Quantification of abemaciclib and metabolites: evolution of bioanalytical methods supporting a novel oncolytic agent

Aim: Bioanalytical methods undergo many revisions and modifications throughout drug development to meet the objectives of the study and development program. Results: Validated LC–MS/MS methodology used to quantify abemaciclib and four metabolites in human plasma is described. The method, initially validated to support the first-in-human study, was successfully modified to include additional metabolites as in vitro and in vivo information about the activity and abundance of human metabolites became available. Consistent performance of the method over time was demonstrated by an incurred sample reanalysis passing rate exceeding 95%, across clinical studies. An overview of the numerous methods involved during the development of abemaciclib, including the quantification of drugs evaluated as combination regimens and used as substrates during drug–drug interaction studies, is presented. Conclusion: Robust bioanalytical methods need to be designed with the flexibility required to support the evolving study objectives associated with registration and post-registration trials.

The Development of Homologous (Canine/Anti-Canine) Antibodies in Dogs with Haemophilia A (Factor VIII Deficiency): A Ten-Year Longitudinal Study

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651541 ◽

1993 ◽

Vol 69 (01) ◽

pp. 021-024 ◽

Cited By ~ 20

Author(s):

Shawn Tinlin ◽

Sandra Webster ◽

Alan R Giles

Keyword(s):

Factor Viii ◽

Canine Model ◽

Significant Inhibition ◽

Human Condition ◽

Haemophilia A ◽

Variable Expression ◽

The Family ◽

SummaryThe development of inhibitors to factor VIII in patients with haemophilia A remains as a serious complication of replacement therapy. An apparently analogous condition has been described in a canine model of haemophilia A (Giles et al., Blood 1984; 63:451). These animals and their relatives have now been followed for 10 years. The observation that the propensity for inhibitor development was not related to the ancestral factor VIII gene has been confirmed by the demonstration of vertical transmission through three generations of the segment of the family related to a normal (non-carrier) female that was introduced for breeding purposes. Haemophilic animals unrelated to this animal have not developed functionally significant factor VIII inhibitors despite intensive factor VIII replacement. Two animals have shown occasional laboratory evidence of factor VIII inhibition but this has not been translated into clinical significant inhibition in vivo as assessed by clinical response and F.VIII recovery and survival characteristics. Substantial heterogeneity of inhibitor expression both in vitro and in vivo has been observed between animals and in individual animals over time. Spontaneous loss of inhibitors has been observed without any therapies designed to induce tolerance, etc., being instituted. There is also phenotypic evidence of polyclonality of the immune response with variable expression over time in a given animal. These observations may have relevance to the human condition both in determining the pathogenetic factors involved in this condition and in highlighting the heterogeneity of its expression which suggests the need for caution in the interpretation of the outcome of interventions designed to modulate inhibitor activity.

In Vitro Activities of Voriconazole in Combination with Three Other Antifungal Agents against Candida glabrata

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.48.9.3317-3322.2004 ◽

2004 ◽

Vol 48 (9) ◽

pp. 3317-3322 ◽

Cited By ~ 21

Author(s):

Francesco Barchiesi ◽

Elisabetta Spreghini ◽

Monia Maracci ◽

Annette W. Fothergill ◽

Isabella Baldassarri ◽

...

Keyword(s):

Candida Glabrata ◽

Antifungal Agents ◽

Beneficial Effects ◽

Synergistic Interactions ◽

Microdilution Method ◽

Broth Microdilution Method ◽

Disk Diffusion Assay ◽

ABSTRACT Candida glabrata has recently emerged as a significant pathogen involved in both superficial and deep-seated infections. In the present study, a checkerboard broth microdilution method was performed to investigate the in vitro activities of voriconazole (VOR) in combination with terbinafine (TRB), amphotericin B (AMB), and flucytosine (5FC) against 20 clinical isolates of C. glabrata. Synergy, defined as a fractional inhibitory concentration (FIC) index of ≤0.50, was observed in 75% of VOR-TRB, 10% of VOR-AMB, and 5% of VOR-5FC interactions. None of these combinations yielded antagonistic interactions (FIC index > 4). When synergy was not achieved, there was still a decrease in the MIC of one or both drugs used in the combination. In particular, the MICs were reduced to ≤1.0 μg/ml as a result of the combination for all isolates for which the AMB MIC at the baseline was ≥2.0 μg/ml. By a disk diffusion assay, the halo diameters produced by antifungal agents in combination were greater that those produced by each drug alone. Finally, killing curves showed that VOR-AMB exhibited synergistic interactions, while VOR-5FC sustained fungicidal activities against C. glabrata. These studies demonstrate that the in vitro activity of VOR against this important yeast pathogen can be enhanced upon combination with other drugs that have different modes of action or that target a different step in the ergosterol pathway. Further studies are warranted to elucidate the potential beneficial effects of such combination regimens in vivo.

Abstract 5402: Two Photon Microscopy Measurements Of Sub-epicardial Sarcomere Length In Perfused Rat Hearts

Circulation ◽

10.1161/circ.118.suppl_18.s_543-c ◽

2008 ◽

Vol 118 (suppl_18) ◽

Author(s):

Patrizia Camelliti ◽

Gil Bub ◽

Daniel J Stuckey ◽

Christian Bollensdorff ◽

Damian J Tyler ◽

...

Keyword(s):

Sarcomere Length ◽

Left Ventricular ◽

Model Systems ◽

Future Research ◽

Fundamental Parameter ◽

Rat Hearts ◽

Perfused Hearts ◽

Sarcomere length (SL) is a fundamental parameter underlying the Frank Starling relation in the heart, as it offers an absolute representation of myocardial stretch. Previous studies addressed the Frank Starling relation by measuring SL in isolated myocytes or muscle strips. Here, we report first data obtained using a novel technique to measure sub-epicardial SL in perfused hearts. Rat hearts were Langendorff perfused (normal Tyrode solution) at a constant pressure of 90mmHg, labeled with the fluorescent membrane marker di-4-ANEPPS, and then arrested with high-K + Tyrode for either 2-photon microscopy (n=4) or MRI (n=4). Image analysis software was developed to extract SL at the cell level from >1,400 2-photon images (Fig 1 ) and correct for cell angle. SL increased by 10±2 % between 30 and 80 min of perfusion (1.98±0.04 to 2.17±0.03 μm; p<0.05; Fig 1 ). Measurements of left ventricular myocardial volume (LVMV) were made in vivo and in perfused hearts using 3D MRI. LVMV increased by 24±7% from in vivo to 30 min of perfusion, and by 11±3 % between 30 and 90 min (539±35; 664±44; 737±49 mm 3 , respectively; p<0.05; Fig 1 ). We show that SL can be measured in isolated perfused hearts. The method allowed monitoring of changes in SL over time, and showed that SL and LVMV increase to a similar extent during 30–80 min perfusion with crystalloid solution, probably due to tissue oedema. This result, together with the increase in LVMV during the first 30 min, highlights the pronounced differences between in vivo , in situ , and in vitro model systems for studies of cardiac physiology and mechanics. Future research will compare changes in SL in healthy hearts and disease models involving contractile dysfunction. Figure 1: Left: 2-photon microscopy image of di-4-ANEPPS labeled myocardium. Right: SL and LVMV changes over time.

Focal adhesion kinase inhibitor TAE226 combined with Sorafenib slows down hepatocellular carcinoma by multiple epigenetics effects

10.21203/rs.3.rs-892488/v1 ◽

2021 ◽

Author(s):

Ilaria Romito ◽

Manuela Porru ◽

Maria Rita Braghini ◽

Luca Pompili ◽

Nadia Panera ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Focal Adhesion Kinase ◽

Focal Adhesion ◽

Kinase Inhibitor ◽

Malignant Tumours ◽

Nurd Complex ◽

Antitumor Effects ◽

Abstract Background Hepatocellular carcinoma (HCC) is one of the most common and lethal malignant tumours worldwide. Sorafenib (SOR) is one of the most effective single-drug systemic therapy against advanced HCC, but the identification of novel combination regimens for a continued improvement in overall survival is a big challenge. Recent studies highlighted the crucial role of focal adhesion kinase (FAK) in HCC growth. The aim of this study was to investigate the antitumor effects of three different FAK inhibitors, alone or in combination with SOR, using in vitro and in vivo models of HCC. Methods The effect of PND1186, PF431396, TAE226 on cell viability was compared to SOR. Among them TAE226, emerging as the most effective FAKi, was then tested alone or in combination with SOR using 2D/3D human HCC cell line cultures and HCC xenograft murine models. The mechanisms of action were assessed by gene/protein expression and imaging approaches, combined with high-throughput methods. Results TAE226 emerged as the more effective FAKi to be combined with SOR against HCC. Combined TAE226 and SOR treatment reduced HCC growth both in vitro and in vivo by affecting tumour-promoting gene expression and inducing epigenetic changes via dysregulation of the nuclear interactome of FAK. We characterized a novel nuclear functional interaction between FAK and the NuRD complex. TAE226-mediated FAK depletion and SOR-promoted MAPK down-modulation causing an increase of histone H3 lysine 27 acetylation, counteracting its trimethylation by decreasing the nuclear amount of HDAC1/2. Conclusions Altogether, our findings provide the first evidence that TAE226 combined with SOR efficiently reduce HCC growth in vitro and in vivo. Our data also highlight that deep analysis of FAK nuclear interactome may lead to the identification of new promising therapeutic approaches for HCC.

Recovery of Aging-Related Size Increase of Skin Epithelial Cells: In vivo Mouse and In vitro Human Study

PLoS ONE ◽

10.1371/journal.pone.0122774 ◽

2015 ◽

Vol 10 (3) ◽

pp. e0122774 ◽

Cited By ~ 4

Author(s):

Igor Sokolov ◽

Natali V. Guz ◽

Swaminathan Iyer ◽

Amy Hewitt ◽

Nina A. Sokolov ◽

...

Keyword(s):

Epithelial Cells ◽

Human Study ◽

Size Increase

Time-Dependent Reduction of Calcium Oscillations in Adipose-Derived Stem Cells Differentiating towards Adipogenic and Osteogenic Lineage

Biomolecules ◽

10.3390/biom11101400 ◽

2021 ◽

Vol 11 (10) ◽

pp. 1400

Author(s):

Enrico C. Torre ◽

Mesude Bicer ◽

Graeme S. Cottrell ◽

Darius Widera ◽

Francesco Tamagnini

Keyword(s):

Stem Cells ◽

Osteogenic Differentiation ◽

Adipogenic Differentiation ◽

Specific Treatment ◽

Stem Cell Differentiation ◽

Calcium Oscillations ◽

Multipotent Stem Cells ◽

Adipose-derived mesenchymal stromal cells (ASCs) are multipotent stem cells which can differentiate into various cell types, including osteocytes and adipocytes. Due to their ease of harvesting, multipotency, and low tumorigenicity, they are a prime candidate for the development of novel interventional approaches in regenerative medicine. ASCs exhibit slow, spontaneous Ca2+ oscillations and the manipulation of Ca2+ signalling via electrical stimulation was proposed as a potential route for promoting their differentiation in vivo. However, the effects of differentiation-inducing treatments on spontaneous Ca2+ oscillations in ASCs are not yet fully characterised. In this study, we used 2-photon live Ca2+ imaging to assess the fraction of cells showing spontaneous oscillations and the frequency of the oscillation (measured as interpeak interval—IPI) in ASCs undergoing osteogenic or adipogenic differentiation, using undifferentiated ASCs as controls. The measurements were carried out at 7, 14, and 21 days in vitro (DIV) to assess the effect of time in culture on Ca2+ dynamics. We observed that both time and differentiation treatment are important factors associated with a reduced fraction of cells showing Ca2+ oscillations, paralleled by increased IPI times, in comparison with untreated ASCs. Both adipogenic and osteogenic differentiation resulted in a reduction in Ca2+ dynamics, such as the fraction of cells showing intracellular Ca2+ oscillations and their frequency. Adipogenic differentiation was associated with a more pronounced reduction of Ca2+ dynamics compared to cells differentiating towards the osteogenic fate. Changes in Ca2+ associated oscillations with a specific treatment had already occurred at 7 DIV. Finally, we observed a reduction in Ca2+ dynamics over time in untreated ASCs. These data suggest that adipogenic and osteogenic differentiation cell fates are associated with specific changes in spontaneous Ca2+ dynamics over time. While this observation is interesting and provides useful information to understand the functional correlates of stem cell differentiation, further studies are required to clarify the molecular and mechanistic correlates of these changes. This will allow us to better understand the causal relationship between Ca2+ dynamics and differentiation, potentially leading to the development of novel, more effective interventions for both bone regeneration and control of adipose growth.

Rifabutin Is Bactericidal against Intracellular and Extracellular Forms of Mycobacterium abscessus

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00363-20 ◽

2020 ◽

Vol 64 (11) ◽

Cited By ~ 2

Author(s):

Matt D. Johansen ◽

Wassim Daher ◽

Françoise Roquet-Banères ◽

Clément Raynaud ◽

Matthéo Alcaraz ◽

...

Keyword(s):

Opportunistic Pathogen ◽

Mycobacterium Abscessus ◽

Zebrafish Model ◽

Content Type ◽

Attractive Option ◽

Conventional Antibiotics ◽

Susceptibility Profiles ◽

ABSTRACT Mycobacterium abscessus is increasingly recognized as an emerging opportunistic pathogen causing severe lung diseases. As it is intrinsically resistant to most conventional antibiotics, there is an unmet medical need for effective treatments. Repurposing of clinically validated pharmaceuticals represents an attractive option for the development of chemotherapeutic alternatives against M. abscessus infections. In this context, rifabutin (RFB) has been shown to be active against M. abscessus and has raised renewed interest in using rifamycins for the treatment of M. abscessus pulmonary diseases. Here, we compared the in vitro and in vivo activity of RFB against the smooth and rough variants of M. abscessus, differing in their susceptibility profiles to several drugs and physiopathologial characteristics. While the activity of RFB is greater against rough strains than in smooth strains in vitro, suggesting a role of the glycopeptidolipid layer in susceptibility to RFB, both variants were equally susceptible to RFB inside human macrophages. RFB treatment also led to a reduction in the number and size of intracellular and extracellular mycobacterial cords. Furthermore, RFB was highly effective in a zebrafish model of infection and protected the infected larvae from M. abscessus-induced killing. This was corroborated by a significant reduction in the overall bacterial burden, as well as decreased numbers of abscesses and cords, two major pathophysiological traits in infected zebrafish. This study indicates that RFB is active against M. abscessus both in vitro and in vivo, further supporting its potential usefulness as part of combination regimens targeting this difficult-to-treat mycobacterium.

Potentials and Safety of Date Palm Fruit against Diabetes: A Critical Review

Foods ◽

10.3390/foods9111557 ◽

2020 ◽

Vol 9 (11) ◽

pp. 1557

Author(s):

Md Al-Tareq Mia ◽

Md Golam Mosaib ◽

Md Ibrahim Khalil ◽

Md Asiful Islam ◽

Siew Hua Gan

Keyword(s):

Date Palm ◽

Antioxidant Activities ◽

Human Study ◽

Insulin Receptors ◽

Β Cells ◽

Palm Fruit ◽

Good Potential ◽

Palm Fruits

Diabetes is a chronic metabolic disorder triggered by disturbances in carbohydrate, protein, and lipid metabolisms, where either reduced secretion or sensitivity of insulin is observed coupled with poor glucose control. Date palm fruits are one of the fruits reported to have good potential in diabetes treatment due to its presence of polyphenols exerting strong antioxidant activities. Other possible mechanisms of action include the polyphenolic compounds, which can inhibit enzymes like α-amylase and α-glucosidase. Flavonoids in dates can stimulate β-cells by increasing the number of islets and β-cells, recovering endocrine pancreatic tissues, reducing β-cell apoptosis, activating insulin receptors following the increase in insulin secretion, in addition to improving diabetes-induced complications. In this review, the in vitro, in vivo, and human study-based evidence of date palm as an anti-diabetic fruit is summarised.

Intraperitoneal pressure: Stability over time and validation of Durand’s measurement method

Peritoneal Dialysis International ◽

10.1177/0896860820973120 ◽

2020 ◽

pp. 089686082097312

Author(s):

Alicia Sobrino-Pérez ◽

Alfonso Pérez-Escudero ◽

Lucila Fernández-Arroyo ◽

Ana Dorado-García ◽

Berta Martín-Alcón ◽

...

Keyword(s):

Peritoneal Dialysis ◽

Venous Pressure ◽

Relevant Parameter ◽

Pressure System ◽

Intraperitoneal Pressure ◽

In Vitro Measurements ◽

Long Timescales ◽

Intraperitoneal pressure (IPP) is gaining consideration as a relevant parameter of peritoneal dialysis (PD) in adults, although many of its aspects are still pending clarification. We address here its stability over time and the validity of the usual method of clinical measurement, as proposed by Durand in 1992 but never specifically validated. We performed this validation by comparing Durand’s method and direct measurements with a central venous pressure system. We performed a total of 250 measurement pairs in 50 patients with different intraperitoneal volumes plus in-vitro measurements with a simulated peritoneum. Absolute differences between the two systems in vivo were 0.87 ± 0.91 cmH2O (range 0–5 cmH2O); only 6.4% of them were ≥3 cmH2O. In vitro results for both methods were identical. We also compared IPP measurements in the same patient separated by 1–4 h (514 measurement pairs in 136 patients), 1 week (92 pairs in 92 patients), and 2 years (34 pairs in 17 patients). Net differences of measurements separated by hours or 1 week were close to 0 cmH2O, with oscillations of 1.5 cmH2O in hours and 2.3 cmH2O in 1 week. IPP measured 2 years apart presented a net decrease of 2.5 ± 4.9 cmH2O, without correlation with body mass index changes or any other usual parameter of PD. In hours, 7% of IPP differences were >3 cmH2O, 22% in 1 week, and 50% in 2 years. In conclusion, Durand’s method is precise enough to measure IPP in peritoneal dialysis. This parameter is not stable over long timescales, so it is necessary to use recent measurements.

In Vitro and In Vivo Activities of Novel Fluoroquinolones Alone and in Combination with Clarithromycin against Clinically Isolated Mycobacterium avium Complex Strains in Japan

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00410-07 ◽

2007 ◽

Vol 51 (11) ◽

pp. 4071-4076 ◽

Cited By ~ 31

Author(s):

Yoshihisa Kohno ◽

Hideaki Ohno ◽

Yoshitsugu Miyazaki ◽

Yasuhito Higashiyama ◽

Katsunori Yanagihara ◽

...

Keyword(s):

Infectious Disease ◽

Mycobacterium Avium ◽

Antimicrobial Agents ◽

Histopathological Examination ◽

Bacterial Counts ◽

Viable Bacteria ◽

The Individual ◽

ABSTRACT The recommended treatments for Mycobacterium avium complex (MAC) infectious disease are combination regimens of clarithromycin (CLR) or azithromycin with ethambutol and rifamycin. However, these chemotherapy regimens are sometimes unsuccessful. Recently developed antimicrobial agents, such as newer fluoroquinolones (FQs) containing C-8 methoxy quinolone (moxifloxacin [MXF] and gatifloxacin [GAT]), are expected to be novel antimycobacterial agents. Here, we evaluated the in vitro and in vivo antimycobacterial activities of three FQs (MXF, GAT, and levofloxacin) and CLR against clinically isolated MAC strains. Subsequently, the in vitro and in vivo synergic activities of FQ-CLR combinations against MAC strains were investigated. CLR and the individual FQs alone showed promising activity against MAC strains in vitro, and the bacterial counts in organs (lungs, liver, and spleen) of MAC-infected mice treated with single agents were significantly reduced compared to control mice. CLR showed the best anti-MAC effect in vivo. When the three FQs were individually combined with CLR in vitro, mild antagonism was observed for 53 to 57% of the tested isolates. Moreover, mice were infected with MAC strains showing mild antagonism for FQ-CLR combinations in vitro, and the anti-MAC effects of the FQ-CLR combinations were evaluated by counting the viable bacteria in their organs and by histopathological examination after 28 days of treatment. Several FQ-CLR combinations exhibited bacterial counts in organs significantly higher than those in mice treated with CLR alone. Our results indicate that the activity of CLR is occasionally attenuated by combination with an FQ both in vitro and in vivo and that this effect seems to be MAC strain dependent. Careful combination chemotherapy using these agents against MAC infectious disease may be required.