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Background: Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive interstitial lung disease (ILD) whose cause and pathogenesis are not yet well understood. Until now, no animal model of lung fibrosis succeeds in recapitulating all IPF features, thus the use of different rodent models is essential for the evaluation and development of new effective pharmacological treatments. Recently, the alveolar epithelial dysfunction has been emphasized in the etiopathogenesis context of IPF. Remarkably, the role of an aberrant basaloid cell type, primarily found in humans and confirmed in mice, seems to be crucial in the establishment and progression of the disease/model. Our work aimed to characterize for the first time this cell population in a rat model of lung fibrosis induced by a double bleomycin (BLM) administration, demonstrating the translational value of the model and its potential use in the testing of effective new drugs., Methods: Rats received an intratracheal BLM administration at day 0 and 4. Animals were sacrificed 21 and 28 days post-BLM. The fibrosis evaluation was carried out through histological (Ashcroft score and automatic image analysis) and immunoenzymatic analysis. Immunofluorescence was used for the characterization of the aberrant basaloid cells markers., Results: Lung histology revealed an increase in severe grades of Ashcroft scores and areas of fibrosis, resulting in a rise of collagen deposition at both the analyzed time-points. Immunofluorescence staining indicated the presence of KRT8+ cells in bronchial epithelial cells from both controls (saline, SAL) and BLM-treated animals. Interesting, KRT8+ cells were found exclusively in the fibrotic parenchyma (confirmed by the alpha-smooth muscle actin (α-SMA) staining for myofibroblasts) of BLM-treated animals. Moreover, KRT8+ cells co-expressed markers as Prosurfactant protein C (Pro-SPC) and Vimentin, suggesting their intermediate state potentially originating from alveolar type II (AT2) cells, and participating to the abnormal epithelial-mesenchymal crosstalk., Conclusion: Previous preclinical studies demonstrated the presence of KRT8+ aberrant basaloid-like cells in murine models of lung fibrosis. This work investigated the same cell population in a different rodent (the rat) model of lung fibrosis triggered by a double administration of BLM. Our results provided a further confirmation that, in rats, the intratracheal administration of BLM induced the appearance of a population of cells compatible with the KRT8+ alveolar differentiation intermediate (ADI) cells, as described previously in the mouse. This piece of work enforces previous evidence and further support the use of a rat model of BLM resembling the alveolar epithelial dysfunction to evaluate new clinical candidates for development in IPF., Competing Interests: Francesca Ruscitti, Vanessa Pitozzi, Silvia Pontis, Paola L. Caruso, Sofia Beghi, Marcello Trevisani are employees of Chiesi Farmaceutici S.p.A, the judgments in data interpretation and writing were not influenced by this relationship. All the remaining authors have not actual and perceived conflicts of interest., (© 2024 The Author(s). Published by IMR Press.)

Unlabelled: A new SERM, CHF 4227.01, given to 6-month-old female rats immediately after ovariectomy, preserved bone mass and bone microarchitecture without affecting uterus weight. It also decreased serum cholesterol and fat mass in estrogen-deficient rats., Introduction: We tested the effect of a new benzopyran derivative, CHF 4227.01, with selective estrogen receptor modulator (SERM) activity on bone mass and biomechanics in ovariectomized (OVX) female rats in comparison with 17alpha-ethinylestradiol (EST), raloxifene (RLX), and lasofoxifene (LFX)., Materials and Methods: Four doses of CHF 4227.01 (0.001, 0.01, 0.1, and 1 mg/kg body weight [bw]/day) were administered in OVX animals daily by gavage 5 days/week for 4 months. EST was administered at a dose of 0.1 mg/kg bw/day, whereas RLX and LSX were administered at doses of 1 and 0.1 mg/kg bw/day, respectively, by gavage. In one group (Sham), rats were operated but the ovaries not removed; another OVX group was treated only with placebo., Results and Conclusions: Treatment with CHF 4227.01 (1.0 and 0.1 mg/kg bw), EST (0.1 mg/kg bw), LFX (0.1 mg/kg bw), or RLX (1.0 mg/kg bw) prevented bone loss on the lumbar spine and the proximal femur assessed in vivo by DXA. Volumetric BMD obtained by pQCT ex vivo confirmed protection from bone loss in the spine and proximal femur among rats treated with CHF 4227.01. This effect was associated with strong inhibition of bone resorption both histologically and biochemically. Furthermore, CHF 4227.01 preserved trabecular microarchitecture, analyzed by muCT, and maintained biomechanical indices of bone strength in the spine and proximal femur, effects also observed for RLX, whereas LSX was less protective of microarchitecture. CHF 4227.01 treatment did not affect uterine weight, prevented the increase in body weight and fat mass seen in OVX animals, and decreased serum cholesterol to below the average of intact animals. In conclusion, CHF 4227.01 exhibits a promising therapeutic and safety profile as a new SERM on both skeletal and extraskeletal outcomes.

The synthesis, binding affinity for estrogen receptor subtypes (ER alpha and ER beta) and pharmacological activity on rat uterus of a new class of potent ligands, characterized by a 3-phenylbenzopyran scaffold with a basic side chain in position 4, are reported. Some of these compounds, endowed with very high receptor affinity, showed potent inhibition of agonist-stimulated uterine growth, with no or limited proliferative effect. Binding affinity mostly depended on the nature and position of substituents at the 3-phenyl ring, while the uterine activity seems to be affected by basic chain length. Compound 9c (CHF4227) showed excellent binding affinity and antagonist activity on the uterus. The docking of benzopyran derivatives explained the structure-affinity relationships observed for 3-phenyl substitution: a small, hydrophobic 4'-substituent could interact with a small accessory binding cavity, while di-substitution at 4' and 3' led to some ER alpha selectivity. This selectivity can be ascribed to differences in amino acid composition and side chain conformation in the region accommodating the 3-phenyl ring at human ER alpha and ER beta ligand-binding domain.

We have identified a new benzopyran derivative, 3-(4-methoxy) phenyl-4-[[4-[2-(1-piperidinyl)ethoxy]phenyl]methyl]-2H-1-benzopyran-7-ol hydrochloride (CHF 4227), with improved in vivo estrogen agonist/antagonist effects. CHF 4227 binds with high affinity to the human estrogen receptor-alpha and -beta (dissociation constant K(i) = 0.017 and 0.099 nM, respectively). In immature rats, oral administration of CHF 4227 for 3 days inhibited the uterotrophic action of 17alpha-ethynyl estradiol (EE2) (ED(50) = 0.016 mg/kg. day); raloxifene was 25 times less potent as estrogen antagonist (ED(50) = 0.39 mg/kg. day), whereas both compounds were found to be devoid of uterotrophic activity. In line with its estrogen antagonist effect, CHF 4227 significantly prevented the development of dimethylbenz[a]anthracene (DMBA)-induced mammary tumors, the incidence being reduced from 87.5 to 26.3% 6 months after DMBA administration. In ovariectomized (OVX) rats treated orally for 4 weeks, CHF 4227 completely inhibited OVX effects on bone density (ED(50) = 0.003 mg/kg. day) and on serum osteocalcin levels. The protective effects on bone were comparable with those achieved with EE2, whereas raloxifene was less efficacious and 100 times less potent. CHF 4227 reduced serum cholesterol (ED(50) = 0.007 mg/kg. day) and had little to no stimulatory effects on uterine weight, uterine peroxidase activity, and endometrium epithelial thickness. In conclusion, CHF 4227 compares favorably in efficacy and potency with raloxifene in preventing bone loss and in antagonizing EE2 stimulation of the uterus. This profile along with the minimal uterine stimulation suggests a therapeutic advantage to CHF 4227 over EE2 or raloxifene for the treatment of postmenopausal women.

We have discovered a new, nonsteroidal, estrogen agonist/antagonist, 3-phenyl-4-[[4-[2-(1-piperidinyl)ethoxy]phenyl] methyl]-2H-1-benzopyran-7-ol (CHF 4056). The aim of this study was to determine the effects of CHF 4056 on a series of parameters (body weight, uteri, serum cholesterol, and bones) that were previously shown to be sensitive to estrogens and to selective estrogen receptor modulators (SERMs). CHF 4056 is a benzopyran derivative that binds with high affinity to the human estrogen receptors alpha and beta (dissociation constant K(i) of 0.041 and 0.157 nM, respectively). In immature rats, CHF 4056 induced a full estrogen antagonism (half-maximal efficacious dose = 0.33 mg/kg x day p.o.) coupled with a lack of uterine stimulatory activity, whereas the structurally related SERM levormeloxifene demonstrated a maximal partial agonist effect of approximately 65% that of 17alpha-ethynyl estradiol (EE2). In ovariectomized (OVX) rats, CHF 4056 (0.1-1 mg/kg x day p.o. for 4 weeks) significantly reduced OVX-induced bone loss in the lumbar spine L1-4 and OVX-induced increase in serum osteocalcin. These protective effects on bone tissue were comparable with those of 0.1 mg/kg x day EE2. In the same experimental conditions, serum cholesterol was significantly lower in the CHF 4056-treated animals, compared with vehicle-treated OVX rats. In line with the results observed in immature rats, also in OVX rats CHF 4056 diverged dramatically from EE2 and levormeloxifene in its lack of significant estrogenic effects on uterine tissue. In conclusion, CHF 4056 is a new SERM that produces beneficial effects on bone and cholesterol levels, while maintaining antagonist effects on the uterus.