-
The Journal of Antimicrobial... May 2018We examined the in vitro pharmacodynamics and cellular accumulation of the standard anti-leishmanial drugs amphotericin B and miltefosine in intracellular Leishmania...
OBJECTIVES
We examined the in vitro pharmacodynamics and cellular accumulation of the standard anti-leishmanial drugs amphotericin B and miltefosine in intracellular Leishmania donovani amastigote-macrophage drug assays.
METHODS
Primary mouse macrophages were infected with L. donovani amastigotes. In time-kill assays infected macrophages were exposed to at least six different concentrations of serially diluted drugs and the percentage of infected macrophages was determined after 6, 12, 24, 48, 72 and 120 h of exposure. Cellular drug accumulation was measured following exposure to highly effective drug concentrations for 1, 6, 24, 48 and 72 h. Data were analysed through a mathematical model, relating drug concentration to the percentage of infected cells over time. Host cell membrane damage was evaluated through measurement of lactate dehydrogenase release. The effect of varying the serum and albumin concentrations in medium on the cellular accumulation levels of miltefosine was measured.
RESULTS
Amphotericin B was more potent than miltefosine (EC50 values of 0.65 and 1.26 μM, respectively) and displayed a wider therapeutic window in vitro. The kinetics of the cellular accumulation of amphotericin B was concentration- and formulation-dependent. At an extracellular concentration of 10 μM miltefosine maximum cellular drug levels preceded maximum anti-leishmanial kill. Miltefosine induced membrane damage in a concentration-, time- and serum-dependent manner. Its cellular accumulation levels increased with decreasing amounts of protein in assay medium.
CONCLUSIONS
We have developed a novel approach to investigate the cellular pharmacology of anti-leishmanial drugs that serves as a model for the characterization of new drug candidates.
Topics: Amphotericin B; Animals; Anti-Bacterial Agents; Antiprotozoal Agents; Cells, Cultured; Female; Leishmania donovani; Macrophages; Mice, Inbred BALB C; Models, Theoretical; Phosphorylcholine
PubMed: 29506127
DOI: 10.1093/jac/dky014 -
Clinical and Translational Medicine Nov 2021Lipid rafts (LRs), cholesterol-enriched microdomains on cell membranes, are increasingly viewed as signalling platforms governing critical facets of cancer progression....
BACKGROUND
Lipid rafts (LRs), cholesterol-enriched microdomains on cell membranes, are increasingly viewed as signalling platforms governing critical facets of cancer progression. The phenotype of cancer stem-like cells (CSCs) presents significant hurdles for successful cancer treatment, and the expression of several CSC markers is associated with LR integrity. However, LR implications in CSCs remain unclear.
METHODS
This study evaluated the biological and molecular functions of LRs in colorectal cancer (CRC) by using an LR-disrupting alkylphospholipid (APL) drug, miltefosine. The mechanistic role of miltefosine in CSC inhibition was examined through normal or tumour intestinal mouse organoid, human CRC cell, CRC xenograft and miltefosine treatment gene expression profile analyses.
RESULTS
Miltefosine suppresses CSC populations and their self-renewal activities in CRC cells, a CSC-targeting effect leading to irreversible disruption of tumour-initiating potential in vivo. Mechanistically, miltefosine reduced the expression of a set of genes, leading to stem cell death. Among them, miltefosine transcriptionally inhibited checkpoint kinase 1 (CHEK1), indicating that LR integrity is essential for CHEK1 expression regulation. In isolated CD44 CSCs, we found that CSCs exhibited stronger therapy resistance than non-CSC counterparts by preventing cell death through CHEK1-mediated cell cycle checkpoints. However, inhibition of the LR/CHEK1 axis by miltefosine released cell cycle checkpoints, forcing CSCs to enter inappropriate mitosis with accumulated DNA damage and resulting in catastrophic cell death.
CONCLUSION
Our findings underscore the therapeutic potential of LR-targeting APLs for CRC treatment that overcomes the therapy-resistant phenotype of CSCs, highlighting the importance of the LR/CHEK1 axis as a novel mechanism of APLs.
Topics: Animals; Antineoplastic Agents; Cell Proliferation; Colorectal Neoplasms; Disease Models, Animal; Membrane Microdomains; Mice; Phosphorylcholine
PubMed: 34841679
DOI: 10.1002/ctm2.552 -
Antimicrobial Agents and Chemotherapy Jan 2006The interaction of miltefosine with amphotericin B, sodium stibogluconate, paromomycin, and sitamaquine was assessed in vitro and additionally for the first three...
The interaction of miltefosine with amphotericin B, sodium stibogluconate, paromomycin, and sitamaquine was assessed in vitro and additionally for the first three combinations in vivo. In vitro interactions were indifferent for miltefosine combined with amphotericin B (mean sums of fractional inhibitory concentrations [mean summation operatorFICs] ranging from 1.22 to 1.51 at the 50% effective concentration [EC50] level and 1.08 to 1.38 at the EC90 level), sitamaquine (mean summation operatorFICs from 1.33 to 1.38 and 1.0 to 1.02, respectively), and paromomycin (mean summation operatorFICs from 0.79 to 0.93 at the EC50 and 0.77 to 1.35 at the EC90 level). Some synergy was observed for miltefosine combined with sodium stibogluconate (mean summation operatorFICs from 0.61 to 0.75 at EC50 and 0.49 to 0.97 at EC90). Different interactions were found in vivo, where the highest potentiation of miltefosine activity was achieved with amphotericin B (activity enhancement index [AEI] of up to 11.3). No significant interaction was observed when miltefosine was combined with sodium stibogluconate (AEI of up to 2.38). The potentiation of miltefosine in vivo was also achieved with the combination of miltefosine and paromomycin (AEI of up to 7.22).
Topics: Amphotericin B; Animals; Antiprotozoal Agents; Cricetinae; Drug Synergism; Leishmania donovani; Leishmaniasis, Visceral; Mice; Mice, Inbred BALB C; Parasitic Sensitivity Tests; Phosphorylcholine
PubMed: 16377670
DOI: 10.1128/AAC.50.1.73-79.2006 -
Molecular Imaging 2014Positive margins after breast conservation surgery represent a significant problem in the treatment of breast cancer. The near-infrared fluorescence agent CLR1502...
Positive margins after breast conservation surgery represent a significant problem in the treatment of breast cancer. The near-infrared fluorescence agent CLR1502 (Cellectar Biosciences, Madison, WI) was studied in a preclinical breast cancer model to determine imaging properties and ability to detect small islands of malignancy. Nude mice bearing human breast cancer flank xenografts were given a systemic injection of CLR1502, and imaging was performed using LUNA (Novadaq Technologies Inc., Richmond, BC) and Pearl Impulse (LI-COR Biosciences, Lincoln, NE) devices. Normal tissues were examined for fluorescence signal, and conventional and fluorescence histology was performed using the Odyssey scanner. Peak tumor to background ratio occurred 2 days after injection with CLR1502. The smallest amount of tumor that was imaged and detected using these devices was 1.9 mg, equivalent to 1.9 × 10⁶ cells. The highest fluorescence signal was seen in tumor and normal lymph node tissue, and the lowest fluorescence signal was seen in muscle and plasma. Human breast cancer tumors can be imaged in vivo with multiple optical imaging platforms using CLR1502. This pilot study supports further investigations of this fluorescent agent for improving surgical resection of malignancies, with the goal of eventual clinical translation.
Topics: Animals; Breast Neoplasms; Cell Line, Tumor; Diagnostic Imaging; Female; Fluorescent Dyes; Humans; Indoles; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Phosphorylcholine; Pilot Projects; Tumor Burden
PubMed: 25743270
DOI: 10.2310/7290.2014.00040 -
The American Journal of Tropical... Nov 2017This study describes the epidemiological and clinical characteristics of leishmaniasis and the pharmacological treatment of this disease in the municipality of Pueblo...
This study describes the epidemiological and clinical characteristics of leishmaniasis and the pharmacological treatment of this disease in the municipality of Pueblo Rico, Risaralda, between January 2010 and December 2014. An observational study was conducted using information from the clinical records and epidemiological reports of patients diagnosed and confirmed with leishmaniasis of any age and sex, including sociodemographic, clinical, and pharmacological variables of the therapy received. Univariate and bivariate analyses were performed. A total of 539 cases of leishmaniasis were confirmed, with 29.5% occurring in children under 5 years of age. The median age was 10 years, with predominance in males (55.5%). The indigenous Emberá (aboriginal Americans) were the most affected (50.8%), and 93.3% of cases occurred in people living in scattered rural areas. All lesions corresponded to cutaneous leishmaniasis, of which 251 patients had compromise of the upper limbs (46.6%), 221 of the face (41.0%), and 139 of the lower limbs (25.8%). Pentavalent antimony salts (n-methyl glucamine and sodium stibogluconate) were prescribed in 77.6% ( = 418) of the cases; miltefosine was the second most frequently prescribed medication (21.5%, = 116). The inhabitants of rural areas and the indigenous communities are at a higher risk of acquiring the infection, particularly among infants, which highlights the importance of the biological, social, and demographic factors involved in the disease. There is a need to seek effective public health actions and further research this disease.
Topics: Adolescent; Antimony Sodium Gluconate; Antiprotozoal Agents; Child; Child, Preschool; Colombia; Female; Humans; Leishmaniasis, Cutaneous; Male; Phosphorylcholine; Prevalence; Retrospective Studies; Socioeconomic Factors; Urban Population; Young Adult
PubMed: 28820714
DOI: 10.4269/ajtmh.17-0233 -
Biochimica Et Biophysica Acta.... Mar 2019Lipid rafts display a lateral heterogeneity forming membrane microdomains that hold a fundamental role on biological membranes and are indispensable to physiological...
Lipid rafts display a lateral heterogeneity forming membrane microdomains that hold a fundamental role on biological membranes and are indispensable to physiological functions of cells. Oxidative stress in cellular environments may cause lipid oxidation, changing membrane composition and organization, thus implying in effects in cell signaling and even loss of homeostasis. The individual contribution of oxidized lipid species to the formation or disruption of lipid rafts in membranes still remains unknown. Here, we investigate the role of different structures of oxidized phospholipids on rafts microdomains by carefully controlling the membrane composition. Our experimental approach based on fluorescence microscopy of giant unilamellar vesicles (GUV) enables the direct visualization of the impact of hydroperoxidized POPC lipid (referred to as POPCOOH) and shortened chain lipid PazePC (1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine) on phase separation. We found that the molecular structure of oxidized lipid is of paramount importance on lipid mixing and/or demixing. The hydrophobic mismatch promoted by POPCOOH coupled to its cylindrical molecular shape favor microdomains formation. In contrast, the conical shape of PazePC causes disarrangement of lipid 2D organized platforms. Our findings contribute to better unraveling how oxidized phospholipids can trigger formation or disruption of lipid rafts. As a consequence, phospholipid oxidation may indirectly affect association or dissociation of key biomolecules in the rafts thus altering cell signaling and homeostasis.
Topics: Lipid Bilayers; Lipid Peroxidation; Membrane Microdomains; Oxidants, Photochemical; Oxidation-Reduction; Phosphatidylcholines; Phosphorylcholine; Unilamellar Liposomes
PubMed: 30605637
DOI: 10.1016/j.bbamem.2018.12.017 -
Science Translational Medicine Jun 2014Many solid tumors contain an overabundance of phospholipid ethers relative to normal cells. Capitalizing on this difference, we created cancer-targeted...
Many solid tumors contain an overabundance of phospholipid ethers relative to normal cells. Capitalizing on this difference, we created cancer-targeted alkylphosphocholine (APC) analogs through structure-activity analyses. Depending on the iodine isotope used, radioiodinated APC analog CLR1404 was used as either a positron emission tomography (PET) imaging ((124)I) or molecular radiotherapeutic ((131)I) agent. CLR1404 analogs displayed prolonged tumor-selective retention in 55 in vivo rodent and human cancer and cancer stem cell models. (131)I-CLR1404 also displayed efficacy (tumor growth suppression and survival extension) in a wide range of human tumor xenograft models. Human PET/CT (computed tomography) and SPECT (single-photon emission computed tomography)/CT imaging in advanced-cancer patients with (124)I-CLR1404 or (131)I-CLR1404, respectively, demonstrated selective uptake and prolonged retention in both primary and metastatic malignant tumors. Combined application of these chemically identical APC-based radioisosteres will enable personalized dual modality cancer therapy of using molecular (124)I-CLR1404 tumor imaging for planning (131)I-CLR1404 therapy.
Topics: Animals; Antineoplastic Agents; Cell Line, Tumor; Female; Humans; Mice; Neoplasms; Phosphorylcholine; Positron-Emission Tomography; Tomography, Emission-Computed, Single-Photon; Tomography, X-Ray Computed; Xenograft Model Antitumor Assays
PubMed: 24920661
DOI: 10.1126/scitranslmed.3007646 -
International Journal of Oral Science Jun 2015Secondary caries due to biofilm acids is a primary cause of dental composite restoration failure. To date, there have been no reports of dental composites that can repel...
Secondary caries due to biofilm acids is a primary cause of dental composite restoration failure. To date, there have been no reports of dental composites that can repel protein adsorption and inhibit bacteria attachment. The objectives of this study were to develop a protein-repellent dental composite by incorporating 2-methacryloyloxyethyl phosphorylcholine (MPC) and to investigate for the first time the effects of MPC mass fraction on protein adsorption, bacteria attachment, biofilm growth, and mechanical properties. Composites were synthesized with 0 (control), 0.75%, 1.5%, 2.25%, 3%, 4.5% and 6% of MPC by mass. A commercial composite was also tested as a control. Mechanical properties were measured in three-point flexure. Protein adsorption onto the composite was determined by the microbicinchoninic acid method. A human saliva microcosm biofilm model was used. Early attachment at 4 h, biofilm at 2 days, live/dead staining and colony-forming units (CFUs) of biofilms grown on the composites were investigated. Composites with MPC of up to 3% had mechanical properties similar to those without MPC and those of the commercial control, whereas 4.5% and 6% MPC decreased the mechanical properties (P<0.05). Increasing MPC from 0 to 3% reduced the protein adsorption on composites (P<0.05). The composite with 3% MPC had protein adsorption that was 1/12 that of the control (P<0.05). Oral bacteria early attachment and biofilm growth were also greatly reduced on the composite with 3% MPC, compared to the control (P<0.05). In conclusion, incorporation of MPC into composites at 3% greatly reduced protein adsorption, bacteria attachment and biofilm CFUs, without compromising mechanical properties. Protein-repellent composites could help to repel bacteria attachment and plaque build-up to reduce secondary caries. The protein-repellent method might be applicable to other dental materials.
Topics: Adsorption; Biofilms; Colony Count, Microbial; Composite Resins; Dental Plaque; Methacrylates; Phosphorylcholine; Proteins
PubMed: 25655010
DOI: 10.1038/ijos.2014.77 -
The American Journal of Tropical... Mar 2012Although civilian physicians in the United States seldom encounter patients with leishmaniasis, therapeutic advances in endemic regions have opened the door to... (Review)
Review
Although civilian physicians in the United States seldom encounter patients with leishmaniasis, therapeutic advances in endemic regions have opened the door to approaches that can be applied in this country. Advances revolve around the use of oral miltefosine in all forms of leishmaniasis and the use of short-course intravenous liposomal amphotericin B in visceral and possibly cutaneous infection. Lengthy, traditional intravenous treatment with pentavalent antimony (sodium stibogluconate) still has a role in the United States; however, although expensive, miltefosine and liposomal amphotericin B are considerably more appealing selections for initial therapy.
Topics: Administration, Oral; Amphotericin B; Antiprotozoal Agents; Humans; Immunocompromised Host; Injections, Intravenous; Leishmaniasis; Phosphorylcholine; United States
PubMed: 22403313
DOI: 10.4269/ajtmh.2012.11-0682 -
Biochimica Et Biophysica Acta.... Feb 2018Dengue virus (DENV) non-structural (NS) 4A is a membrane protein essential for viral replication. The N-terminal region of NS4A contains several helices interacting with...
Dengue virus (DENV) non-structural (NS) 4A is a membrane protein essential for viral replication. The N-terminal region of NS4A contains several helices interacting with the cell membrane and the C-terminal region consists of three potential transmembrane regions. The secondary structure of the intact NS4A is not known as the previous structural studies were carried out on its fragments. In this study, we purified the full-length NS4A of DENV serotype 4 into dodecylphosphocholine (DPC) micelles. Solution NMR studies reveal that NS4A contains six helices in DPC micelles. The N-terminal three helices are amphipathic and interact with the membrane. The C-terminal three helices are embedded in micelles. Our results suggest that NS4A contains three transmembrane helices. Our studies provide for the first time structural information of the intact NS4A of DENV and will be useful for further understanding its role in viral replication.
Topics: Amino Acid Sequence; Dengue Virus; Lipid Bilayers; Magnetic Resonance Spectroscopy; Membrane Proteins; Micelles; Models, Molecular; Phosphorylcholine; Protein Binding; Protein Structure, Secondary; Viral Nonstructural Proteins
PubMed: 29055659
DOI: 10.1016/j.bbamem.2017.10.016