In Vitro Pharmacodynamic Parameters of Sordarin Derivatives in Comparison with Those of Marketed Compounds against Pneumocystis carinii Isolated from Rats

ABSTRACT Pneumocystis carinii pneumonia remains one of the most serious complications of immunosuppressed patients. In this study, the in vitro pharmacodynamic parameters of four sordarin derivatives (GM 191519, GM 237354, GM 193663, and GM 219771) have been evaluated by a new quantitative approach and compared with the commercially available drugs pentamidine, atovaquone, and trimethoprim-sulfamethoxazole (TMP-SMX). In vitro activities and in vivo therapeutic efficacies of sordarin derivatives against P. carinii were also evaluated. In vitro activity was determined by the broth microdilution technique, comparing the total number of microorganisms in treated and drug-free cultures by using Giemsa staining. The in vitro maximum effect (E max), the drug concentrations to reach 50% of E max(EC50), and the slope of the dose-response curve were then estimated by the Hill equation (E max sigmoid model). Sordarin derivatives were the most potent agents againstP. carinii, with EC50s of 0.00025, 0.0007, 0.0043, and 0.025 μg/ml for GM 191519, GM 237354, GM 193663, and GM 219771, respectively. The EC50s of pentamidine, atovaquone, and TMP-SMX were 0.025, 0.16, and 26.7/133.5 μg/ml, respectively. The results obtained with this approach showed GM 237354 and GM 191519 to be approximately 35- and 100-fold more active in vitro than pentamidine, the most active marketed compound. All sordarin derivatives tested were at least 5,000-fold more active in vitro than TMP-SMX. The three sordarin derivatives tested in vivo—GM 191519, GM 237354, and GM 219771—showed a marked therapeutic efficacy, defined as reduction of cyst forms per gram of lung. GM 191519 was the most potent (daily dose reducing 50% of the P. carinii burden in the lungs [ED50], 0.05 mg/kg/day) followed by GM 237354 and GM 219771 (ED50s, 0.30 and 0.49 mg/kg/day, respectively). Good agreement between in vitro parameters and in vivo outcome was obtained when P. carinii pneumonia in rats was treated with sordarin derivatives.

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Continuous infusion of DL-alpha-difluoromethylornithine and improved efficacy against a rat model of Pneumocystis carinii pneumonia.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.40.10.2318 ◽

1996 ◽

Vol 40 (10) ◽

pp. 2318-2320 ◽

Cited By ~ 8

Author(s):

K Chin ◽

S Merali ◽

M Sarić ◽

A B Clarkson

Keyword(s):

Drinking Water ◽

Continuous Infusion ◽

Rat Model ◽

Pneumocystis Carinii ◽

Bolus Administration ◽

Drug Concentrations ◽

Water Administration ◽

Standard Animal

The rapid depletion of Pneumocystis carinii polyamines caused by in vitro exposure to DL-alpha-difluoromethylornithine (DFMO; also called eflornithine or Ornidyl) and the rapid repletion following removal of this drug suggested that the in vivo efficacy of DFMO against P. carinii pneumonia (PCP) may be limited by troughs in drug concentration resulting from the schedule of administration. This led to the prediction that, compared with the response to the standard animal protocol of administering DFMO in drinking water, the response of a rat model of PCP to DFMO would be lessened by bolus administration and improved by continuous infusion. These predictions were confirmed. Intraperitoneal bolus administration of up to 3 g of DFMO kg of body weight-1 was completely ineffective, although this dose has been shown to be effective when given in the drinking water. Conversely, continuous infusion improved the response against PCP seven- to ninefold over the response to drinking water administration. These findings suggest that, compared with the standard clinical investigational protocol for treatment of PCP with DFMO given in four divided daily doses, continuous infusion combined with monitoring of drug concentrations in plasma may improve efficacy and/or reduce the already low rate of adverse effects.

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OP0244 A PRECLINICAL TESTING TOOL: THE IN VITRO 3D FRACTURE GAP MODEL

Annals of the Rheumatic Diseases ◽

10.1136/annrheumdis-2020-eular.3546 ◽

2020 ◽

Vol 79 (Suppl 1) ◽

pp. 154.1-154

Author(s):

M. Pfeiffenberger ◽

A. Damerau ◽

P. Hoff ◽

A. Lang ◽

F. Buttgereit ◽

...

Keyword(s):

Fracture Healing ◽

Bone Healing ◽

Inflammatory Markers ◽

Initial Phase ◽

Gap Model ◽

Osteogenic Markers ◽

Immunosuppressed Patients ◽

The Impact

Background:Approximately 10% of fractures lead to significant fracture healing disorders, with a tendency to further increase due to the aging population. Of note, especially immunosuppressed patients with ongoing inflammation show difficulties in the correct course of fracture healing leading to fracture healing disorders. Most notably, invading immune cells and secreted cytokines are considered to provide an inflammatory microenvironment within the fracture gap, primarily during the initial phase of fracture healing. Current research has the focus on small animal models, facing the problem of translation towards the human system. In order to improve the therapy of fracture healing disorders, we have developed a human cell-basedin vitromodel to mimic the initial phase of fracture healing adequately. This model will be used for the development of new therapeutic strategies.Objectives:Our aim is to develop anin vitro3D fracture gap model (FG model) which mimics thein vivosituation in order to provide a reliable preclinical test system for fracture healing disorders.Methods:To assemble our FG model, we co-cultivated coagulated peripheral blood and primary human mesenchymal stromal cells (MSCs) mimicking the fracture hematoma (FH model) together with a scaffold-free bone-like construct mimicking the bony part of the fracture gap for 48 h under hypoxic conditions (n=3), in order to reflect thein vivosituation after fracture most adequately. To analyze the impact of the bone-like construct on thein vitroFH model with regard to its osteogenic induction capacity, we cultivated the fracture gap models in either medium with or without osteogenic supplements. To analyze the impact of Deferoxamine (DFO, known to foster fracture healing) on the FG model, we further treated our FG models with either 250 µmol DFO or left them untreated. After incubation and subsequent preparation of the fracture hematomas, we evaluated gene expression of osteogenic (RUNX2,SPP1), angiogenic (VEGF,IL8), inflammatory markers (IL6,IL8) and markers for the adaptation towards hypoxia (LDHA,PGK1) as well as secretion of cytokines/chemokines using quantitative PCR and multiplex suspension assay, respectively.Results:We found via histology that both the fracture hematoma model and the bone-like construct had close contact during the incubation, allowing the cells to interact with each other through direct cell-cell contact, signal molecules or metabolites. Additionally, we could show that the bone-like constructs induced the upregulation of osteogenic markers (RUNX2, SPP1) within the FH models irrespective of the supplementation of osteogenic supplements. Furthermore, we observed an upregulation of hypoxia-related, angiogenic and osteogenic markers (RUNX2,SPP1) under the influence of DFO, and the downregulation of inflammatory markers (IL6,IL8) as compared to the untreated control. The latter was also confirmed on protein level (e.g. IL-6 and IL-8). Within the bone-like constructs, we observed an upregulation of angiogenic markers (RNA-expression ofVEGF,IL8), even more pronounced under the treatment of DFO.Conclusion:In summary, our findings demonstrate that our establishedin vitroFG model provides all osteogenic cues to induce the initial bone healing process, which could be enhanced by the fracture-healing promoting substance DFO. Therefore, we conclude that our model is indeed able to mimic correctly the human fracture gap situation and is therefore suitable to study the influence and efficacy of potential therapeutics for the treatment of bone healing disorders in immunosuppressed patients with ongoing inflammation.Disclosure of Interests:Moritz Pfeiffenberger: None declared, Alexandra Damerau: None declared, Paula Hoff: None declared, Annemarie Lang: None declared, Frank Buttgereit Grant/research support from: Amgen, BMS, Celgene, Generic Assays, GSK, Hexal, Horizon, Lilly, medac, Mundipharma, Novartis, Pfizer, Roche, and Sanofi., Timo Gaber: None declared

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A Selective ATP-Binding Cassette Subfamily G Member 2 Efflux Inhibitor Revealed via High-Throughput Flow Cytometry

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057112456875 ◽

2012 ◽

Vol 18 (1) ◽

pp. 26-38 ◽

Cited By ~ 15

Author(s):

J. Jacob Strouse ◽

Irena Ivnitski-Steele ◽

Hadya M. Khawaja ◽

Dominique Perez ◽

Jerec Ricci ◽

...

Keyword(s):

Molecular Probes ◽

Atp Binding ◽

Specific Substrate ◽

Tumor Resistance ◽

Drug Concentrations ◽

Efflux Inhibition ◽

Substrate Inhibitor ◽

Atp Binding Cassette

Chemotherapeutics tumor resistance is a principal reason for treatment failure, and clinical and experimental data indicate that multidrug transporters such as ATP-binding cassette (ABC) B1 and ABCG2 play a leading role by preventing cytotoxic intracellular drug concentrations. Functional efflux inhibition of existing chemotherapeutics by these pumps continues to present a promising approach for treatment. A contributing factor to the failure of existing inhibitors in clinical applications is limited understanding of specific substrate/inhibitor/pump interactions. We have identified selective efflux inhibitors by profiling multiple ABC transporters against a library of small molecules to find molecular probes to further explore such interactions. In our primary screening protocol using JC-1 as a dual-pump fluorescent reporter substrate, we identified a piperazine-substituted pyrazolo[1,5-a]pyrimidine substructure with promise for selective efflux inhibition. As a result of a focused structure-activity relationship (SAR)–driven chemistry effort, we describe compound 1 (CID44640177), an efflux inhibitor with selectivity toward ABCG2 over ABCB1. Compound 1 is also shown to potentiate the activity of mitoxantrone in vitro as well as preliminarily in vivo in an ABCG2-overexpressing tumor model. At least two analogues significantly reduce tumor size in combination with the chemotherapeutic topotecan. To our knowledge, low nanomolar chemoreversal activity coupled with direct evidence of efflux inhibition for ABCG2 is unprecedented.

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Amitriptyline and imipramine inhibit the release of acetylcholine from parasympathetic nerve terminals in the rat iris

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y84-143 ◽

1984 ◽

Vol 62 (7) ◽

pp. 857-859 ◽

Cited By ~ 2

Author(s):

J. S. Richardson ◽

T. G. Mattio ◽

E. Giacobini

Keyword(s):

Nerve Terminals ◽

Dependent Manner ◽

Parasympathetic Nerve ◽

Drug Concentrations ◽

Release Of Acetylcholine ◽

Rat Iris ◽

Anticholinergic Side Effects ◽

Dose Dependent ◽

Drug Free

The electrically stimulated release of [3H]acetylcholine from the parasympathetic nerve terminals of the rat iris in vitro is increased in a dose-dependent manner by scopolamine but is decreased by the tricyclic antidepressants amitriptyline and imipramine. The increased release in the presence of scopolamine seems to be due to the blockade of a presynaptic muscarinic autoreceptor that, in the drug-free state, inhibits the release of acetylcholine. However, at drug concentrations that should have comparable antimuscarinic potency, the antidepressants inhibit the release of acetylcholine. This suggests that the anticholinergic side effects of the antidepressants may be due to the reduced release of acetylcholine from parasympathetic nerve terminals as well as a possible direct postsynaptic muscarinic receptor blocking action. Whatever the mechanism of this action, the antidepressants do not have the same effect as scopolamine at the presynaptic muscarinic autoreceptor in the rat iris.

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Correlation of In Vitro Activity, Serum Levels, and In Vivo Efficacy of Posaconazole against Rhizopus microsporus in a Murine Disseminated Infection

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01026-09 ◽

2009 ◽

Vol 53 (12) ◽

pp. 5022-5025 ◽

Cited By ~ 26

Author(s):

M. Mar Rodríguez ◽

F. Javier Pastor ◽

Enrique Calvo ◽

Valentina Salas ◽

Deanna A. Sutton ◽

...

Keyword(s):

Serum Levels ◽

Rhizopus Microsporus ◽

In Vitro Susceptibility ◽

In Vivo Efficacy ◽

Susceptibility Data ◽

Microdilution Method ◽

Drug Concentrations ◽

Broth Microdilution Method

ABSTRACT A broth microdilution method was used to evaluate the in vitro activities of seven antifungal agents against 15 clinical strains of Rhizopus microsporus. Amphotericin B (AMB) and posaconazole (POS) were the most active drugs. In a model of disseminated R. microsporus infection in immunosuppressed mice, we studied the efficacy of POS administered once or twice daily against four of the strains previously tested in vitro and compared it with that of liposomal AMB (LAMB). LAMB was the most effective treatment for the two strains with intermediate susceptibility to POS. For the two POS-susceptible strains, LAMB and POS at 20 mg/kg of body weight twice a day orally showed similar efficacies. The in vivo efficacy of POS administered twice a day orally correlated with the in vitro susceptibility data and the serum drug concentrations.

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The potential of microdialysis to estimate rifampicin concentrations in the lung of guinea pigs

PLoS ONE ◽

10.1371/journal.pone.0245922 ◽

2021 ◽

Vol 16 (1) ◽

pp. e0245922

Author(s):

Faye Lanni ◽

Neil Burton ◽

Debbie Harris ◽

Susan Fotheringham ◽

Simon Clark ◽

...

Keyword(s):

Drug Development ◽

Guinea Pigs ◽

Sampling Technique ◽

Tissue Fluid ◽

Experimental Conditions ◽

Continuous Sampling ◽

Drug Concentrations ◽

The Impact

Optimised pre-clinical models are required for TB drug development to better predict the pharmacokinetics of anti-tuberculosis (anti-TB) drugs to shorten the time taken for novel drugs and combinations to be approved for clinical trial. Microdialysis can be used to measure unbound drug concentrations in awake freely moving animals in order to describe the pharmacokinetics of drugs in the organs as a continuous sampling technique. The aim of this work was to develop and optimise the microdialysis methodology in guinea pigs to better understand the pharmacokinetics of rifampicin in the lung. In vitro experiments were performed before progressing into in vivo studies because the recovery (concentration of the drug in the tissue fluid related to that in the collected dialysate) of rifampicin was dependent on a variety of experimental conditions. Mass spectrometry of the dialysate was used to determine the impact of flow rate, perfusion fluid and the molecular weight cut-off and membrane length of probes on the recovery of rifampicin at physiologically relevant concentrations. Following determination of probe efficiency and identification of a correlation between rifampicin concentrations in the lung and skeletal muscle, experiments were conducted to measure rifampicin in the sacrospinalis of guinea pigs using microdialysis. Lung concentrations of rifampicin were estimated from the rifampicin concentrations measured in the sacrospinalis. These studies suggest the potential usefulness of the microdialysis methodology to determine drug concentrations of selected anti-TB drugs to support new TB drug development.

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SSB facilitates fork substrate discrimination by PriA

10.1101/2020.11.23.394411 ◽

2020 ◽

Author(s):

Hui Yin Tan ◽

Piero R. Bianco

Keyword(s):

Catalytic Efficiency ◽

Maximum Effect ◽

Replication Forks ◽

Replication Process ◽

Replication Restart ◽

Species Specific ◽

The Right ◽

Gene 32

AbstractPriA is a member of the SuperFamily 2 helicase family. Its role in vivo is to reload the primosome onto stalled replication forks resulting in the restart of the previously stalled DNA replication process. SSB is known to play key roles in mediating activities at replication forks and it is known to bind to PriA. To gain mechanistic insight into the PriA-SSB interaction, a coupled spectrophotometric assay was utilized to characterize the ATPase activity of PriA in vitro in the presence of fork substrates. The results demonstrate that SSB enhances the ability of PriA to discriminate between fork substrates 140-fold. This is due to a significant increase in the catalytic efficiency of the helicase induced by DNA-bound SSB. This interaction is species-specific as bacteriophage gene 32 protein cannot substitute for the E.coli protein. SSB, while enhancing the activity of PriA on its preferred fork, both decreases the affinity of the helicase for other forks and decreases catalytic efficiency. Central to the stimulation afforded by SSB is the unique ability of PriA to bind with high affinity to the 3’-OH placed at the end of the nascent leading strand at the fork. When both the 3’-OH and SSB are present, the maximum effect is observed. This ensures that PriA will only load onto the correct fork, in the right orientation, thereby ensuring that replication restart is directed to only the template lagging strand.

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Predicting absorption and pharmacokinetic profile of carbamazepine from controlled-release tablet formulation in humans using rabbit model

Veterinarski glasnik ◽

10.2298/vetgl1102071h ◽

2011 ◽

Vol 65 (1-2) ◽

pp. 71-81

Author(s):

Irena Homsek ◽

Dragica Popadic ◽

Slobodanka Simic ◽

Slavica Ristic ◽

Katarina Vucicevic ◽

...

Keyword(s):

Controlled Release ◽

Rabbit Model ◽

Tablet Formulation ◽

Human Model ◽

In Vitro Dissolution ◽

Pharmacokinetic Parameters ◽

Pharmacokinetic Data ◽

Drug Concentrations

Controlled-release (CR) pharmaceutical formulations offer several advantages over the conventional, immediate release dosage forms of the same drug, including reduced dosing frequency, decreased incidence and/or intensity of adverse effects, greater selectivity of pharmacological activity, reduced drug plasma fluctuation, and better compliance. After a drug product has been registered, and is already on market, minor changes in formulation might be needed. At the same time, the product has to remain effective and safe for patients that could be confirmed via plasma drug concentrations and pharmacokinetic characteristics. It is challenging to predict human absorption and pharmacokinetic characteristics of a drug based on the in vitro dissolution test and the animal pharmacokinetic data. Therefore, the objective of this study was to establish correlation of the pharmacokinetic parameters of carbamazepine (CBZ) CR tablet formulation between the rabbit and the human model, and to establish in vitro in vivo correlation (IVIVC) based on the predicted fractions of absorbed CBZ. Although differences in mean plasma concentration profiles were notified, the data concerning the predicted fraction of drug absorbed were almost superimposable. Accordingly, it can be concluded that rabbits may be representative as an in vivo model for predicting the pharmacokinetics of the CR formulation of CBZ in humans.

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Increased Bone Mass in Mice Lacking the Adipokine Apelin

Endocrinology ◽

10.1210/en.2012-2034 ◽

2013 ◽

Vol 154 (6) ◽

pp. 2069-2080 ◽

Cited By ~ 22

Author(s):

Lalita Wattanachanya ◽

Wei-Dar Lu ◽

Ramendra K. Kundu ◽

Liping Wang ◽

Marcia J. Abbott ◽

...

Keyword(s):

Bone Mass ◽

Bone Cells ◽

Physiological Role ◽

In Vitro Study ◽

Maximum Effect ◽

Dependent Manner ◽

Primary Mouse ◽

Skeletal Homeostasis

Abstract Adipose tissue plays an important role in skeletal homeostasis, and there is interest in identifying adipokines that influence bone mass. One such adipokine may be apelin, a ligand for the Gi-G protein-coupled receptor APJ, which has been reported to enhance mitogenesis and suppress apoptosis in MC3T3-E1 cells and primary human osteoblasts (OBs). However, it is unclear whether apelin plays a physiological role in regulating skeletal homeostasis in vivo. In this study, we compared the skeletal phenotypes of apelin knockout (APKO) and wild-type mice and investigated the direct effects of apelin on bone cells in vitro. The increased fractional cancellous bone volume at the distal femur was observed in APKO mice of both genders at 12 weeks of age and persisted until the age of 20. Cortical bone perimeter at the femoral midshaft was significantly increased in males and females at both time points. Dynamic histomorphometry revealed that APKO mice had increased rates of bone formation and mineral apposition, with evidences of accelerated OB proliferation and differentiation, without significant alteration in osteoclast activity. An in vitro study showed that apelin increased proliferation of primary mouse OBs as well as suppressed apoptosis in a dose-dependent manner with the maximum effect at 5nM. However, it had no effect on the formation of mineralized nodules. We did not observed significantly altered in osteoclast parameters in vitro. Taken together, the increased bone mass in mice lacking apelin suggested complex direct and paracrine/endocrine effects of apelin on bone, possibly via modulating insulin sensitivity. These results indicate that apelin functions as a physiologically significant antianabolic factor in bone in vivo.

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Absence of the Macrophage Mannose Receptor in Mice Does Not Increase Susceptibility to Pneumocystis carinii Infection In Vivo

Infection and Immunity ◽

10.1128/iai.71.11.6213-6221.2003 ◽

2003 ◽

Vol 71 (11) ◽

pp. 6213-6221 ◽

Cited By ~ 79

Author(s):

Steve D. Swain ◽

Sena J. Lee ◽

Michel C. Nussenzweig ◽

Allen G. Harmsen

Keyword(s):

Pneumocystis Carinii ◽

Hydrolytic Enzymes ◽

Mannose Receptor ◽

Opportunistic Pathogen ◽

Cell Types ◽

Surface Expression ◽

Wild Type ◽

Macrophage Mannose Receptor

ABSTRACT Host defense against the opportunistic pathogen Pneumocystis carinii requires functional interactions of many cell types. Alveolar macrophages are presumed to be a vital host cell in the clearance of P. carinii, and the mechanisms of this interaction have come under scrutiny. The macrophage mannose receptor is believed to play an important role as a receptor involved in the binding and phagocytosis of P. carinii. Although there is in vitro evidence for this interaction, the in vivo role of this receptor in P. carinii clearance in unclear. Using a mouse model in which the mannose receptor has been deleted, we found that the absence of this receptor is not sufficient to allow infection by P. carinii in otherwise immunocompetent mice. Furthermore, when mice were rendered susceptible to P. carinii by CD4+ depletion, mannose receptor knockout mice (MR-KO) had pathogen loads equal to those of wild-type mice. However, the MR-KO mice exhibited a greater influx of phagocytes into the alveoli during infection. This was accompanied by increased pulmonary pathology in the MR-KO mice, as well as greater accumulation of glycoproteins in the alveoli (glycoproteins, including harmful hydrolytic enzymes, are normally cleared by the mannose receptor). We also found that the surface expression of the mannose receptor is not downregulated during P. carinii infection in wild-type mice. Our findings suggest that while the macrophage mannose receptor may be important in the recognition of P. carinii, in vivo, this mechanism may be redundant, and the absence of this receptor may be compensated for.

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