Antitumor activity of the PI3K δ-sparing inhibitor MEN1611 in PIK3CA mutated, trastuzumab-resistant HER2 + breast cancer.

PURPOSE: Dysregulation of the PI3K pathway is one of the most common events in breast cancer. Here we investigate the activity of the PI3K inhibitor MEN1611 at both molecular and phenotypic levels by dissecting and comparing its profile and efficacy in HER2 + breast cancer models with other PI3K inhibitors. METHODS: Models with different genetic backgrounds were used to investigate the pharmacological profile of MEN1611 against other PI3K inhibitors. In vitro studies evaluated cell viability, PI3K signaling, and cell death upon treatment with MEN1611. In vivo efficacy of the compound was investigated in cell line- and patient-derived xenografts models. RESULTS: Consistent with its biochemical selectivity, MEN1611 demonstrated lower cytotoxic activity in a p110δ-driven cellular model when compared to taselisib, and higher cytotoxic activity in the p110ß-driven cellular model when compared to alpelisib. Moreover, MEN1611 selectively decreased the p110α protein levels in PIK3CA mutated breast cancer cells in a concentration- and proteasome-dependent manner. In vivo, MEN1611 monotherapy showed significant and durable antitumor activity in several trastuzumab-resistant PIK3CA-mutant HER2 + PDX models. The combination of trastuzumab and MEN1611 significantly improved the efficacy compared to single agent treatment. CONCLUSIONS: The profile of MEN1611 and its antitumoral activity suggest an improved profile as compared to pan-inhibitors, which are limited by a less than ideal safety profile, and isoform selective molecules, which may potentially promote development of resistance mechanisms. The compelling antitumor activity in combination with trastuzumab in HER2 + trastuzumab-resistant, PIK3CA mutated breast cancer models is at the basis of the ongoing B-Precise clinical trial (NCT03767335).

NETosis in Acute Thrombotic Disorders.

The release of extracellular traps by neutrophils (NETs) represents a novel active mechanism of cell death that has been recently implicated in the pathogenesis of thrombotic disorders. The aim of this study was to investigate the generation of NETs in different groups of patients with acute thrombotic events (ATEs) and to establish whether NETs markers can predict the risk of new cardiovascular events. We performed a case-control study of patients with ATE, including acute coronary syndrome (n = 60), cerebrovascular accident (n = 50), and venous thromboembolism (n = 55). Control subjects (n = 70) were identified among patients admitted for acute chest pain and in which a diagnosis of ATE was excluded. Serum levels of NET markers and neutrophil activation, such as myeloperoxidase (MPO)-DNA complexes, neutrophil gelatinase-associated lipocalin, polymorphonuclear neutrophil elastase, lactoferrin, and MPO, were measured in each patient. We found that circulating levels of MPO-DNA complexes were significantly increased in patients with ATE (p < 0.001) compared with controls and that this association remained significant even after fully adjustment for traditional risk factors (p = 0.001). A receiver operating characteristics analysis of circulating MPO-DNA complexes in discriminating between controls and patients with ATE showed a significant area under the curve of 0.76 (95% confidence interval: 0.69-0.82). After a median follow-up of 40.7 (± 13.8) months, 24 out of the 165 patients with ATE presented a new cardiovascular event and 18 patients died. None of the markers under investigation influenced survival or the incidence of new cardiovascular events. In conclusion, we found that increase of markers of NETosis can be observed in acute thrombotic conditions, occurring both on the arterial and venous site. Nevertheless, the level of neutrophil markers measured during the ATE is not predictive of future risk of mortality and cardiovascular events.

Business-community relations under COVID-19: A study of micro and small firms.

In considering the premises of social capital and stakeholder theory, this study examines the extent to which firm-community relationships were affected during the COVID-19 crisis, and the significance of firms for their community during this unprecedented event. Responses from 107 Italian micro and small firms were gathered through an online questionnaire. The findings first reveal a strengthening of relations, particularly between firms and other businesses in their community; however, participants' comments also recognise no changes or even weakening relations. Second, three dimensions highlighting the significance of firms' survival during the crisis emerged: the community context, underlining firms' socioeconomic and symbolic contributions, the immediate stakeholders, emphasising contributions towards employment and support of local businesses, and the broader community-society context, underscoring firms' contribution towards consumer and societal needs. The study proposes a conceptual framework illustrating various relationships between the findings and the considered conceptual underpinnings and suggests various implications.

Overcoming the unprecedented: Micro, small and medium hospitality enterprises under COVID-19.

The sudden irruption of COVID-19 has paralysed, even devastated, numerous industries. Academic and industry publications also convey the destructive impacts of this phenomenon on hospitality and tourism businesses. While business owners and managers are still constrained by unpredictability, restrictions, and ongoing uncertainty, those vying to continue will need to build their adaptive skill repertoire to cope with the crisis-related regime. This study is primarily concerned with businesses' adaptation phase from owners/managers' viewpoints, including how they manage and envision a future coexistence with COVID-19 threats. Drawing on an international sample of owners/managers of hospitality and tourism businesses, and considering the foundations of the dynamic capabilities framework, eight dimensions emerged from the findings. Five of these, persevering, dynamic, austere restrictions, business environment, and stakeholder, strongly suggest the relevance of reconfiguring, a cluster of dynamic capabilities. Together, the dimensions demonstrate participants' strong commitment to navigate through the threat while pursuing socioeconomic sustainability.

Targeting CD205 with the antibody drug conjugate MEN1309/OBT076 is an active new therapeutic strategy in lymphoma models.

Antibody drug conjugates represent an important class of anti-cancer drugs in both solid tumors and hematological cancers. Here, we report preclinical data on the anti-tumor activity of the first-in-class antibody drug conjugate MEN1309/OBT076 targeting CD205. The study included preclinical in vitro activity screening on a large panel of cell lines, both as single agent and in combination and validation experiments on in vivo models. CD205 was first shown frequently expressed in lymphomas, leukemias and multiple myeloma by immunohistochemistry on tissue microarrays. Anti-tumor activity of MEN1309/OBT076 as single agent was then shown across 42 B-cell lymphoma cell lines with a median IC50 of 200 pM and induction of apoptosis in 25/42 (59.5%) of the cases. The activity appeared highly correlated with its target expression. After in vivo validation as the single agent, the antibody drug conjugate synergized with the BCL2 inhibitor venetoclax, and the anti-CD20 monoclonal antibody rituximab. The first-in-class antibody drug targeting CD205, MEN1309/OBT076, demonstrated strong pre-clinical anti-tumor activity in lymphoma, warranting further investigations as a single agent and in combination.

COVID-19, aftermath, impacts, and hospitality firms: An international perspective.

Drawing on the theory of resilience, and on an international sample of 45 predominantly small hospitality businesses, this exploratory study extends knowledge about the key concerns, ways of coping, and the changes and adjustments undertaken by these firms' owners and managers during the COVID-19 outbreak. The various emergent relationships between the findings and the considered conceptual underpinnings of the literature on resilience, revealed nine theoretical dimensions. These dimensions critically illuminate and extend understanding concerning the actions and alternatives owners-managers resorted to when confronted with an extreme context. For instance, with financial impacts and uncertainty being predominant issues among participants, over one-third indicated actioning alternative measures to create much-needed revenue streams, and preparing for a new post-COVID-19 operational regime, respectively. Furthermore, 60 percent recognised making changes to the day-to-day running of the business to respond to initial impacts, or biding time in anticipation of a changing business and legal environment.

Managing the COVID-19 Pandemic: Experience of Managers in Healthcare: A Narrative Inquiry.

The pandemic represented a challenge for hospital managers at different levels, required to reorganise services without compromising care. This study aimed to analyse the experiences of hospital managers during the COVID-19 pandemic. A narrative inquiry was conducted in a multisite acute hospital. Data were collected through narratives and open-ended questions. Direct and non-direct-care managers were invited to participate. Data were analyzed considering Clandinin & Connelly's (2000) framework and Braun & Clarke, (2006). Thirty-six narratives and open-ended question responses were analysed. Participants were nurses (n = 20), doctors, technicians, administrative and hospitality service managers. Themes were grouped into three narrative dimensions: (1) personal-"the emergency engulfed us", (2) practical/professional-"managing the pandemic", (3) social-"the strength of the team and people". Different narrative threads were identified between direct-care and non direct-care managers. Problems faced, factors helpful for management and suggestions for improvement were also reported. The pandemic had an important impact on managers and their roles, in terms of the need for clear concise information, staff support, and adequate professional and technical resources. A sense of unity and belonging facilitated management.

Biomarkers of Neutrophil Activation in Patients with Symptomatic Chronic Peripheral Artery Disease Predict Worse Cardiovascular Outcome.

Neutrophils play a role in cardiovascular (CV) disease. However, relatively scant evidence exists in the setting of peripheral artery disease (PAD). The aims of this study were to measure biomarkers of neutrophil activation in patients with symptomatic chronic PAD compared with healthy controls, to assess their association with PAD severity, and to evaluate their prognostic value in patients with PAD. The following circulating markers of neutrophil degranulation were tested: polymorphonuclear neutrophil (PMN) elastase, neutrophil gelatinase-associated lipocalin (NGAL), and myeloperoxidase (MPO). Neutrophil extracellular traps (NETs) were quantified by measuring circulating MPO-DNA complexes. Patients with PAD underwent a comprehensive series of vascular tests. The occurrence of 6-month major adverse CV (MACE) and limb events (MALE) was assessed. Overall, 110 participants were included, 66 of which had PAD. After adjustment for conventional CV risk factors, PMN-elastase (adjusted odds ratio [OR]: 1.008; 95% confidence interval [CI]: 1.002-1.015; p = 0.006), NGAL (adjusted OR: 1.045; 95%CI: 1.024-1.066; p < 0.001), and MPO (adjusted OR: 1.013; 95%CI: 1.001-1.024; p = 0.028) were significantly associated with PAD presence. PMN-elastase (adjusted hazard ratio [HR]: 1.010; 95%CI: 1.000-1.020; p = 0.040) and MPO (adjusted HR: 1.027; 95%CI: 1.004-1.051; p = 0.019) were predictive of 6-month MACE and/or MALE. MPO displayed fair prognostic performance on receiver operating characteristic (ROC) curve analyses, with an area under the curve (AUC) of 0.74 (95%CI: 0.56-0.91) and a sensitivity and specificity of 0.80 and 0.65, respectively, for a cut-off of 108.37 ng/mL. MPO-DNA showed a weak inverse correlation with transcutaneous oximetry (TcPO2) on proximal foot (adjusted ρ -0.287; p = 0.032). In conclusion, in patients with symptomatic chronic PAD, enhanced neutrophil activity may be associated with an increased risk of acute CV events, rather than correlate with disease severity. Further research is needed to clarify the role of neutrophils in PAD natural history.

Six Sigma can significantly reduce costs of poor quality of the surgical instruments sterilization process and improve surgeon and operating room personnel satisfaction.

Operating room (OR) management is a complex multidimensional activity combining clinical and managerial aspects. This longitudinal observational study aimed to assess the impact of Six-Sigma methodology to optimize surgical instrument sterilization processes. The project was conducted at the operating theatre of our tertiary regional hospital during the period from July 2021 to December 2022. The project was based on the surgical instrument supply chain analysis. We applied the Six Sigma lean methodology by conducting workshops and practical exercises and by improving the surgical instrument process chain, as well as checking stakeholders' satisfaction. The primary outcome was the analysis of Sigma improvement. Through this supply chain passed 314,552 instruments in 2022 and 22 OR processes were regularly assessed. The initial Sigma value was 4.79 ± 1.02σ, and the final one was 5.04 ± 0.85σ (SMD 0.60, 95%CI 0.16-1.04, p = 0.010). The observed improvement was estimated in approximately $19,729 of cost savings. Regarding personnel satisfaction, 150 questionnaires were answered, and the overall score improved from 6.6 ± 2.2 pts to 7.0 ± 1.9 pts (p = 0.013). In our experience the application of the Lean Six Sigma methodology to the process of handling the surgical instruments from/to the OR was cost-effective, significantly decreased the costs of poor quality and increased internal stakeholder satisfaction.

Design and synthesis of novel sulfonamide-containing bradykinin hB(2) receptor antagonists. Synthesis and structure-relationships of α,α-tetrahydropyranylglycine.

A series of α,α-cycloalkylglycine sulfonamide compounds of general formula 1 has previously been identified by our group as selective human B(2)(hB(2)) receptor antagonists. Here we report the in vitro and in vivo BK antagonist activity of a further evolution of the series, consisting in compounds of the general formula 2, containing either an alkyl piperazine or a 4-alkyl piperidine ring bearing various positively charged groups (R'). These studies unexpectedly revealed quite a flat nanomolar/subnanomolar SAR for the binding affinity, while differences were seen in the in vitro functional activities. We propose that variations in the residence time may explain these results.

Reducing Changeover Time Between Surgeries Through Lean Thinking: An Action Research Project.

Background: Maximizing the utilization of the operating room suite by safely and efficiently changing over patients is an opportunity to deliver more value to patients and be more efficient in the operating suite. Lean Thinking is a concept that focuses on the waste inadvertently generated during organization and development of an activity, which should maximize customer value while minimizing waste. It has been widely applied to increase process efficiency and foster continuous improvement in healthcare and in the operating room environment. The objective of this paper is to provide insight on how healthcare professionals can be engaged in continuous improvement by embracing Lean Thinking and ultimately reducing changeover time between surgeries. Methods: Using an action research approach, Lean methodology such as Gemba walks, Process Mapping, Root-Cause-Analysis, and the Single Minute Exchange of Dies (SMED) system was applied to understand the causes of variability and wastes concerning changeovers and improve processes in the context of gynecological- and general surgery. Data were collected and analyzed through observations and video recordings. Problem and issue have been raised to management team attention and included in the annual balanced scorecard of the hospital. This initiative has been also made relevant to the team working in the operating suite and related processes before and after the entry of the patient in the operating suite. Results: Improved patient flow and inter-professional collaboration through standardized and safer work enabled effective parallel processing and allowed the hospital to reduce changeover time between operations by 25% on average, without changes in terms of infrastructure, technology or resources. Conclusion: Lean thinking allowed the team to re-evaluate how the whole operating suite performs as a system, by starting from a sub-process as changeover. It is fundamental in order to improve further and obtain sustainable results over time, to act on a system level by defining a common goal between all stakeholders supported by a management and leading system such as visual/weekly management, optimizing planning, implementing standard-works to be followed by every associate and guaranteeing the role of the surgeon as process driver who pull performances.

PCSK9 promotes arterial medial calcification.

BACKGROUND AND AIMS: A complex interplay among chronic kidney disease (CKD), lipid metabolism and aortic calcification has been recognized. Here we investigated the influence of kidney function on PCSK9 levels and its potential direct action on smooth muscle cells (SMCs) calcification. METHODS AND RESULTS: In a cohort of 594 subjects, a negative association between glomerular filtration rate and plasma PCSK9 was found. Atherosclerotic cardiovascular disease, as co-morbidity, further increased PCSK9 plasma levels. Diet-induced uremic condition in rats led to aortic calcification and increased total cholesterol and Pcsk9 levels in plasma, livers, and kidneys. Both human and rat SMCs overexpressing human PCSK9 (SMCsPCSK9), cultured in a pro-calcific environment (2.0 mM or 2.4 mM inorganic phosphate, Pi) showed a significantly higher extracellular calcium (Ca2+) deposition compared to control SMCs. The addition of recombinant human PCSK9 did not increase the extracellular calcification of SMCs, suggesting the involvement of intracellular PCSK9. Accordingly, the further challenge with evolocumab did not affect calcium deposition in hSMCsPCSK9. Under pro-calcific conditions, SMCsPCSK9 released a higher number of extracellular vesicles (EVs) positive for three tetraspanin molecules, such as CD63, CD9, and CD81. EVs derived from SMCsPCSK9 tended to be more enriched in calcium and alkaline phosphatase (ALP), compared to EVs from control SMCs. In addition, PCSK9 has been detected in SMCsPCSK9-derived EVs. Finally, SMCsPCSK9 showed an increase in pro-calcific markers, namely bone morphogenetic protein 2 and ALP, and a decrease in anti-calcific mediator matrix GLA protein and osteopontin. CONCLUSIONS: Our study reveals a direct role of PCSK9 on vascular calcification induced by higher inorganic phosphate levels associated with renal impairment. This effect appears to be mediated by a switch towards a pro-calcific phenotype of SMCs associated with the release of EVs containing Ca2+ and ALP.

Cholesterol-Lowering Action of a Novel Nutraceutical Combination in Uremic Rats: Insights into the Molecular Mechanism in a Hepatoma Cell Line.

Appropriate nutraceutical combinations may represent a valid approach to prevent vascular calcification associated with chronic kidney disease (CKD). In the present study, we tested the effect of a new nutraceutical combination named RenaTris®, containing MK-7, magnesium carbonate, and Sucrosomial® Iron, on vascular calcification in uremic rats. Rats were randomly divided into three groups, i.e. control (high-phosphate diet), uremic (high-phosphate diet containing 0.5% adenine), and supplemented uremic diet (0.5% adenine, MK-7, magnesium carbonate, and Sucrosomial® Iron). After six weeks, sera and vascular calcification were examined. The uremic diet increased creatinine and phosphate levels and induced extensive vascular calcification. The uremic condition also induced a mild hypercholesterolemic condition (+52% of total cholesterol; p < 0.05). The supplemented uremic diet did not reduce creatinine, phosphate levels, or vascular calcification, however, we observed a significant hypocholesterolemic effect (-18.9% in supplemental uremic vs. uremic diet; p < 0.05). Similar to simvastatin, incubation of cultured human hepatoma cells (Huh7) with MK-7 significantly reduced cholesterol biosynthesis (-38%) and induced 3-hydroxy-3-methyl-glutaryl-CoA (HMG-CoA) reductase and low-density lipoprotein receptor (LDLR) at both mRNA and protein levels. The effect of MK-7 on LDLR was counteracted by the co-incubation with squalene. Unlike simvastatin, MK-7 reduced PCSK9 in Huh7. These results indicated that the new nutraceutical combination significantly impacts cholesterol metabolism and its supplementation may help to control mild hypercholesterolemic conditions in CKD patients.

Antitumor activity of delimotecan against human metastatic melanoma: pharmacokinetics and molecular determinants.

Delimotecan (MEN 4901/T-0128) is a new cytotoxic prodrug constituted by a camptothecin analog (T-2513) bound to carboxymethyl dextran through a triglycine linker. A significant antitumor activity of delimotecan against human metastatic melanoma xenograft model Me15392 is reported. Dacarbazine, the drug approved for the treatment of metastatic melanoma, was ineffective in this melanoma model. Pharmacokinetic studies, together with the expression analysis of mRNA for enzymes involved in delimotecan metabolism, showed that T-2513 and other cytotoxic metabolites of delimotecan (SN 38 and T-0055) are generated in greater quantities in the tumor tissue than in toxicity target tissues, such as liver, thus accounting for the antitumoral activity. Moreover, we demonstrated that human metastatic melanoma cells are able to phagocytose delimotecan and cleave it to release the cytotoxic moieties T-2513 in the tumoral environment. Further flow cytometric analysis showed a higher recruitment of macrophages in xenografted human metastatic melanoma, when compared with other human tumors. Thus, the antitumoral activity of delimotecan exerted on metastatic melanoma is due to several factors: (i) the ability of melanoma cells to phagocytose and metabolise delimotecan; (ii) the accumulation of delimotecan in tumoral mass; (iii) the recruitment of macrophage cells to the melanoma nodule and (iv) the expression in melanoma cells of a pattern of enzymes that converts delimotecan into cytotoxic metabolites. Based on these results, delimotecan might be exploited as a new anticancer agent for the therapy of metastatic melanoma because of its high efficacy and good selectivity, and therefore clinical trials for this indication are warranted.

MEN1309/OBT076, a First-In-Class Antibody-Drug Conjugate Targeting CD205 in Solid Tumors.

CD205 is a type I transmembrane glycoprotein and is a member of the C-type lectin receptor family. Analysis by mass spectrometry revealed that CD205 was robustly expressed and highly prevalent in a variety of solid malignancies from different histotypes. IHC confirmed the increased expression of CD205 in pancreatic, bladder, and triple-negative breast cancer (TNBC) compared with that in the corresponding normal tissues. Using immunofluorescence microscopy, rapid internalization of the CD205 antigen was observed. These results supported the development of MEN1309/OBT076, a fully humanized CD205-targeting mAb conjugated to DM4, a potent maytansinoid derivate, via a cleavable N-succinimidyl-4-(2-pyridyldithio) butanoate linker. MEN1309/OBT076 was characterized in vitro for target binding affinity, mechanism of action, and cytotoxic activity against a panel of cancer cell lines. MEN1309/OBT076 displayed selective and potent cytotoxic effects against tumor cells exhibiting strong and low to moderate CD205 expression. In vivo, MEN1309/OBT076 showed potent antitumor activity resulting in durable responses and complete tumor regressions in many TNBC, pancreatic, and bladder cancer cell line-derived and patient-derived xenograft models, independent of antigen expression levels. Finally, the pharmacokinetics and pharmacodynamic profile of MEN1309/OBT076 was characterized in pancreatic tumor-bearing mice, demonstrating that the serum level of antibody-drug conjugate (ADC) achieved through dosing was consistent with the kinetics of its antitumor activity. Overall, our data demonstrate that MEN1309/OBT076 is a novel and selective ADC with potent activity against CD205-positive tumors. These data supported the clinical development of MEN1309/OBT076, and further evaluation of this ADC is currently ongoing in the first-in-human SHUTTLE clinical trial.

Design and synthesis of novel sulfonamide-containing bradykinin hB2 receptor antagonists. 2. Synthesis and structure-activity relationships of alpha,alpha-cycloalkylglycine sulfonamides.

Recently we reported on the design and synthesis of a novel class of selective nonpeptide bradykinin (BK) B2 receptor antagonists (J. Med. Chem. 2006, 3602-3613). This work led to the discovery of MEN 15442, an antagonist with subnanomolar affinity for the human B2 receptor (hB2R), which also displayed significant and prolonged activity in vivo (for up to 210 min) against BK-induced bronchoconstriction in the guinea-pig at a dose of 300 nmol/kg (it), while demonstrating only a slight effect on BK-induced hypotension. Here we describe the further optimization of this series of compounds aimed at maximizing the effect on bronchoconstriction and minimizing the effect on hypotension, with a view to developing topically delivered drugs for airway diseases. The work led to the discovery of MEN 16132, a compound which, after intratracheal or aerosol administration, inhibited, in a dose-dependent manner, BK-induced bronchoconstricton in the airways, while showing minimal systemic activity. This compound was selected as a preclinical candidate for the topical treatment of airway diseases involving kinin B2 receptor stimulation.

Sabarubicin (MEN10755)-induced apoptosis is independent from mtDNA in A2780 human ovarian tumor cells.

BACKGROUND: The role of mitochondrial DNA (mtDNA) in anthracycline-induced apoptosis is controversial. Sabarubicin accumulates in the mitochondria of A2780 human ovarian tumor cells. The effects of this new anthracycline on the structure and the functionality of mtDNA, as well as on the apoptosis of mtDNA-depleted cells have been investigated. MATERIALS AND METHODS: Sabarubicin-induced mtDNA cleavage was detected by Southern blotting and mitochondrial mRNA expression was analyzed by real-time PCR. Apoptosis was studied in mtDNA-depleted (theta0) and parental (theta+) A2780 cells detecting nuclear DNA fragmentation using ELISA and cytofluorimetrically using Annexin V/PI staining. Mitochondrial membrane potential was studied using the cyanine dye JC-1. RESULTS: Sabarubicin induced mtDNA cleavage in the A2780 cells, but this damage did not affect mitochondrial mRNA expression. Apoptosis was induced by sabarubicin in theta0 as well as in theta+ cells. CONCLUSION: The results showed that mtDNA did not influence anthracycline-induced apoptosis in A2780 cells.

Design and synthesis of novel sulfonamide-containing bradykinin hB2 receptor antagonists. 1. Synthesis and SAR of alpha,alpha-dimethylglycine sulfonamides.

We recently published the extensive in vivo pharmacological characterization of MEN 16132 (J. Pharmacol. Exp. Ther. 2005, 616-623; Eur. J. Pharmacol. 2005, 528, 7), a member of the sulfonamide-containing human B(2) receptor (hB(2)R) antagonists. Here we report, in detail, how this family of compounds was designed, synthesized, and optimized to provide a group of products with subnanomolar affinity for the hB(2)R and high in vivo potency after topical administration to the respiratory tract. The series was designed on the basis of indications from the X-ray structures of the key structural motifs A and B present in known antagonists and is characterized by the presence of an alpha,alpha-dialkyl amino acid. The first lead (17) of the series was submitted to extensive chemical work to elucidate the structural requirements to increase hB(2) receptor affinity and antagonist potency in bioassays expressing the human B(2) receptor (hB(2)R). The following structural features were selected: a 2,4-dimethylquinoline moiety and a piperazine linker acylated with a basic amino acid. The representative lead compound 68 inhibited the specific binding of [(3)H]BK to hB(2)R with a pKi of 9.4 and antagonized the BK-induced inositolphosphate (IP) accumulation in recombinant cell systems expressing the hB(2)R with a pA(2) of 9.1. Moreover, compound 68 when administered (300 nmol/kg) intratracheally in the anesthetized guinea pig, was able to significantly inhibit BK-induced bronchoconstriction for up to 120 min after its administration, while having a lower and shorter lasting effect on hypotension.

MEN16132, a novel potent and selective nonpeptide antagonist for the human bradykinin B2 receptor. In vitro pharmacology and molecular characterization.

The pharmacological characterization of the novel nonpeptide antagonist for the B2 receptor, namely MEN16132 (4-(S)-Amino-5-(4-{4-[2,4-dichloro-3-(2,4-dimethyl-8-quinolyloxymethyl)phenylsulfonamido]-tetrahydro-2H-4-pyranylcarbonyl}piperazino)-5-oxopentyl](trimethyl)ammonium chloride hydrochloride) is presented. The affinity of MEN16132 for the bradykinin B2 receptor has been investigated by means of competition studies at [3H]bradykinin binding to membranes prepared from Chinese Hamster Ovary (CHO) cells expressing the human bradykinin B2 receptor (pKi 10.5), human lung fibroblasts (pKi 10.5), guinea pig airways (pKi 10.0), guinea pig ileum longitudinal smooth muscle (pKi 10.2), or guinea pig cultured colonic myocytes (pKi 10.3). In all assays MEN16132 was as potent as the peptide antagonist Icatibant, and from 3- to 100-fold more potent than the reference nonpeptide antagonists FR173657 or LF16-0687. The selectivity for the bradykinin B2 receptor was checked at the human bradykinin B1 receptor (pKi<5), and at a panel of 26 different receptors and channels. The antagonist potency was measured in functional assays, i.e., in blocking the bradykinin induced inositolphosphates (IP) accumulation at the human (CHO: pKB 10.3) and guinea pig (colonic myocytes: pKB 10.3) B2 receptor, or in antagonizing the bradykinin induced contractile responses in human (detrusor smooth muscle: pKB 9.9) and guinea pig (ileum longitudinal smooth muscle: pKB 10.1) tissues. In both functional assay types MEN16132 exerted a different antagonist pattern, i.e., surmountable at the human and insurmountable at the guinea pig bradykinin B2 receptors. Moreover, the receptor determinants important for the high affinity interaction of MEN16132 with the human bradykinin B2 receptor were investigated by means of radioligand binding studies performed at 24 point-mutated receptors. The results obtained revealed that residues in transmembrane segment 2 (W86A), 3 (I110A), 6 (W256A), and 7 (Y295A, Y295F but not much Y295W), were crucial for the high affinity of MEN16132. In conclusion, MEN16132 is a new, potent, and selective nonpeptide bradykinin B2 receptor antagonist.

Expression and characterization of biologically active human Fas ligand produced in CHO cells.

We describe an expression system for high-yield production of recombinant soluble human FasL (rsh- FasL) in CHO cells. After one round of selection for gene amplification, cell lines producing rsh-FasL up to 60 microg/L x 10(6) cells in 24 h were obtained. Cell lines were grown in protein-free medium as suspension cultures. The protein secreted into growth medium was purified by immunoaffinity. The rsh-FasL thus obtained was further fractionated by gel filtration and a form of approx 140 kDa was isolated and characterized. Mass spectral analysis yielded a main peak of 28,321.15 Da, while, although to a lesser extent, dimeric and trimeric forms were also detected according to the described oligomerized state of native FasL. Our procedure permits consistent production of biologically active rsh-FasL as shown in tests on FasL-sensitive cells and in in vitro binding assays.