nucleotide concentration in Crohn's disease patients after beginning azathioprine 2 mg/kg per day by mouth. A , received an intravenous loading dose of azathioprine; 0 » received a placebo intravenous loading dose. Reproduced with permission from Sandborn WJ et al. Gastroenterology 1999;
117:527-35.
One of the pathways of biotransformation that is available to 6-mercaptopurine is metabolized by a polymorphically expressed enzyme, thiopurine methyltransferase [131]. Erythrocyte thiopurine methyltransferase activity correlates well with the enzyme's activity in other cells and tissues. The majority of the Caucasian population, ^ 9 0 % , have an erythrocyte thiopurine methyltransferase activity of about 14-25 U/ml erythrocytes, and these indivi- duals are homozygous for two wild-type TPMT alleles. Approximately 10% of this population have an enzyme activity that is detectable but below 14 U/
ml erythrocytes, and are heterozygous for one active and one inactive copy of TMPT. One percent or less of individuals have no detectable thiopurine methyl- transferase activity and have 2 inactive TTMralleles [131, 132]. The erythrocyte 6-thioguanine nucleotide concentration varies considerably among patients being treated with azathioprine or 6-mercaptopur- ine, and appears to be dependent on the individuals' inherent, genetically determined thiopurine methyl- transferase activity [132, 133]. Thus, patients with low thiopurine methyltransferase activity have greater 6-thioguanine nucleotide concentrations, presumably because more 6-mercaptopurine is shunted into the hypoxanthine phosphoribosyl transferase pathway.
Adverse effects
Two categories of adverse effects to azathioprine and 6-mercaptopurine have been described: allergic.
occurring early during treatment; and dose-related, generally occurring later. Allergy to these drugs can manifest as pancreatitis, fever, rash, nausea, diar- rhea, and hepatitis. Approximately 5% of the IBD population beginning treatment with azathioprine or 6-mercaptopurine will experience an allergic reac- tion to the drug [134], which can be confirmed by re- challenge if necessary. Dose-related toxicities of azathioprine or 6-mercaptopurine include bone marrow depression leading to leukopenia, anemia or thrombocytopenia. If severe, leukopenia can potentially cause profound immune suppression, and thus predispose to opportunistic infections or neoplasms. Cytopenias have been reported to develop from 2 weeks to 11 years after beginning therapy, at a cumulative frequency of about 10%
[126, 135, 136]. Although the numbers are insuffi- cient for statistical analysis, there are enough reports of fatal or life-threatening opportunistic infections in leukopenic IBD patients treated with azathioprine or 6-mercaptopurine to dispel any tendency towards complacency [134, 135]. If leukopenia and a serious infection develop in a patient receiving azathioprine or 6-mercaptopurine, reversal of bone marrow suppression is usually possible with granulocyte colony-stimulating factor.
It is not completely clear if azathioprine or 6- mercaptopurine therapy confers an excess risk of malignancy. In the New York series, a wide spectrum of malignant neoplasms developed in IBD patients who had at some point received 6-mercaptopurine, but the overall rate of 3.1 % was low and probably not greater than the general population [134]. In the British series, of 755 patients treated with azathiopr- ine for a median duration of 12.5 years at a dose of 2 mg/kg per day, 31 developed a cancer compared to 24.3 expected cases [137]. The difference was signifi- cant only when considering colorectal adenocarci- nomas, a recognized complication of IBD. Thus, the data are quite reassuring, but isolated and anecdotal reports of unusual lymphomas arising in IBD patients during or after azathioprine or 6-mercapto- purine therapy continue to generate some concern [136, 138]. Nevertheless, if there is an increased cancer risk a t t r i b u t a b l e to these d r u g s , t h e magnitude of the increased risk is small.
Optimizing ttierapy
In some studies of patients being treated with
azathioprine or 6-mercaptopurine for diseases other
than IBD, low thiopurine methyltransferase activity.
resulting in greater 6-thioguanine nucleotide concen- tration, has been associated with the development of leukopenia [123, 139]. Conversely, patients with higher thiopurine methyltransferase activity may be undertreated with standard doses of azathioprine or 6-mercaptopurine. For example, in childhood acute leukemia the risk of relapse during therapy with 6- mercaptopurine is greatest in those patients with the highest erythrocyte thiopurine methyltransferase activity, because they inactivate the drug more efficiently and have lower concentrations of active 6- thioguanine nucleotides [140, 141].
In IBD patients it is currently uncertain if signifi- cant associations exist between azathioprine or 6- mercaptopurine metabolism and the occurrence of clinical response or toxicity. A number of studies that were performed in CD patients selected without regard to thiopurine methyltransferase activity have suggested that 6-mercaptopurine pharmacogenetics may in fact be clinically relevant. In a group of pediatric CD patients receiving 6-mercaptopurine therapy, one study found that disease severity was least in those with the highest erythrocyte 6-thiogua- nine nucleotide concentration [142]. Another found that 65% of patients with inactive disease had 6- thioguanine nucleotide levels greater than 230 p m o l / 8 x 10^ erythrocytes, compared to 28% of patients with active CD [143]. Similar studies in adults with CD and UC have documented a greater mean 6-thioguanine nucleotide concentration among responders to azathioprine or 6-mercapto- purine than among patients with persistent active disease [144, 145].
In contrast, two prospective studies of azathiopr- ine in CD arrived at different conclusions. These clinical trials examined the relationships between thiopurine methyltransferase activity and erythro- cyte 6-thioguanine nucleotide concentration, and the clinical outcomes of leukocyte count and efficacy [126, 130]. To minimize the potential for toxicity in these studies, individuals were excluded if thiopurine methyltransferase activity was in the heterozygous or homozygous low range (approximately 20% of the screened population, unpublished observation, W.J.
Sandborn). No significant correlations were found between thiopurine methyltransferase activity or erythrocyte 6-thioguanine nucleotide concentration, and leukocyte count or clinical efficacy. The exclu- sion of individuals with intermediate or low thiopur- ine methyltransferase activity probably decreased the power of these studies to detect pharmacogenetic differences. Furthermore, a cross-sectional study of
IBD patients that included individuals with inter- mediate-range thiopurine methyltransferase activity who were receiving azathioprine or 6-mercapto- purine also found no significant associations between thiopurine methyltransferase activity or erythrocyte 6-thioguanine nucleotide concentration and disease severity [146].
Based on these data the benefit of monitoring erythrocyte 6-thioguanine nucleotide levels during azathioprine or 6-mercaptopurine therapy of IBD remains uncertain. Prospective studies that examine if a clinical benefit derives from using erythrocyte 6- thioguanine nucleotide concentrations to guide ther- apy in IBD are needed. It is our practice to measure thiopurine methyltransferase activity before begin- ning therapy with one of these drugs, to exclude the rare patient that is completely deficient in this enzyme. Individuals who have intermediate thiopur- ine methyltransferase activity can probably be trea- ted safely with azathioprine or 6-mercaptopurine, but at half the standard dose. We currently do not routinely use erythrocyte 6-thioguanine nucleotide concentration for therapeutic drug monitoring, but this practice is commonplace at some centers.
T h e r a p e u t i c trials have demonstrated that azathioprine should be given at a dose of 2-2.5 mg/
kg per day, and 6-mercaptopurine at 1.5 mg/kg per day. There is no role for an intravenous loading dose to hasten the onset of clinical response. We counsel patients about the risk of leukopenia, and check a complete blood count every month during therapy.
The results of one retrospective study suggested that
the development of mild leukopenia, an indicator of
intensity of treatment with 6-mercaptopurine, identi-
fied CD patients with a greater likelihood of a
favorable response [147]. Based on this observation,
one group of investigators has tried dose escalation
in C D p a t i e n t s refractory to s t a n d a r d - d o s e
azathioprine. In this uncontrolled study, after
increasing the dose of azathioprine from a mean of
2 mg/kg per day to 2.7 mg/kg per day, 14 of 18
patients improved and were able to discontinue
steroids [148]. Although no significant leukopenia
was observed, two patients developed serious cyto-
megalovirus infection. Other studies that have exam-
ined the relationship between leukocyte count and
clinical response during azathioprine and 6-
mercaptopurine therapy of IBD have not supported
the concept that leukopenia is associated with effi-
cacy [126, 146]. For these reasons we do not recom-
mend use of greater than standard doses of these
agents.
Methotrexate
Chemistry and metabolism
Methotrexate is an antimetabolite anticancer drug that, like azathioprine, has been found to have beneficial effects in a number of chronic inflamma- tory diseases. Structurally, methotrexate is the 4- amino, N-10 methyl analog of folic acid. Like folic acid, methotrexate is a substrate for intracellular enzyme folylpolyglutamate synthase, which adds up to five glutamic acid residues to these compounds [149]. This modification promotes intracellular retention of methotrexate and increases the drug's affinity for a number of target enzymes [150, 151].
Over 90% of a dose of parenteral methotrexate is excreted unchanged in the urine [152]. In the liver a minor metabolic pathway of methotrexate produces small quantities of the 7-hydroxy derivative.
IVIechanism of action
The principal biochemical action of methotrexate, and the mechanism that is believed to be responsible for the drug's cytotoxic activity, is inhibition of dihydrofolate reductase. This enzyme is critical for regenerating the fully reduced folate co-factors that are required for reactions involving transfer of one- carbon fragments, such as the production of thymi- dylate and purines. Methotrexate acts as a reversible, competitive inhibitor of dihydrofolate reductase. The polyglutamated metabolites of methotrexate are important not only because they are retained intra- cellularly, but also because they are potent inhibitors of enzymes that lie downstream of dihydrofolate reductase in the metabolic pathway of folic acid [151, 153]. Inhibition of these enzymes, including thymidylate synthase and 5-aminoimidazole-4-car- boxamide ribotide transformylase, further impairs folate-dependent synthetic reactions.
At the high doses used in cancer chemotherapy, methotrexate blocks de-novo synthesis of DNA pre- cursors leading to inhibition of cellular proliferation and cytotoxicity. Although methotrexate is beneficial in a number of chronic inflammatory diseases, cyto- toxic or antiproliferative effects of folic acid analog therapy are difficult to invoke as the anti-inffamma- tory mechanism of action because clinically effica- cious doses of methotrexate do not decrease circulat- ing neutrophil, lymphocyte or platelet numbers [154, 155]. The occurrence of cytopenias or mucositis, clinical evidence of cytotoxicity, are in fact indica-
tions to lower the dose of methotrexate in chronic inflammatory diseases. Also, administration of folic acid ameliorates some of the toxicities of metho- trexate that are attributable to its antiprohferative eff*ects, without impairing its anti-inflammatory action [156].
In efforts to identify the mechanism of action of methotrexate in inflammatory diseases the effects of this drug on a number of mediators of inffammation have been examined, principally in patients with rheumatoid arthritis. Methotrexate has been found to decrease neutrophil production of leukotriene B4 [157], to decrease IL-1 concentration in synovial fluid [158] and to lower plasma IL-6 and soluble IL- 2 receptor concentrations [159]. However, other agents such as glucocorticoids produce similar effects, so these changes may reffect general improve- ment rather than methotrexate-specific phenomena.
It has alternatively been proposed that the anti- inflammatory action of methotrexate may be mediated by increased release of the endogenous anti-inflammatory autocoid, adenosine. Experi- ments in tissue culture and animal models of acute inflammation have demonstrated that methotrexate can promote extracellular release of adenosine, which inhibits neutrophil adherence to endothelial cells and accumulation at sites of inflammation [160, 161]. In animal experiments, methotrexate has also been shown to decrease leukocyte-endothelial cell interactions through release of adenosine and occupancy of adenosine A2 receptors [162].
To determine if the methotrexate-induced eleva- tions of extracellular adenosine concentration that were demonstrated in tissue culture and animal models of acute inflammation were relevant to the use of this drug in humans with IBD, the effect of methotrexate on rectal and plasma adenosine was studied. In 10 IBD patients with varying degrees of rectal involvement, adenosine was measured in the rectal lumen using in-vivo rectal dialysis [163]. The concentration of adenosine in the rectum tended to correlate with the severity of proctitis. After a sub- cutaneous injection of methotrexate 15 or 25 mg the mean rectal adenosine concentration did not change.
Furthermore, the plasma adenosine concentration did not differ significantly from baseline at any time during the 24 h after the methotrexate injection.
These data suggest that the in-vivo anti-inffammatory actions of methotrexate are not related to alterations of adenosine metabohsm.
Activated T lymphocytes form an important part
of the inflammatory infiltrate in IBD, and the major
508
contribution of T lymphocytes in the pathogenesis of mucosal inflammation is highlighted by the many animal models of IBD that occur with various perturbations of T lymphocyte function [164]. The effect of methotrexate on T lymphocyte survival has been studied [165]. Methotrexate-treated T lympho- cytes were found to be primed to die by apoptosis when stimulated with mitogens, by a Fas-indepen- dent mechanism. Furthermore, the inhibitory effect of methotrexate was confined to cells that were activated at the time of drug exposure. This process was inhibited by fully reduced folate and thymidine, indicating that methotrexate-induced lymphocyte apoptosis was dependent on inhibition of dihydrofo- late reductase and thymidylate synthase. These results demonstrate that methotrexate inhibits the activation of T lymphocytes, a process that is likely to be important to this drug's in-vivo anti-inflamma- tory actions.
Pharmacokinetics
For the treatment of chronic inflammatory diseases, such as psoriasis and rheumatoid arthritis, metho- trexate has traditionally been given once weekly, because the incidence of hepatotoxicity appeared to be less with this approach than with daily adminis- tration [166]. The doses that have been used range from 7.5 to 25 mg per week, usually given orally or by intramuscular injection. When methotrexate was first tried for IBD, this therapeutic regime was empirically adopted [167].
The bioavailability of oral methotrexate is dose- dependent, because the pathways of absorption are saturable, and at higher doses methotrexate induces malabsorption through its antiproliferative effects on epithelia [168]. Reported oral bioavailability at the low doses used in chronic inflammatory diseases range from 50% to 90% [169-171]. Intramuscular and subcutaneous methotrexate exhibit near-com- plete bioavailability [152, 172]; thus, considering ease of administration, the subcutaneous route is preferred.
After administration, methotrexate is widely dis- tributed and rapidly cleared, mostly in unchanged form by the kidney. However, a small fraction of the drug is concentrated in many cell types as polygluta- mated methotrexate. In IBD patients receiving weekly injections of methotrexate, the drug has been shown to gradually accumulate in erythrocytes, reaching a plateau after about 8 weeks (Fig. 6) [173].
Methotrexate also accumulates in many other
o E
UJ
2 4 6 8 10 12 14 Weeks after starting methotrexate n(15
n(25 mg) 10 mg) 5
10 5
10 5
Figure 6. Mean erythrocyte methotrexate concentrations after starting methotrexate 15 mg/week ( # ) or 25 mg/week ( O ) by subcutaneous injection. Reproduced with permission from Egan U et ai, Aliment Pharmacol Ther 1999; 13:1597-604.
300 n
200 H
-S 100 o
• Pre-methotrexate D Post-methotrexate
M.
300
200
MOO
I
o E
0)
Dialysate Biopsy
Figure 7. In IBD patients receiving weekly injections of methotrexate the drug can be detected in the rectal lumen and mucosa 7 days after the last dose. The concentration is higher in biopsy material than in dialysate, indicating intracellular concentration of methotrexate. Reproduced with permission from Egan LJ et a/., Clin Pharmacol Ther 1999; 65:
29-39.
