Synthesis of 14 C- and 2 H-labelled CHF6001: A new potent PDE4 inhibitor.

An 8-step preparation of 14 C-labelled CHF6001, a potent phosphodiesterase 4 inhibitor in the treatment of respiratory diseases, is described. An overall yield of approximately 9% was obtained starting from copper[14 C]cyanide. The synthesis of a stable labelled version of CHF6001 is also reported using the commercially available trideuterated bromomethylcyclopropane as starting material.

Development and validation of a bioanalytical method for the quantification of CHF6550 and its metabolite (CHF6671) in rat plasma and lung homogenate using LC-MS/MS.

A selective and sensitive liquid chromatography-tandem mass spectrometry method was developed and validated for accurate determination of CHF6550 and its main metabolite in rat plasma and lung homogenate samples. All biological samples were prepared by simple protein precipitation method using deuterated internal standards. The analytes were separated on a HSS T3 analytical column with 3.2 min run time at flow rate of 0.5 mL/min. The detection was performed on a triple-quadrupole tandem mass spectrometer equipped with positive-ion electrospray ionization by selected-reaction monitoring of the transitions at m/z 735.3 â†’ 98.0 for CHF6550 and m/z 638.3 â†’ 319.2 and 638.3 â†’ 376.2 for CHF6671. The calibration curves for plasma samples were linear between 50 and 50000 pg/mL for both analytes. The calibration curves for lung homogenate samples were linear within 0.1-100 ng/mL for CHF6550 and 0.3-300 ng/mL for CHF6671. The method was successfully applied to a 4-week toxicity study.

Bronchodilator activity of (3R)-3-[[[(3-fluorophenyl)[(3,4,5-trifluorophenyl)methyl]amino] carbonyl]oxy]-1-[2-oxo-2-(2-thienyl)ethyl]-1-azoniabicyclo[2.2.2]octane bromide (CHF5407), a potent, long-acting, and selective muscarinic M3 receptor antagonist.

The novel quaternary ammonium salt (3R)-3-[[[(3-fluorophenyl)[(3,4,5-trifluorophenyl)methyl]amino]carbonyl]oxy]-1-[2-oxo-2-(2-thienyl)ethyl]-1-azoniabicyclo[2.2.2]octane bromide (CHF5407) showed subnanomolar affinities for human muscarinic M1 (hM1), M2 (hM2), and M3 (hM3) receptors and dissociated very slowly from hM3 receptors (t(½) = 166 min) with a large part of the receptorial complex (54%) remaining undissociated at 32 h from radioligand washout. In contrast, [(3)H]CHF5407 dissociated quickly from hM2 receptors (t(½) = 31 min), whereas [(3)H]tiotropium dissociated slowly from both hM3 (t(½) = 163 min) and hM2 receptor (t(½) = 297 min). In the guinea pig isolated trachea and human isolated bronchus, CHF5407 produced a potent (pIC(50) = 9.0-9.6) and long-lasting (up to 24 h) inhibition of M3 receptor-mediated contractile responses to carbachol. In the guinea pig electrically driven left atrium, the M2 receptor-mediated inhibitory response to carbachol was recovered more quickly in CHF5407-pretreated than in tiotropium-pretreated preparations. CHF5407, administered intratracheally to anesthetized guinea pigs, potently inhibited acetylcholine (Ach)-induced bronchoconstriction with an ED(50) value of 0.15 nmol/kg. The effect was sustained over a period of 24 h, with a residual 57% inhibition 48 h after antagonist administration at 1 nmol/kg. In conscious guinea pigs, inhaled CHF5407 inhibited Ach-induced bronchoconstriction for at least 24 h as did tiotropium at similar dosages. Cardiovascular parameters in anesthetized guinea pigs were not significantly changed by CHF5407, up to 100 nmol/kg i.v. and up to 1000 nmol/kg i.t. In conclusion, CHF5407 shows a prolonged antibronchospastic activity both in vitro and in vivo, caused by a very slow dissociation from M3 receptors. In contrast, CHF5407 is markedly short-acting at M2 receptors, a behavior not shared by tiotropium.

Role of efflux transporters in the absorption, distribution and elimination in rodents of a novel PDE4 inhibitor, CHF6001.

CHF6001 is a new and potent PDE4 inhibitor for the treatment of human lung diseases, designed for topical administration by inhalation. In preclinical assessment CHF6001 appeared safe and devoid of emetic effect, which is typical side effect of PDE4 inhibitors in humans. CHF6001 absorption, distribution and excretion were evaluated in rats by PO and IV administration of [14C]CHF6001; additionally the role of transporters was investigated by using transfected cells expressing either human transporters or MDR1 and BCRP KO mice. [14C]CHF6001 intravenously administered as bolus distributed in all the tissues (with very low levels in brain and fetus) and it was mainly eliminated in bile. Following oral administration [14C]CHF6001 about half of the dose was absorbed through the gut. In vitro, CHF6001 was a substrate of human membrane transporters MDR1 and BCRP. In wild and BCRP KO mice CHF6001 was not detectable in brain, whereas it was measurable in Mdr1a/b KO mice. Therefore, in animal species Mdr1a/b plays a significant role in CHF6001 disposition, limiting its distribution into brain and contributing to the safety profile observed in preclinical evaluation. This behavior was confirmed by the results of the first human studies, where CHF6001 was safe and with no emetic effect at all the evaluated doses.

Discovery of a novel isoxazoline derivative of prednisolone endowed with a robust anti-inflammatory profile and suitable for topical pulmonary administration.

A novel glucocorticoids series of (GCs), 6α,9α-di-Fluoro 3-substituted C-16,17-isoxazolines was designed, synthesised and their structure-activity relationship was evaluated with glucocorticoid receptor (GR) binding studies together with GR nuclear translocation cell-based assays. This strategy, coupled with in silico modelling analysis, allowed for the identification of Cpd #15, an isoxazoline showing a sub-nanomolar inhibitory potency (IC50=0.84 nM) against TNFα-evoked IL-8 release in primary human airways smooth muscle cells. In Raw264.7 mouse macrophages, Cpd #15 inhibited LPS-induced NO release with a potency (IC50=6 nM)>10-fold higher with respect to Dexamethasone. Upon intratracheal (i.t.) administration, Cpd #15, at 0.1 µmol/kg significantly inhibited and at 1 µmol/kg fully counteracted eosinophilic infiltration in a model of allergen-induced pulmonary inflammation in rats. Moreover, Cpd #15 proved to be suitable for pulmonary topical administration given its sustained lung retention (t1/2=6.5h) and high pulmonary levels (>100-fold higher than plasma levels) upon intratracheal administration in rats. In summary, Cpd #15 displays a pharmacokinetic and pharmacodynamic profile suitable for topical treatment of conditions associated with pulmonary inflammation such as asthma and COPD.

Determination of a new polymer-bound paclitaxel derivative (PNU 166945), free paclitaxel and 7-epipaclitaxel in dog plasma and urine by reversed-phase high-performance liquid chromatography with UV detection.

A sensitive and selective high-performance liquid chromatographic method for the determination of PNU 166945, a new polymer-bound paclitaxel derivative, free paclitaxel and 7-epipaclitaxel in dog plasma and urine has been developed. The method involves a solid-phase extraction of free paclitaxel and its possible degradation product 7-epipaclitaxel from plasma and urine, previously buffered with an equal volume of 0.05 M or 1 M KH2PO4 respectively, on 1-ml cyanopropyl columns. Cartridges elution was performed with the mobile phase, 0.05 M (pH 4.6) monobasic potassium phosphate-acetonitrile mixture (45:55, v/v). The samples were chromatographed on a reversed-phase octyl 4-microns column with UV detection at 229 nm. The retention times of paclitaxel and 7-epipaclitaxel were about 14 and 22 min, respectively. Determination of total paclitaxel (free + polymer-bound) was performed after release of paclitaxel from the polymeric carrier by chemical hydrolysis at room temperature (22 degrees C) for 20 h. After addition of 0.5 ml of methanol-0.1 M KH2PO4 mixture (50:50, v/v, pH = 7.5) to 0.5 ml of plasma or urine, paclitaxel was analysed as described above. PNU 166945 concentration was then determined by subtraction of free from total paclitaxel. The linearity, precision, accuracy and recovery of the method were evaluated. The limit of quantitation of the method was 5 ng/ml for biological fluid for paclitaxel and 7-epipaclitaxel and 20 ng/ml for PNU 166945 (as paclitaxel equivalent).

Determination of PNU-248686A, a novel matrix metalloproteinase inhibitor, in human plasma by liquid chromatography-tandem mass spectrometry, following protein precipitation in the 96-well plate format.

A sensitive, specific and high-throughput analytical method for the quantitation of PNU-248686A (I), in human plasma has been developed. I, sodium (2R)-3-[[(4'-chloro(1,1'-biphenyl)-4-yl]sulfonyl]-2-hydroxy-2-[(phenylsulfanyl)methyl] propanoate, is an orally active matrix metalloproteinase (MMP) inhibitor developed for the treatment of solid tumors over-expressing MMPs. Concentrations of I, as free acid, were determined in human plasma by LC-MS-MS after plasma protein precipitation in the 96-well plate format. Aliquots of plasma (50 microl) were placed into the plates and 0.2 ml of methanol was added. The plates were shaken for 5 min and centrifuged at 1500 g for 10 min. Aliquots of 10 microl of the supernatants were then directly injected into the LC-MS-MS system. A Symmetry Shield C. column (50 x 2.1 mm, 3.5 microm) was used to perform the chromatographic analysis. The mobile phase was 5 mM ammonium formate buffer solution pH 5.0-acetonitrile (60:40. v/v) with a flow-rate of 0.3 ml/min. Retention time of I was about 1.2 min. Total cycle time was 2.5 min. MS detection used the Applied Biosystems-MDS Sciex API 3000 with TurbolonSpray interface and single reaction monitoring (461 --> 251 m/z transition) operated in negative ion mode. Calibration curves were constructed by plotting the area of the compound (y) against its concentration (x). A weighed linear regression (weighting factor 1/x(2)) was used to calculate I concentrations in quality control and unknown samples. The method was fully validated over the range of 5.0-5000 ng/ml. The suitability and robustness of the method for in vivo samples was confirmed by analysis of plasma samples from a pilot clinical study.

LC-MS-MS determination of exemestane in human plasma with heated nebulizer interface following solid-phase extraction in the 96 well plate format.

A sensitive, specific and rapid analytical method for the quantitation of exemestane (EXE) in human plasma has been developed. EXE, 6-methylen-androsta-1,4-diene-3,17-dione, is an orally active irreversible steroidal aromatase inhibitor used for the therapy of metastatic postmenopausal breast cancer, with estrogen-dependent pathological conditions. The method involves extraction of EXE from human plasma by solid phase extraction using C2 endcapped sorbent in the 96 well plate format (50 mg/2 ml). After conditioning of the sorbent with 1 ml of acetonitrile (x2) the plates were rinsed with 1 ml of water (x2). The prepared samples (0.5 ml plasma, spiked with [13C3] EXE as internal standard (IS) and diluted with 0.5 ml water) were loaded and drawn through the plate with a minimum of vacuum. The plates were then washed with 1 ml acetonitrile:water (10:90) followed by a drying step for 30 min at full vacuum. Elution was by 0.15 ml of 0.1% trifluoracetic acid in acetonitrile (x2) under a minimum of vacuum. Aliquots of 80 microl were finally injected into the LC-MS-MS system. A Zorbax SB C8 column (4.6 x 150 mm, 5 microm) was used to perform the chromatographic separation; the mobile phase was 100% acetonitrile. MS detection used the heated nebulizer interface, with multiple reaction monitoring (MRM) (297-->121 m/z for EXE and 300-->123 m/z for IS) operated in positive ion mode. A weighed linear regression analysis (weighing factor 1/x2) was used to calculate EXE concentration in standard and unknown samples. The method was fully validated in the concentration range 0.05-25 ng ml(-1).

Clindamycin/cefonicid in head and neck oncologic surgery: one-day prophylaxis is as effective as a three-day schedule.

The aim of our study was to evaluate the optimal duration of antibiotic prophylaxis in major oncologic surgery of the head and neck using a novel broad spectrum drug combination: clindamycin and cefonicid. A prospective randomized study was carried out on 126 evaluable patients undergoing clean-contaminated (skin to mucosa) surgery for cancer of larynx, pharynx or oral cavity. Cases at high surgical risk (because of need of pedicled or microvascular free flaps reconstruction), were excluded from the study. Within 20 days after surgery, only one case of wound infection was recorded among the 62 patients treated with the one-day schedule, versus three cases registered among the 64 subjects receiving three-day chemoprophylaxis. Episodes of systemic infections and eventual wound complications occurring in the first 20 days after surgery have also been recorded. The role of potential risk factors for postoperative complications has been evaluated. According to our findings, a three-day antibiotic regimen is not more effective than a short-term (one-day) schedule in preventing wound or systemic infection in clean-contaminated head and neck cancer surgery without flap reconstruction.

Epithelial-myoepithelial carcinoma of the parotid gland: a clinico-pathologic and immunohistochemical study of seven cases.

Seven cases of epithelial-myoepithelial carcinoma of the parotid gland are reported. Immunohistochemical evidence for the dual (glandular secretory and myoepithelial) differentiation of the cells composing these lesions is presented. Three of the cases recurred locally and two gave rise to metastases. The biologic behavior does not appear to be correlated with the histologic features that constitute the morphologic spectrum of epithelial-myoepithelial carcinoma.

CHF5074, a novel gamma-secretase modulator, attenuates brain beta-amyloid pathology and learning deficit in a mouse model of Alzheimer's disease.

BACKGROUND AND PURPOSE: We evaluated the effects of 1-(3',4'-dichloro-2-fluoro[1,1'-biphenyl]-4-yl)-cyclopropanecarboxylic acid (CHF5074), a new gamma-secretase modulator, on brain beta-amyloid pathology and spatial memory in transgenic mice expressing the Swedish and London mutations of human amyloid precursor protein (hAPP). EXPERIMENTAL APPROACH: Sixty 6-month-old hAPP mice were treated for 6 months with CHF5074 or ibuprofen (375 ppm in the diet) or standard diet. Twenty-one wild-type mice received standard diet. KEY RESULTS: Compared with transgenic controls, CHF5074 treatment significantly reduced the area occupied by plaques in cortex (P = 0.003) and hippocampus (P = 0.004). The number of plaques were also reduced by CHF5074 in both cortex (P = 0.022) and hippocampus (P = 0.005). Plaque-associated microglia in CHF5074-treated animals was lower than in transgenic controls in cortex (P = 0.008) and hippocampus (P = 0.002). Ibuprofen treatment significantly reduced microglia area in cortex and hippocampus but not beta-amyloid burden. On the last day of the Morris water maze, transgenic controls performed significantly worse than the non-transgenic animals and the CHF5074-treated transgenic mice, on the swimming path to reach the hidden platform. Ibuprofen-treated animals did not perform significantly better than transgenic controls. CONCLUSIONS AND IMPLICATIONS: Chronic CHF5074 treatment reduced brain beta-amyloid burden, associated microglia inflammation and attenuated spatial memory deficit in hAPP mice. This novel gamma-secretase modulator is a promising therapeutic agent for Alzheimer's disease.

A transmissible cytotoxic activity isolated from a patient with brain ischemia causes microglial cell activation and dysfunction.

1. Microglial cell activation occurs during brain injury, ischemia, and in several neurologic disorders. Recently, we isolated a transmissible cytotoxic activity (TCA) from the cerebrospinal fluid of a patient with brain ischemia. Such a TCA, associated with one or more protein(s) that supposedly had undergone in vivo misfolding, causes apoptosis in vitro in different cell lines, including microglial cells. The TCA producing cells and the potential in vivo role of such cytotoxic activity remains to be elucidated. Here, we investigated the in vitro effects of TCA on microglial cell immune functions.2. The murine microglial cell line RR4 was exposed to TCA, and then its response was evaluated as: (a) phagocytosis and antifungal activity against Candida albicans; (b) secretory pattern; and (c) levels of p38 phosphorylation.3. Unlike mock-treated controls, microglial cells exposed to TCA showed an increase in phagocytic activity. Unexpectedly, their capability to kill the ingested fungi significantly diminished. Moreover, TCA-treated cells produced amounts of macrophage inflammatory protein 1-alpha, tumor necrosis factor-alpha, and nitric oxide significantly higher than mock-treated cells. Finally, phosphorylation of p38 mitogen-activated protein kinase (MAPK) was detected in TCA-treated but not in mock-treated controls as early as 30 min after treatment.4. Overall, these results indicate that TCA causes a rapid molecular response in microglial cells, by the time, leading to an intriguing effector and secretory dysfunction.

[Intraparotid lesions in HIV infection]. / Lesioni intraparotidee in infezione da HIV.

HIV-related lesions of the parotid region. A case of parotid enlargement in a 43-year-old male with unsuspected HIV infection is reported. Lesions consisted in lymphonodal changes, epi-myoepithelial islands and cysts lined by squamous epithelium. It is suggested that it is possible to recognize an unsuspected HIV infection, when the first clinical involvement is in the parotid region.

Short-term versus long-term antimicrobial prophylaxis in oncologic head and neck surgery.

BACKGROUND: Although antimicrobial prophylaxis is mandatory in major clean-contaminated oncologic surgery of the head and neck, both the choice of specific antimicrobial compounds and the treatment duration are still discussed. METHODS: A prospective, randomized trial was carried out to compare efficacy and tolerability of clindamycin-cefonicid administered for 1 day versus 3 days in reducing the rate of wound and systemic infections. The following potential risk factors for surgical wound infection were evaluated: type of surgery, stage of disease, preoperative tracheostomy, preoperative radiotherapy, and diabetes mellitus. RESULTS: One-hundred sixty-two patients were evaluable; 81 received 1-day chemoprophylaxis, while the remaining 81 were treated according to the 3-day schedule. During the first 20 days after surgery, wound infections occurred in 2 (2.5%) and 3(3.7%) patients, respectively, in the 1-day and 3-day treatment groups, so that no significant difference was found among the two evaluated chemoprophylaxis schedules. CONCLUSION: A 3-day schedule did not prove useful in preventing wound and systemic infections. All presumed risk factors were not associated with an increased rate of wound infections, although preoperative radiotherapy was associated with a greater severity of infections and a higher risk of late wound complications.