5-HT2B receptor antagonists attenuate myofibroblast differentiation and subsequent fibrotic responses in vitro and in vivo.

Pulmonary fibrosis is characterized by excessive accumulation of connective tissue, along with activated extracellular matrix (ECM)-producing cells, myofibroblasts. The pathological mechanisms are not well known, however serotonin (5-HT) and 5-HT class 2 (5-HT2) receptors have been associated with fibrosis. The aim of the present study was to investigate the role of 5-HT2B receptors in fibrosis, using small molecular 5-HT2B receptor antagonists EXT5 and EXT9, with slightly different receptor affinity. Myofibroblast differentiation [production of alpha-smooth muscle actin (α-SMA)] and ECM synthesis were quantified in vitro, and the effects of the receptor antagonists were evaluated. Pulmonary fibrosis was also modeled in mice by subcutaneous bleomycin administrations (under light isoflurane anesthesia), and the effects of receptor antagonists on tissue density, collagen-producing cells, myofibroblasts and decorin expression were investigated. In addition, cytokine expression was analyzed in serum. Lung fibroblasts displayed an increased α-SMA (P < 0.05) and total proteoglycan production (P < 0.01) when cultured with TGF-ß1 together with 5-HT, which were significantly reduced with both receptor antagonists. Following treatment with EXT5 or EXT9, tissue density, expression of decorin, number of collagen-producing cells, and myofibroblasts were significantly decreased in vivo compared to bleomycin-treated mice. Receptor antagonization also significantly reduced systemic levels of TNF-α and IL-1ß, indicating a role in systemic inflammation. In conclusion, 5-HT2B receptor antagonists have potential to prevent myofibroblast differentiation, in vitro and in vivo, with subsequent effect on matrix deposition. The attenuating effects of 5-HT2B receptor antagonists on fibrotic tissue remodeling suggest these receptors as novel targets for the treatment of pulmonary fibrosis.

The effect of a long term epidural infusion of ropivacaine on CYP2D6 activity.

BACKGROUND: Ropivacaine and one of its metabolites, pipecoloxylidide, inhibit CYP2D6 in. human liver microsomes in vitro with K(i) values of 5 microM (1.4 mg/L) and 13 microM (3.6 mg/L), respectively. We investigated the effect of a 50 h continuous epidural infusion of ropivacaine 2 mg/mL at a rate of 14 mL/h on CYP2D6 activity. METHODS: Nineteen patients (41-85 yr) undergoing hip or knee replacement, all extensive metabolizers with respect to CYP2D6 activity, were included. Medications known to inhibit or be metabolized by CYP2D6, or known to be strong inhibitors/inducers of CYP1A2 or CYP3A4 were not allowed. Patients received 10 mg debrisoquine (a marker for CYP2D6 activity) before surgery and after 40 h epidural infusion. The metabolic ratio (MR) for debrisoquine hydroxylation was calculated as the amount of debrisoquine/amount of 4-OH-debrisoquine excreted in 0-10 h urine. RESULTS: The median (range) of MR before and after ropivacaine were 0.54 (0.1-3.4) and 1.79 (0.3-6.7), respectively. The Hodges Lehman estimate of the ratio MR after/MR before ropivacaine was 2.2 with a 95% confidence interval 1.9-2.7 (P < 0.001). CONCLUSION: A continuous epidural infusion of ropivacaine inhibits CYP2D6 activity in patients who are extensive metabolizers resulting in a twofold increase in the MR for debrisoquine hydroxylation. However, since none of the patients was converted into a functional poor metabolizer (MR >12.6), the effect on the metabolism of other drugs metabolized by CYP2D6 is unlikely to be of major clinical importance.

High airway-to-blood transport of an opioid tetrapeptide in the isolated rat lung after aerosol delivery.

The airway-to-blood absorption of the mu-selective opioid tetrapeptide agonist Tyr-D-Arg-Phe-Phe-NH(2) (MW 631) was investigated in the isolated, perfused, and ventilated rat lung model. The lung metabolism of the peptide was compared after airway and vascular delivery. The concentrations of the parent tetrapeptide and five of its metabolites in the perfusate and in bronchoalveolar lavage fluid were analyzed by LC-MS. The metabolism of the peptide was higher after delivery to the airways compared to vascular delivery. However, the tetrapeptide was highly transported from the air-to-blood side to an extent of 47.8 +/- 10.7% in 2 h. In conclusion, the results prompt investigations of the pulmonary route as a successful alternative to parenteral delivery for this tetrapeptide.

Regional differences in bioavailability of an opioid tetrapeptide in vivo in rats after administration to the respiratory tract.

TArPP (Tyr-D-Arg-Phe-Phe-NH(2)), 1-10 micromol/kg, was administered to anesthetized rats by nasal microinfusion, intratracheal microinfusion, intratracheal nebulization, aerosol inhalation, and i.v. bolus and infusion. Plasma concentrations of TArPP and its deamidated metabolite were determined by LC-MS-MS. Regional differences in bioavailability (F), first-pass metabolism, and absorption rate were found for TArPP after delivery to the respiratory tract. Absorption was rapid after both pulmonary and nasal administration (t(max) approximately 10-20 min). After nasal microinfusion, F was 52 +/- 9%. For all the pulmonary groups, F was higher (72-114%). First-pass metabolism of TArPP was lower in the lung than in the nasal cavity. It is evident that the pulmonary route is attractive for successful systemic delivery of small, hydrophilic and enzymatic susceptible peptides.