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Crohn's disease is a chronic inflammatory bowel disease characterized by inflammation of both the small and large intestines. Methotrexate (MTX), a classical dihydrofolate reductase (DHFR) inhibitor, has been used as a therapeutic agent in the treatment of patients with Crohn's disease in recent years. We sought to develop antifolates similar in structure to MTX that would be effective in reducing inflammation in a mouse disease model of colitis. Four classical DHFR inhibitors encompassing ester bridges in the central parts of the molecules were synthesized. These antifolates were efficient inhibitors of the DHFR enzyme derived from rat. They were also tested in vitro for their ability to inhibit induced proliferation of lymphocytes from mouse spleen. Inhibition of cell proliferation was achieved only in the micromolar range, whereas MTX was effective at low nanomolar concentrations. One of the DHFR inhibitors (1), with an IC50 value for rlDHFR approximately 8 times higher than that of methotrexate, was selected for in vivo experiments in an experimental colitis model in mice. This compound demonstrated a clear antiinflammatory effect after topical administration, comparable to the effect achieved with the glucocorticoid budesonide. Three parameters were evaluated in this model:  myeloperoxidase activity, colon weight, and inflammation scoring. A favorable in vivo effect of compound 1 (15 mg/(kg·day)) was observed in all three inflammatory parameters. However, the results cannot be explained fully by DHFR inhibition or by inhibition of lymphocyte cell proliferation, suggesting that other yet unidentified mechanisms enable reduction of inflammation in the colitis model. The mechanism of action of methotrexate analogues encompassing a bridging ester group is not well understood in vivo but seems to lend itself well to further development of similar compounds.

A high airway concentration might be required for the antiasthmatic efficacy of inhaled glucocorticosteroids (GCS). The topical uptake and retention of GCS in airway tissue were compared for GCS of the inhaled type [budesonide (BUD), fluticasone propionate (FP), and beclomethasone dipropionate (BDP)] and of the noninhaled type (dexamethasone and hydrocortisone). 3H-labeled GCS solutions were administered into rat airways by either perfusion of trachea in vivo, intratracheal instillation, or inhalation. Radioactivity was determined in the airway tissue, lung parenchyma, and plasma 20 min to 24 hr after exposure. Ethanol extracts of exposed tracheas were analyzed by HPLC. Exposed tracheas were also incubated in vitroin buffer, and the released radioactivity was analyzed by HPLC. BUD, FP, and BDP were equally well taken up into the airway tissue; their uptake was 25–130 times greater than that of dexamethasone and hydrocortisone. BUD was shown to form very lipophilic intracellular fatty acid esters (at carbon 21) in the airway and lung tissue after topical application. In large airways 20 min after administration, approximately 70–80% of retained BUD was conjugated. BUD stored in esterified form in the tissue was retained in large airways for a prolonged time, compared with FP and BDP, which do not form such conjugates. The fatty acid conjugation of BUD is reversible in vivo; BUD conjugates are gradually hydrolyzed and free BUD is regenerated. This reversible conjugation may improve airway selectivity, as well as prolong the local anti-inflammatory action of BUD in the airways and might be one explanation for why BUD is efficacious in the treatment of mild asthma when inhaled once daily.

Recognition of the therapeutic value of glucocorticosteroids in the treatment of inflammation has preceded awareness of the mechanism(s) of action of these drugs. We recently showed that coculture of human T84 epithelial monolayers for 2 days with anti-CD3 activated peripheral blood mononuclear cells (A-PBM) led to impaired ion transport responses and reduced barrier function. We tested the hypothesis that budesonide, as a member of the new generation of more topically selective steroids, could prevent these immune-mediated epithelial abnormalities. Budesonide added to the coculture system dose-dependently inhibited the following functional T84 abnormalities measured in Ussing chambers: reduced transport responses (decreased short-circuit current changes to carbachol (raises [Ca2+]i) and forskolin (raises cAMP, cyclic adenosine monophosphate(i)); and increased permeability (decreased resistance and increased fluxes of 3H-mannitol and 51CrEDTA). For the beneficial effects of budesonide to be observed, PBM pretreatment (> or = 3 hr) and daily addition (for 2 days) to the coculture system was necessary. Budesonide (10(-7) M) dramatically reduced A-PBM proliferation (measured by 3H-thymidine incorporation) and cytokine (IL-1beta, IL-2, IL-6, IFN-gamma, TNF-alpha) production, but was not cytotoxic to immune cells. Budesonide treatment of T84 epithelial cells alone did not directly affect epithelial physiology, nor did it prevent epithelial abnormalities evoked by subsequent exposure to A-PBM or conditioned media from immune cells. Our studies showed that budesonide prevents epithelial dysfunction in this model by inhibiting activation of both T cells and monocytes.

The affinity for the glucocorticoid receptor in rat skeletal muscle of some glucocorticoids with a new type of 16 alpha, 17 alpha-acetal substituent has been estimated and correlated to the glucocorticoid activities in three in vivo systems in rats. Budesonide (an approximately 1:1 mixture of the C(22) epimers of 11 beta, 21-dihydroxy-16 alpha, 17 alpha-[(22R,S)-propylmethylenedioxy]-pregna-1,4-diene-3,20-dione) and the isolated (22R)- and (22S)-epimers bound to the same binding site as the potent glucocorticoids dexamethasone (DEX) or triamcinolone 16 alpha, 17 alpha-acetonide (TA), but with even higher affinity than DEX or TA, despite the lack of a 9 alpha-fluoro atom in budesonide and its epimers. The (22R)-epimer was twice as active as the (22S)-epimer, 4 times more active than TA, and 14 times more active than DEX. The introduction of a 9 alpha-fluoro atom slightly decreased the binding affinity of the (22R)-epimer of budesonide, in contrast to the positive effect of 9 alpha-fluorination of, e.g., 16 alpha, 17 alpha-acetonides. The negative influence of 9 alpha-fluorination of the (22R)-epimer was partially reversed in the 6 alpha, 9 alpha-difluorinated (22R)-epimer. Nevertheless, the fluorinated compounds were more active than DEX and TA (8 and 11 times more active than DEX, and 2 and 3 times more active than TA, in case of the 9 alpha-fluoro- and 6 alpha, 9 alpha-difluoro-derivatives of the (22R)-epimer, respectively). Budesonide is metabolized mainly to 16 alpha-hydroxyprednisolone (11 beta, 16 alpha, 17 alpha, 21-tetrahydroxy-pregna-1,4-diene-3,20-dione) and 6 beta-hydroxy-budesonide. Both metabolites were very weak competitors for the ligand-binding sites on the receptor (3% and 6% of the affinity of DEX, respectively). The affinity for the receptor in vitro was closely correlated to the topical glucocorticoid activity in vivo for the 12 steroids compared (r = 0.98; R = 0.98), which supports the contention that in vitro tests for receptor affinity are useful when screening for agonists among steroids with the present type of structures. The results on receptor-ligand interaction are in accordance with X-ray crystallographic data available for some steroids.