Concentration-dependent Inhibition Research Articles

Biomimetic on-chip assay reveals the anti-metastatic potential of a novel thienopyrimidine compound in triple-negative breast cancer cell lines.

Introduction: This study presents a microfluidic tumor microenvironment (TME) model for evaluating the anti-metastatic efficacy of a novel thienopyrimidines analog with anti-cancer properties utilizing an existing commercial platform. The microfluidic device consists of a tissue compartment flanked by vascular channels, allowing for the co-culture of multiple cell types and providing a wide range of culturing conditions in one device. Methods: Human metastatic, drug-resistant triple-negative breast cancer (TNBC) cells (SUM159PTX) and primary human umbilical vein endothelial cells (HUVEC) were used to model the TME. A dynamic perfusion scheme was employed to facilitate EC physiological function and lumen formation. Results: The measured permeability of the EC barrier was comparable to observed microvessels permeability in vivo. The TNBC cells formed a 3D tumor, and co-culture with HUVEC negatively impacted EC barrier integrity. The microfluidic TME was then used to model the intravenous route of drug delivery. Paclitaxel (PTX) and a novel non-apoptotic agent TPH104c were introduced via the vascular channels and successfully reached the TNBC tumor, resulting in both time and concentration-dependent tumor growth inhibition. PTX treatment significantly reduced EC barrier integrity, highlighting the adverse effects of PTX on vascular ECs. TPH104c preserved EC barrier integrity and prevented TNBC intravasation. Discussion: In conclusion, this study demonstrates the potential of microfluidics for studying complex biological processes in a controlled environment and evaluating the efficacy and toxicity of chemotherapeutic agents in more physiologically relevant conditions. This model can be a valuable tool for screening potential anticancer drugs and developing personalized cancer treatment strategies.

Chronic exposure to bisphenol A induces behavioural, neurochemical, histological, and ultrastructural alterations in the ganglia tissue of the date mussels Lithophaga lithophaga.

Bisphenol A (BPA), a common plastic additive, has been demonstrated mechanistically to be a potential endocrine disruptor and to affect a variety of body functions in organisms. Although previous research has shown that BPA is toxic to aquatic organisms, the mechanism of neurotoxic effects in marine bivalves remains unknown. The current study aimed to elucidate the neurotoxic effects of BPA when administered at different concentrations (0.25, 1, 2, and 5 µg/L) for twenty-eight days in the ganglia of a bivalve model, the Mediterranean mussel (Lithophaga lithophaga), which is an ecologically and economically important human food source of bivalve species in the Mediterranean Sea. Our findings revealed an increase in behavioural disturbances and malondialdehyde levels in treated mussel ganglia compared to the control group. Furthermore, superoxide dismutase activity increased in the ganglia of L. lithophaga treated with 0.25 and 2 µg/L. However, at BPA concentrations of 1 and 5 µg/L, SOD activity was significantly reduced, as was total glutathione concentration. BPA causes neurotoxicity, as evidenced by concentration-dependent inhibition of acetylcholinesterase, dopamine, and serotonin. After chronic exposure to BPA, neurons showed distortion of the neuronal cell body and varying degrees of pyknosis. The ultrastructure changes in BPA-treated groups revealed the lightening of the nucleoplasm and a shrunken nuclear envelope. Overall, our findings suggest that BPA exposure altered antioxidation, neurochemical biomarkers, histopathological, and ultrastructural properties, resulting in behavioural changes. As a result, our findings provide a basis for further study into the toxicity of BPA in marine bivalves.

Novel Potent Autophagy Inhibitor Ka-003 Inhibits Dengue Virus Replication.

Every year, dengue virus (DENV) affects millions of people. Currently, there are no approved drugs for the treatment of DENV infection. Autophagy is a conserved degradation process that was shown to be induced by DENV infection and required for optimal DENV replication. The modulation of autophagy is, therefore, considered an attractive target to treat DENV infection. This study carried out a high-content image screen analysis using Crispr-Cas9 GFP-LC3 knocked-in HeLa cells of a compound library synthesized from or inspired by natural products and their biocongener precursors to discover novel autophagy inhibitors. The screen identified Ka-003 as the most effective compound for decreasing the number of autophagic vacuoles inside cells upon autophagy induction. Ka-003 could inhibit autophagy in a dose-dependent manner at low micromolar concentrations. More importantly, Ka-003 demonstrated the concentration-dependent inhibition of DENV production in Crispr-Cas9 GFP-LC3 knocked-in THP-1 monocytes. The core structure of Ka-003, which is a methyl cyclohexene derivative, resembles those found in mulberry plants, and could be synthetically prepared in a bioinspired fashion. Taken together, data indicate that Ka-003 hampered autophagy and limited DENV replication. The low cytotoxicity of Ka-003 suggests its therapeutic potential, which warrants further studies for the lead optimization of the compound for dengue treatment.

Phytochemical derivatives and secondary metabolites rich Rhizophora mucronata as an active anti-oxidant and anti-bacterial agent against multi drug resistant bacteria

In the present study, the phytochemicals and anti-oxidant ability rich marine mangrove plant Rhizophora mucronata (R. mucronata) was evaluated. The UV-bio spectrometer result of O.D value of phenolic content was shown 0.986 as well as flavonoid content were shown 0.994 at 100 µg/mL concentration. The high yields of phenol and flavonoid contents were observed compared with standard gallic acid and rutin. Concisely, the more phenols and flavonoids and some bioactive derivatives were clearly exhibited by LC-MS scanning report. In addition, the anti-oxidant activity result shown with 85 % and DPPH scavenging assay result shown 66% in crude extract of R. mucronata were observed and it was very higher than the rate of standard gallic acid and ascorbic acid. Further, the liquid–liquid extraction of available bioactive compounds were purified and shown excellent anti-bacterial activity against multi drug resistant bacteria. The anti-microbial activity result indicated that the 250 µg/mL of R. mucronata extract was shown 24 and 26 mm zone of inhibition against K. pneumoniae and A. baumannii. Then, the concentration dependent inhibition of R. mucronata extract was shown against both the tested pathogens and 250 µg/m concentrations was fixed as a minimum inhibition concentration range. Finally, the induction of outer cellular layer morphology effect and damaged size and shapes of the R. mucronata extract was shown against K. pneumoniae and A. baumannii. Altogether, the present study results were deliberately recommended that the R. mucronata extract as potential anti-oxidant and anti-bacterial agent. Hence, the natural marine mangrove plant R. mucronata is the safe, eco-friendly, low cost source for discovery of potential drug against various infections.

An in vitro evaluation of kratom (Mitragyna speciosa) on the catalytic activity of carboxylesterase 1 (CES1)

Kratom, (Mitragyna Speciosa Korth.) is a plant indigenous to Southeast Asia whose leaves are cultivated for a variety of medicinal purposes and mostly consumed as powders or tea in the United States. Kratom use has surged in popularity with the lay public and is currently being investigated for possible therapeutic benefits including as a treatment for opioid withdrawal due to the pharmacologic effects of its indole alkaloids. A wide array of psychoactive compounds are found in kratom, with mitragynine being the most abundant alkaloid. The drug-drug interaction (DDI) potential of mitragynine and related alkaloids have been evaluated for effects on the major cytochrome P450s (CYPs) via in vitro assays and limited clinical investigations. However, no thorough assessment of their potential to inhibit the major hepatic hydrolase, carboxylesterase 1 (CES1), exists. The purpose of this study was to evaluate the in vitro inhibitory potential of kratom extracts and its individual major alkaloids using an established CES1 assay and incubation system. Three separate kratom extracts and the major kratom alkaloids mitragynine, speciogynine, speciociliatine, paynantheine, and corynantheidine displayed a concentration-dependent reversible inhibition of CES1. The experimental Ki values were determined as follows for mitragynine, speciociliatine, paynantheine, and corynantheidine: 20.6, 8.6, 26.1, and 12.5 μM respectively. Speciociliatine, paynantheine, and corynantheidine were all determined to be mixed-type reversible inhibitors of CES1, while mitragynine was a purely competitive inhibitor. Based on available pharmacokinetic data, determined Ki values, and a physiologically based inhibition screen mimicking alkaloid exposures in humans, a DDI mediated via CES1 inhibition appears unlikely across a spectrum of doses (i.e., 2–20g per dose). However, further clinical studies need to be conducted to exclude the possibility of a DDI at higher and extreme doses of kratom and those who are chronic users.

Isolation and Characterization of Anti-Inflammatory Compounds from Ficus microcarpa L.f. Stem Bark.

The anti-inflammatory effect of the ethyl acetate extract of F. microcarpa bark (EAFMB) was investigated in acute and chronic (21 days) inflammation induced in Wistar albino rats. EAFMB (200 mg/kg b.w.) exhibited comparable anti-inflammatory effects to the reference drug, with a reduction of 59.48% at 4 h in acute inflammation and 83.96% on day 21 in chronic inflammation. Bioassay-guided fractionation using DPPH radical scavenging activity led to isolating and identifying three compounds from EAFMB: oleanolic acid, catechin, and p-hydroxycinnamic acid. All these compounds demonstrated the concentration-dependent inhibition of COX enzymes and the protection of egg albumin from heat-induced denaturation. Catechin exhibited the highest COX inhibition (COX-1 and COX-2 IC50 = 9.02 and 50.38 μM, respectively) and anti-denaturation effect (IC50 = 27.13 μg/mL) compared to oleanolic acid and p-hydroxycinnamic acid. These isolated compounds are likely responsible for the anti-inflammatory activities of F. microcarpa bark.

Evaluation of in vitro Antioxidant, Antimicrobial and Cytotoxic Activities of Crude Ethanol Extract and Fractions of Achillea sintenisii Hub. Mor.

Objective: The Achillea species have been used to treat various ailments due to its anti-inflammatory, hemostatic, spasmolytic and cholagogue effects in the Turkish traditional medicine. However, there is no biological activity studies on some Achillea species except for the well-knowns. This work aimed to determine the antioxidant, antimicrobial and cytotoxic activities of the crude ethanolic extracts and fractions of Achillea sintenisii using in vitro methods. 
 Methods: The antioxidant activity was investigated by DPPH (1,1´-diphenyl- 2-picrylhydrazyl), ABTS (2,2´-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) radical scavenging, total phenol and total flavonoid content, and iron chelating methods. Antimicrobial activity evaluated by micro-plate dilution method against five test organisms. Cytotoxicity was determined by MTT method using MCF-7 breast cancer cell line and PC-3 prostate cancer cell line. Apoptosis was also measured by AO/EB staining. 
 Results: The n-Hexane fractions showed the highest antimicrobial and cytotoxic activities, respectively. Administration of the extracts on the cancer cells showed a concentration dependent inhibition on cell proliferation. The anti-proliferation effect could be via apoptosis and associated with the cell death. 
 Conclusion: The results showed that the extracts demonstrated antioxidant, antimicrobial, and cytotoxic activity, also supports the claims of traditional usage.

Aβ dissociation by pectolinarin may counteract against Aβ-induced synaptic dysfunction and memory impairment

Alzheimer's disease (AD) is a degenerative brain disorder characterised by various neurological symptoms, including memory impairment and mood disorders, associated with the abnormal accumulation of amyloid b(Aβ) and tau proteins in the brain. There is still no definitive treatment available for AD, and the Aβ antibody drugs, which are expected to be approved by the FDA, have many limitations. Therefore, there is an urgent need to develop low-molecular-weight therapeutic agents for the management of AD. In this study, we investigated whether pectolinarin, a flavonoid, regulates Aβ aggregation and Aβ-induced toxicity. Pectolinarin demonstrated concentration-dependent inhibition of Aβ aggregation and had the ability to break down pre-formed Aβ aggregates, thereby reducing their neurotoxicity. Furthermore, pectolinarin suppressed Aβ aggregates-induced reduction in long-term potentiation (LTP) in the hippocampus. Oral administration of pectolinarin in experimental animals inhibited memory impairment and LTP deficits induced by Aβ injection in the hippocampus. These results indicate that pectolinarin may reduce toxic Aβ species and Aβ-induced memory impairments and synaptic dysfunction.

Abstract P318: INDOLE-3 ACETIC ACID: A NOVEL DUAL-ACTION INHIBITOR OF THE ENDOTHELIN SYSTEM FOR TREATING HYPERTENSION

Elevated levels of endothelin-1 (ET-1) stimulate potent vasoconstriction, free radical production, oxidative stress, and vascular inflammation, which are often present in salt-sensitive hypertension, pulmonary hypertension, drug-resistant hypertension, and hypertension among African Americans. There is currently no known drug that simultaneously targets both ET-1 receptors (ETRs) and the ET-1 synthesizing enzyme ECE-1, even though dual targeting of the ET system could lead to a new generation of drug molecules with improved therapeutic outcome. Using an integrated in vitro, in vivo and in silico approaches, here we report the discovery of indole-3 acetic acid (IAA) as an inhibitor of both ET-1 receptors and ECE-1. IAA is produced in the human body via tryptophan metabolism by gut microbiota. Our data demonstrates that IAA concentration-dependently inhibits ET-1-evoked vasoconstriction in rat resistance mesenteric arteries and ET-1-stimulated reactive oxygen species (ROS) production in smooth muscle cells (SMCs). Furthermore, IAA suppresses ET-1-induced endothelial cell (EC) activation, adhesion molecule overexpression, and leukocyte adhesion. IAA inhibits ET-1-stimulated, ET A R- and ET B R-mediated increases in [Ca 2+ ] i . Intriguingly, IAA produces a concentration-dependent inhibition of purified human ECE-1. Treatment of Dahl salt-sensitive (DSS) hypertensive rats, a model of augmented ET-1 signaling, with an oral extended-release formulation of IAA significantly inhibits blood pressure elevation in high-salt diet-fed (HSD) DSS rats. IAA-treated DSS arteries show intact acetylcholine- and sodium nitroprusside-induced vasodilation, suggesting that IAA preserves endothelial and smooth muscle function, respectively. IAA treatment also prevents arterial remodeling, oxidative stress, and kidney dysfunction in HSD DSS rats. Overall, our study identifies IAA as the first-in-class dual-action inhibitor of ETRs and ECE-1, which produces profound antihypertensive effect in vivo. IAA could also be therapeutically useful for other cardiovascular diseases in which ET-1 signaling is implicated.

Spermidine-capped carbon dots as potent antimicrobial nanomaterials against Escherichia coli

The food industry must prevent food contamination caused by pathogenic Escherichia coli, as they pose a severe public health threat globally, hence the need to explore new antimicrobial materials. In this paper, spermidine capped carbon dots (S-PCDs) were synthesized by hydrothermal reaction, and the antibacterial effects against E. coli and multi-drug resistant E. coli (MREC) was determined, so as to explore their potential application as antibacterial nanomaterials. The minimum inhibitory concentration values of S-PCDs against E. coli and MREC were 64 μg/mL and 128 μg/mL, respectively, showing strong antibacterial activity. The antibacterial zone was increased significantly with the increasing content of S-PCDs (p < 0.05). The results of bacteriostatic curve and bactericidal curve of S-PCDs on E. coli and MREC also showed that S-PCDs had a concentration-dependent inhibition on bacteria. In addition, the intrinsic antibacterial mechanism indicated that the antibacterial action of S-PCDs may involve cell membrane damage and reactive oxygen species production. During 30 days of exposure, S-PCDs showed persistent antibacterial effect against both E. coli and MREC without detectable resistance, especially without triggering secondary resistance in MREC. S-PCDs has potential application value in foodborne diseases caused by complex bacteria and even drug-resistant bacteria.

Investigation of In-vitro Antiproliferative activity of Ampelocissus latifolia root extract on MCF-7 breast cancer cells Ampelocissus latifolia root extract against MCF-7 breast cancer cells.

Background: Breast cancer is one of the leading causes of cancer related death in women worldwide. Treatment of cancer has been plagued with toxic side effects of anticancer drugs. The need of the hour is the development of novel compounds with maximum cytotoxic effect on cancer cells with minimal toxicity to normal cells. The current direction of researchers worldwide is to identify anticancer compounds from natural sources. In India, traditional medicine has employed use of herbaceous climbers of grape family for treatment of ailments ranging from snakebites to diabetes. In the current study, an attempt has been made to explore the in vivo antiproliferative property of ethanolic extract of root of Ampelocissus latifolia against MCF-7 breast cancer cell line. Materials and Methods: The antiproliferative effect of Ethanolic extract of root of Ampelocissus latifolia (ERAL) was estimated by 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl--tetrazolium bromide (MTT) assay. The MCF -7 cell viability in various concentrations of ERAL including 7.8mcg/ml, 15.6mcg/ml, 31.2mcg/ml, 62.5mcg/ml, 125mcg/ml, 250mcg/ml, 500mcg/ml and 1000mcg/ml was tested. The IC50 value was calculated. All the experiments were done in triplicates. This was followed up with DNA fragmentation assay and fluorescent staining and microscopy. Results: The MCF -7 cell viability in various concentration of ERAL including 7.8mcg/ml, 15.6mcg/ml, 31.2mcg/ml ,62.5mcg/ml, 125mcg/ml, 250mcg/ml, 500mcg/ml and 1000mcg/ml was found to be 69.01%, 62.39%, 55.04%, 48.00%, 41.17%, 33.82%, 26.78% and 20.06% respectively .The IC50 concentration was found to be in the range of 62.5mcg/ml.The cell viability was found to be dose dependant.DNA fragmentation assay and DAPI and PI staining of cells treated with IC50 concentration of ERAL were indicative of significant cell death. Conclusion: The concentration dependent inhibition of MCF-7 cells supported by DNA fragmentation and fluorescent staining indicate that Ampelocissus latifolia can be a source of novel anticancer molecules.

Cell-free production of fluorescent proteins for the discovery of novel ribosome-targeting antibiotics

Various issues including the overuse of antibiotics has led to the development of threatening multidrug-resistant bacterial strains urging development of novel anti-infectives. One quarter of current clinical phase III antibiotic drug candidates address ribosomal protein translation as a target. Here, we describe an effective cell-free in vitro screening system for inhibitors of bacterial ribosome activity with direct fluorescence read-out. Using ribosomal S30 extracts from Escherichia coli, Salmonella enterica, and Pseudomonas putida, the validity of this system is demonstrated by concentration-dependent inhibition of translation by a set of different classes of translation-targeting drugs. The single-compartment cell-free translation reaction is compatible with multi-well formats. Fluorophore formation of green fluorescent protein or monomeric NeonGreen occurs in an hour time frame without the need of adding reagents for secondary enzymatic detection saving handling time, and prohibiting false positives. As label-free readout, the dose response further allows for IC50 determination in the same setup. Together, we show that cell-free production of fluorescent proteins for the discovery of ribosome-targeting antibiotics is feasible and amenable to high-throughput applications.

Targeting FAK/PYK2 with SJP1602 for Anti-Tumor Activity in Triple-Negative Breast Cancer.

Triple-negative breast cancer (TNBC) presents significant challenges due to its aggressive nature and limited treatment options. Focal adhesion kinase (FAK) has emerged as a critical factor promoting tumor growth and metastasis in TNBC. Despite encouraging results from preclinical and early clinical trials with various FAK inhibitors, none have yet achieved clinical success in TNBC treatment. This study investigates the therapeutic potential of a novel dual inhibitor of FAK and PYK2, named SJP1602, for TNBC. In vitro experiments demonstrate that SJP1602 effectively inhibits FAK and PYK2 activities, showing potent effects on both kinases. SJP1602 shows concentration-dependent inhibition of cell growth, migration, invasion, and 3D spheroid formation in TNBC cell lines, surpassing the efficacy of other FAK inhibitors. Pharmacokinetic studies in rats indicate favorable bioavailability and sustained plasma concentrations of SJP1602, supporting its potential as a therapeutic agent. Furthermore, in TNBC xenograft models, SJP1602 exhibits significant dose-dependent inhibition of tumor growth. These promising results emphasize the potential of SJP1602 as a potent dual inhibitor of FAK and PYK2, deserving further investigation in clinical trials for TNBC treatment.

Immunomodulatory and Antioxidant Properties of Ipomoea batatas Flour and Extracts Obtained by Green Extraction.

Sweet potato (SP), Ipomoea batatas Lam, belongs to the Convolvulaceae family. It produces edible storage roots. Currently, orange varieties contribute to improving food systems and managing vitamin A deficiency. Processing of this food crop into flour allows better conservation. However, nutrition health data regarding SP flour obtained by green extraction remains scarce. In this study, we therefore explored its phytochemistry and its associated bioactivity potential for human health. We analyzed the nutritional composition of orange flesh sweet potato (OFSP) flour and assessed the antioxidant (free radical scavenging) and immunomodulatory (on inflammatory murine macrophages) properties of the extract. More specifically, we measured the impact of OFSP flour extract on mediators such as Nitric Oxide (NO) and the production of pro-inflammatory cytokines such as Interleukin-6 (IL-6), Tumor Necrosis Factor alpha (TNF-alpha), Monocyte Chemoattractant Protein-1 (MCP-1), and Prostaglandin-E2 (PGE-2). Our results indicated significant fiber, mineral, beta-carotene, and polyphenols content in the extracts, and antioxidant and immunomodulatory bioactivities were also demonstrated with a concentration-dependent inhibition of cytokine production. Taken together, our results suggest that Ipomoea batatas flour could, in addition to being a good source of energy and beta-carotene provitamin A, constitute a food of interest for the prophylaxis of metabolic diseases associated with an underlying low-grade inflammatory state.

Comparative study of the antibacterial and anti-inflammatory activities of the seed coat vs. seed kernel extracts from the plant Mangifera indica L. in inflammatory acne treatment

Introduction: The present study was developed to investigate and compare the extraction yield, the contents of polyphenols and mangiferin, the activities against Propionibacterium acnes, Staphylococcus aureus, and Escherichia coli bacteria, and the anti-inflammatory activities of ethanol extracts of mango seed kernels vs. seed coats. Methods: Mango seed kernels and seed coats were extracted using ethanol as the solvent and tested against microorganisms using the disc diffusion method. The minimum inhibitory concentration (MIC) levels of extracts were determined by the agar dilution method. The anti-inflammatory activities were assessed both in vitro and in vivo by albumin denaturation method and carrageenan-induced paw edema test, respectively. Results: Both extracts yielded high contents of mangiferin and phenolic compounds. The antibacterial activities of both extracts showed inhibition of the tested microorganisms Propionibacterium acnes and Staphylococcus aureus but not Escherichia coli. Seed kernel extract (0.2 g/kg) reduced paw edema by 44.8% at 3 hours after λ-carrageenan administration. Meanwhile, 0.5 g/kg seed coat extract reduced paw edema less than the seed kernel extract (23.1% vs. 44.8%). Mango seed kernel extract, mango seed coat extract, and diclofenac sodium displayed concentration-dependent inhibition of heat-induced protein denaturation with IC50 values of 137.23 μg/mL, 292.12 μg/mL, and 6.64 μg/mL, respectively. Conclusion: The obtained results confirmed the antibacterial and anti-inflammatory potential of mango seed kernel and seed coat extracts. The mango seed kernel extract was proven to be more effective than the mango seed coat extract and thus can be used in cosmetics as an anti-inflammatory and antibacterial agent.

Blocking the major inflammatory pathways by newly synthesized thiadiazine derivatives via in-vivo, in-vitro and in-silico mechanism

A series of new thiadiazine derivatives including 2-(5-alkyl/aryl-6-thioxo-1,3,5-thiadiazinan-3-yl) propanoic acids (a) and 4-methyl-2-(5-alkyl/aryl-6-thioxo-1,3,5-thiadiazinan-3-yl) pentanoic acids (b) were synthesized by reacting primary alkyl/aryl amines with CS2, followed by reaction with formaldehyde and amino acids. The chemical structures of synthesized compounds were confirmed by 13C- NMR and 1H- NMR techniques. The inhibitory potential of major inflammatory enzymes, COX-2 and 5-LOX was examined. Moreover, anti-nociceptive and anti-inflammatory activities were evaluated in the in vivo thermally induced nociceptive, and carrageenan induced paw edema models in mice. The in-vitro results reflect that these compounds exhibited concentration dependent inhibition of COX-2 and 5-LOX. The tested compounds at 50 mg/kg showed significant effect on thermally induced pain, and reduced latency time (seconds) as compared to the vehicle treated animals. Moreover, tested compounds exhibited percent inhibition of paw edema in the carrageenan induced paw edema model in mice. Furthermore, the binding modes of the most active COX-2 and 5-LOX inhibitors were determined through computational methods. The computational study reflects that the docked compounds have high binding affinities for COX-2 and 5-LOX enzymes, which leads to inhibition of these enzymes.

BI 1291583: a novel selective inhibitor of cathepsin C with superior in vivo profile for the treatment of bronchiectasis

BackgroundAirway inflammation in chronic inflammatory lung diseases (e.g. bronchiectasis) is partly mediated by neutrophil-derived serine protease (NSP)/antiprotease imbalance. NSPs are activated during neutrophil myelopoiesis in bone marrow by cathepsin C (CatC; DPP1). CatC is therefore an attractive target to reduce NSP activity in the lungs of patients with bronchiectasis, restoring the protease/antiprotease balance. We report results from the preclinical pharmacological assessment of the novel CatC inhibitor BI 1291583.MethodsBinding kinetics of BI 1291583 to human CatC were determined by surface plasmon resonance. In vitro inhibition of human CatC activity was determined by CatC-specific fluorescent assay, and selectivity was assessed against related cathepsins and unrelated proteases. Inhibition of NSP neutrophil elastase (NE) production was assessed in a human neutrophil progenitor cell line. In vivo inhibition of NE and NSP proteinase 3 (PR3) in bronchoalveolar lavage fluid (BALF) neutrophils after lipopolysaccharide (LPS) challenge and distribution of BI 1291583 was determined in a mouse model.ResultsBI 1291583 bound human CatC in a covalent, reversible manner, selectively and fully inhibiting CatC enzymatic activity. This inhibition translated to concentration-dependent inhibition of NE activation in U937 cells and dose-dependent, almost-complete inhibition of NE and PR3 activity in BALF neutrophils in an in vivo LPS-challenge model in mice. BI 1291583 exhibited up to 100 times the exposure in the target tissue bone marrow compared with plasma.ConclusionBI 1291583-mediated inhibition of CatC is expected to restore the protease–antiprotease balance in the lungs of patients with chronic airway inflammatory diseases such as bronchiectasis.

Effects of orally administered pimobendan on platelet function in healthy adult cats.

Several phosphodiesterase inhibitors have demonstrable antiplatelet actions when administered to human patients. Concentration-dependent inhibition of feline platelet aggregation by pimobendan has been previously demonstrated in vitro. However, there are no published reports characterizing the effect of oral pimobendan, administered at therapeutic doses, on platelet function in cats. This study aimed to evaluate the effect of orally administered pimobendan on platelet function in healthy adult cats. 6 healthy purpose-bred adult cats. Cats were administered pimobendan orally at a dosage of 0.625 mg/cat (low-dose) twice daily for 1 week, followed by 1.25 mg/cat (high-dose) twice daily for 1 week. Venous blood sampling for platelet testing and plasma drug concentration occurred at baseline, 1 hour postdose on the eighth day of treatment with low-dose pimobendan, 1 hour postdose on the eighth day of treatment with high-dose pimobendan, and after a 1-week washout period. Platelet function was assessed by whole blood aggregometry and by use of a platelet function analyzer (PFA-100®). Friedman tests were used to compare platelet function parameters among the 4 sampling timepoints. After 1 week of treatment, median (range) plasma pimobendan concentrations were 15.1 ng/mL (6.89-20.2 ng/mL) and 32.8 ng/mL (23.3-44.8 ng/mL) in cats receiving low-dose and high-dose pimobendan, respectively. No significant differences in PFA closure time or any aggregometry variable were found among the treatment conditions. Pimobendan was not associated with measurable inhibition of platelet function when administered orally to healthy adult cats at 2 clinically relevant dosages.

Fractionation and Antibacterial Evaluation of the Surface Compounds from the Leaves of Combretum zeyheri on Selected Pathogenic Bacteria.

Combretum zeyheri is traditionally used for the treatment of many infections, including bacterial infections. The aim of this study was to fractionate and evaluate antibacterial activity of the crude extract of C. zeyheri, as well as the surface compounds from the leaves of C. zeyheri, in two pathogenic bacteria, Staphylococcus aureus and Pseudomonas aeruginosa. The antibacterial activities of fractions obtained from chromatographic separations were determined using broth microdilution assay on the laboratory and clinical strains of S. aureus and P. aeruginosa. The fractionation of the compounds on the leaf surface yielded 262 fractions. The fractionated compounds with similar TLC profiles were pooled together to yield 47 pools. The extract and pooled fractions CZSC151154, CZSC155160, and CZSC209213 showed significant antibacterial activity with MIC values ranging from 12.5 μg/ml to 100 μg/ml. The clinical strain of S. aureus had MIC greater than 100 μg/ml for CZSC151154 and CZSC155160. The minimum bactericidal concentration values for these fractions were also in the range of 12.5 μg/ml to 100 μg/ml. The extract and fractions CZSC151154, CZSC155160, and CZSC209213 showed a concentration-dependent inhibition of growth in S. aureus. Analyses of the CZSC209213 pool by LC-MS showed the presence of nine compounds which are (3R,7R)-1,3,7-octanetriol, (-)-tortuosamine, 11-aminoundecanoic acid, 1-piperidinecarboxaldehyde, 3-hydroxy-4-isopropylbenzyl alcohol 3-glucoside, hydroxy-isocaproic acid, oleamide, palmitic amide, phytospingosine, and sphinganine. In conclusion, C. zeyheri leaf surface compounds exhibited antibacterial activity. The crude extract and the pooled fractions showed concentration-dependent inhibition of growth on S. aureus. Results from this study indicate the potential of C. zeyheri as a source of lead compounds that may be further developed into antibacterial drugs.

Comparative evaluation of the potential anti-spasmodic activity of Piper longum, Piper nigrum, Terminalia bellerica, Terminalia chebula, and Zingiber officinale in experimental animals

BackgroundSpasm of muscle is one of the frequent complaints seen by most of the population worldwide. The present study evaluated the efficacy of some of the commonly used herbal extracts against known spasmogens, such as histamine and 5-hydroxytryptamine (5-HT). Material and methodsThe study was conducted on isolated guinea pig ileum and rat uterus preparations using histamine and 5-HT, respectively. Five herbal extracts such as Piper longum (P.L), Piper nigrum (P.N), Terminalia bellerica (T.B), Terminalia chebula (T.C), and Zingiber officinale (Z.O) were tested. Herbal extracts at doses 50, 150, 500, 1500, and 5000 mcg/ml were pretreated to the isolated tissue preparation, and the contractile response of histamine and 5-HT was recorded. The efficacy and the inhibitory concentration (IC50) were calculated and statistically analyzed by one-way ANOVA. ResultsThe study indicated that all five herbal extracts produced a concentration-dependent suppression of histamine and 5-HT-induced responses. A significant (p < 0.05) non-competitive antagonism was observed against the known spasmogen induced smooth muscle contraction for P.L, P.N, T.B, and Z.O in both guinea pigs and rat uterus preparation. Moreover, P.L and P.N completely abolished (100%) the contractile response induced by histamine and 5-HT. Although, T.C produced a concentration-dependent reduction in known spasmogen-induced contraction but the response was found to be statistically non-significant (p greater than 0.05). ConclusionThe finding suggested that P.L. and P.N. have better activity in terms of reducing the spasmogenic contractions compared to other extracts. Additionally, T.B. and Z.O. can lessen the uterine and intestinal contractions brought on by spasmogens. Although P.L and P.N demonstrated better efficacy against the spasmogenic activity of histamine and 5-HT, more research, particularly on isolated phytochemicals of the extracts and involving different experimental models, is required before establishing the precise safety and efficacy against spasmogenic-induced disorders.