scholarly journals Assessment of Malaria In Vitro Drug Combination Screening and Mixed-Strain Infections Using the Malaria Sybr Green I-Based Fluorescence Assay

2009 ◽  
Vol 53 (6) ◽  
pp. 2557-2563 ◽  
Author(s):  
Edgie-Mark A. Co ◽  
Richard A. Dennull ◽  
Drew D. Reinbold ◽  
Norman C. Waters ◽  
Jacob D. Johnson
Keyword(s):  
Drug Combination ◽  
Antimalarial Drug ◽  
Fixed Ratio ◽  
Drug Combinations ◽  
Sybr Green ◽  
Sybr Green I ◽  
The Individual ◽  
Parasite Populations ◽  
Concentration Curves

ABSTRACT Several drug development strategies, including optimization of new antimalarial drug combinations, have been used to counter malaria drug resistance. We evaluated the malaria Sybr green I-based fluorescence (MSF) assay for its use in in vitro drug combination sensitivity assays. Drug combinations of previously published synergistic (atovaquone and proguanil), indifferent (chloroquine and azithromycin), and antagonistic (chloroquine and atovaquone) antimalarial drug interactions were tested against Plasmodium falciparum strains D6 and W2 using the MSF assay. Fifty percent inhibitory concentrations (IC50s) were calculated for individual drugs and in fixed ratio combinations relative to their individual IC50s. Subsequent isobologram analysis and fractional inhibitory concentration determinations demonstrated the expected drug interaction pattern for each combination tested. Furthermore, we explored the ability of the MSF assay to examine mixed parasite population dynamics, which are commonly seen in malaria patient isolates. Specifically, the capacity of the MSF assay to discern between single and mixed parasite populations was determined. To simulate mixed infections in vitro, fixed ratios of D6 and W2 strains were cocultured with antimalarial drugs and IC50s were determined using the MSF assay. Dichotomous concentration curves indicated that the sensitive and resistant parasites composing the genetically heterogeneous population were detectable. Biphasic analysis was performed to obtain subpopulation IC50s for comparison to those obtained for the individual malaria strains alone. In conclusion, the MSF assay allows for reliable antimalarial drug combination screening and provides an important method to discern between homogenous and heterogeneous parasite populations.

scholarly journals Adaptación y optimización de un método de lectura por fluorometría en el modelo farmacológico in vitro de cultivo de Plasmodium falciparum

2016 ◽  
Vol 45 (1) ◽  
pp. 127-146 ◽  
Author(s):  
María Helena Arias Marciales ◽  
Yinneth Victoria Rodríguez Novoa ◽  
Giovanny Garavito Cárdenas
Keyword(s):  
Plasmodium Falciparum ◽  
Sybr Green ◽  
Sybr Green I

<p>El modelo farmacológico de cultivo in vitro de P. falciparum es crucial en el tamizaje inicial de sustancias o extractos de plantas con posible actividad antiplasmodial. La densidad parasitaria puede determinarse mediante variadas metodologías, sin embargo, se han descrito numerosas ventajas y desventajas asociadas a cada una de ellas. Se evaluaron el tiempo de incubación necesario para la tinción y el uso de cultivos asincrónicos o sincrónicos en busca de valores óptimos; evidenciando un tiempo óptimo de 2 h, y límites de detección y cuantificación, menores en cultivos asincrónicos. Empleando las cepas FCR3 y FCB2 se evidenció un ruido de fondo de 12% y 38% respectivamente; la linealidad mostró una buena correlación, r2 de 0,9644 (FCR3) y 0,9841 (FCB2) y una pendiente de 1761,8 y 852,4 respectivamente. Además, se comprobó había concordancia entre los métodos, fluorométrico con SYBR Green I (SYBRG I) y microscópico con Giemsa, con diferencia media de 0,00002% y 0,09109% para FCR3 y FCB2 respectivamente. Los límites de detección y cuantificación fueron 0,5% y 1,5% de parasitemia. El factor Z con FCB2 fue 0,376, en tanto que con FCR3 alcanzó 0,702. La concentración inhibitoria 50 (CI50) frente a P. falciparum FCR3, generada por cloroquina (CQ) fue 0,37 mcg/mL por microscopía y 0,35 mcg/mL por fluorometría. Nuestros hallazgos sugieren que el ensayo de fluorescencia con SYBRG I, empleando fluorómetros comúnmente disponibles en muchos laboratorios, es preciso, robusto, rápido y exacto; para la evaluación in vitro de sustancias o extractos con posible actividad antiplasmodial.</p>

scholarly journals A Chemical Rescue Screen Identifies a Plasmodium falciparum Apicoplast Inhibitor Targeting MEP Isoprenoid Precursor Biosynthesis

2014 ◽  
Vol 59 (1) ◽  
pp. 356-364 ◽  
Author(s):  
Wesley Wu ◽  
Zachary Herrera ◽  
Danny Ebert ◽  
Katie Baska ◽  
Seok H. Cho ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Antimalarial Drug ◽  
Drug Targets ◽  
Specific Activity ◽  
Drug Candidate ◽  
Content Type ◽  
Chemical Rescue ◽  
Growth Inhibitory ◽  
Parasite Populations

ABSTRACTThe apicoplast is an essential plastid organelle found inPlasmodiumparasites which contains several clinically validated antimalarial-drug targets. A chemical rescue screen identified MMV-08138 from the “Malaria Box” library of growth-inhibitory antimalarial compounds as having specific activity against the apicoplast. MMV-08138 inhibition of blood-stagePlasmodium falciparumgrowth is stereospecific and potent, with the most active diastereomer demonstrating a 50% effective concentration (EC50) of 110 nM. Whole-genome sequencing of 3 drug-resistant parasite populations from two independent selections revealed E688Q and L244I mutations inP. falciparumIspD, an enzyme in the MEP (methyl-d-erythritol-4-phosphate) isoprenoid precursor biosynthesis pathway in the apicoplast. The active diastereomer of MMV-08138 directly inhibited PfIspD activityin vitrowith a 50% inhibitory concentration (IC50) of 7.0 nM. MMV-08138 is the first PfIspD inhibitor to be identified and, together with heterologously expressed PfIspD, provides the foundation for further development of this promising antimalarial drug candidate lead. Furthermore, this report validates the use of the apicoplast chemical rescue screen coupled with target elucidation as a discovery tool to identify specific apicoplast-targeting compounds with new mechanisms of action.

Phase 1 study of CPX-1, a fixed ratio formulation of irinotecan (IRI) and floxuridine (FLOX), in patients with advanced solid tumors

2006 ◽  
Vol 24 (18_suppl) ◽  
pp. 2014-2014 ◽  
Author(s):  
G. Batist ◽  
K. Chi ◽  
W. Miller ◽  
S. Chia ◽  
F. Hasanbasic ◽  
...  
Keyword(s):  
Dose Escalation ◽  
Phase 1 ◽  
Fixed Ratio ◽  
Compression Fracture ◽  
Drug Combinations ◽  
Molar Ratio ◽  
Open Label ◽  
Dose Escalation Study ◽  
Grade 3

2014 Background: In vitro studies have shown that varying the ratio of individual agents in drug combinations can result in synergistic, additive or antagonistic activity against tumor cells. CPX-1 is a liposomal formulation of IRI and FLOX in a fixed 1:1 molar ratio which was selected as optimal in vitro and confirmed to be synergistic in vivo in preclinical tumor models. CPX-1 overcomes the dissimilar pharmacokinetics (PK) of the individual drugs, enables sustained maintenance of this ratio after IV administration, and was evaluated in a Phase I open-label, dose-escalation study. Methods: Starting dose was 30 U/m2 (1 Unit of CPX-1 contains 1 mg IRI + 0.36 mg FLOX) given on day 1 and 15 of each 28-day cycle. Dose escalation was by modified Fibonacci with 4 subjects/cohort. Eligibility included: ≥ 18 yo; advanced solid tumor; ECOG PS ≤ 2; adequate bone marrow/liver/renal function. PK analysis was done on day 1 and 15 of the first cycle. Results: 26 subjects (16M:10F), median age 54.5 y (21–72), all with prior therapy, enrolled in 6 cohorts with the 5th cohort expanded to 6 subjects. Diagnoses: 8 colorectal, 3 pancreatic, 3 ovarian, 2 breast, 2 gastric, 2 esophageal, 2 sarcomas, 1 renal cell, 1 prostate, 1 NSCLC and 1 sphenoid sinus. Response: 20 subjects evaluable: 2 confirmed PRs (NSCLC 8+ wks; Colon 13+ wks, in a patient with prior IRI exposure) and 13 with SD (8–24+wks). Safety: DLTs were observed at the 6th dose level: 4 subjects with DLTs: 3 diarrhea (one resulting in death due to dehydration/ARF) and one neutropenia. Other possibly related grade 3 and 4 events included one each of: grade 3 diarrhea, grade 3 vomiting, grade 3 neutropenia, grade 3 fatigue, grade 3 compression fracture and arthralgia and pulmonary embolism grade 4. PK: In all 14 subjects analyzed to date the 1:1 molar ratio of IRI to FLOX was maintained for 24 hours and metabolites 5-FU and SN-38 were present in the plasma. Conclusions: CPX-1 represents a new approach to developing drug combinations in which drug ratios are pre-selected in vitro based on optimal antitumor activity and maintained systemically through pharmacokinetic control. Phase 2 studies are planned with a recommended dose of 210U/m2 of CPX-1. [Table: see text]

scholarly journals Syrosingopine sensitizes cancer cells to killing by metformin

2016 ◽  
Vol 2 (12) ◽  
pp. e1601756 ◽  
Author(s):  
Don Benjamin ◽  
Marco Colombi ◽  
Sravanth K. Hindupur ◽  
Charles Betz ◽  
Heidi A. Lane ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Drug Combination ◽  
Therapeutic Strategy ◽  
Synthetic Lethality ◽  
Glycolytic Enzyme ◽  
Transformed Cells ◽  
Monoamine Transporters ◽  
Vesicular Monoamine Transporters ◽  
The Individual

We report that the anticancer activity of the widely used diabetic drug metformin is strongly potentiated by syrosingopine. Synthetic lethality elicited by combining the two drugs is synergistic and specific to transformed cells. This effect is unrelated to syrosingopine’s known role as an inhibitor of the vesicular monoamine transporters. Syrosingopine binds to the glycolytic enzyme α-enolase in vitro, and the expression of the γ-enolase isoform correlates with nonresponsiveness to the drug combination. Syrosingopine sensitized cancer cells to metformin and its more potent derivative phenformin far below the individual toxic threshold of each compound. Thus, combining syrosingopine and codrugs is a promising therapeutic strategy for clinical application for the treatment of cancer.

scholarly journals Assessment and Continued Validation of the Malaria SYBR Green I-Based Fluorescence Assay for Use in Malaria Drug Screening

2007 ◽  
Vol 51 (6) ◽  
pp. 1926-1933 ◽  
Author(s):  
Jacob D. Johnson ◽  
Richard A. Dennull ◽  
Lucia Gerena ◽  
Miriam Lopez-Sanchez ◽  
Norma E. Roncal ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Culture Condition ◽  
Microtiter Plate ◽  
Growth Conditions ◽  
Drug Susceptibility ◽  
Sybr Green ◽  
Sybr Green I ◽  
Phenotypic Correlation ◽  
In Vitro Susceptibility

ABSTRACT Several new fluorescence malaria in vitro drug susceptibility microtiter plate assays that detect the presence of malarial DNA in infected erythrocytes have recently been reported, in contrast to traditional isotopic screens that involve radioactive substrate incorporation to measure in vitro malaria growth inhibition. We have assessed and further characterized the malaria SYBR Green I-based fluorescence (MSF) assay for its ability to monitor drug resistance. In order to use the MSF assay as a drug screen, all assay conditions must be thoroughly examined. In this study we expanded upon the capabilities of this assay by including antibiotics and antifolates in the drug panel and testing folic acid-free growth conditions. To do this, we evaluated a more expansive panel of antimalarials in combination with various drug assay culture conditions commonly used in drug sensitivity screening for their activity against Plasmodium falciparum strains D6 and W2. The detection and quantitation limits of the MSF assay were 0.04 to 0.08% and ∼0.5% parasitemia, respectively. The MSF assay quality was significantly robust, displaying a Z′ range of 0.73 to 0.95. The 50% inhibitory concentrations for each drug and culture condition combination were determined by using the MSF assay. Compared to the standard [3H]hypoxanthine assay, the MSF assay displayed the expected parasite drug resistance patterns with a high degree of global and phenotypic correlation (r 2 ≥ 0.9238), regardless of which culture condition combination was used. In conclusion, the MSF assay allows for reliable one-plate high-throughput, automated malaria in vitro susceptibility testing without the expense, time consumption, and hazard of other screening assays.

In-vitro culture of Plasmodium falciparum: Utility of modified (RPNI) medium for drug-sensitivity studies using SYBR Green I assay

2011 ◽  
Vol 127 (1) ◽  
pp. 318-321 ◽  
Author(s):  
Shubhra Singh ◽  
Rajeev Kumar Srivastava ◽  
Mukesh Srivastava ◽  
S.K. Puri ◽  
K. Srivastava
Keyword(s):  
Plasmodium Falciparum ◽  
In Vitro Culture ◽  
Drug Sensitivity ◽  
Sybr Green ◽  
Sybr Green I ◽  
Sensitivity Studies

scholarly journals In Vitro Evaluation of Antifungal Drug Combinations against Multidrug-Resistant Candida auris Isolates from New York Outbreak

2020 ◽  
Vol 64 (4) ◽  
Author(s):  
Brittany O’Brien ◽  
Sudha Chaturvedi ◽  
Vishnu Chaturvedi
Keyword(s):  
New York ◽  
Drug Combination ◽  
Multidrug Resistant ◽  
Drug Combinations ◽  
Health Care Facilities ◽  
Antifungal Drugs ◽  
Candida Auris ◽  
Pathogenic Yeast ◽  
Content Type

ABSTRACT Since 2016, New York hospitals and health care facilities have faced an unprecedented outbreak of the pathogenic yeast Candida auris. We tested over 1,000 C. auris isolates from affected facilities and found high resistance to fluconazole (MIC > 256 mg/liter) and variable resistance to other antifungal drugs. Therefore, we tested if two-drug combinations are effective in vitro against multidrug-resistant C. auris. Broth microdilution antifungal combination plates were custom manufactured by TREK Diagnostic System. We used 100% inhibition endpoints for the drug combination as reported earlier for the intra- and interlaboratory agreements against Candida species. The results were derived from 12,960 readings, for 15 C. auris isolates tested against 864 two-drug antifungal combinations for nine antifungal drugs. Flucytosine (5FC) at 1.0 mg/liter potentiated the most combinations. For nine C. auris isolates resistant to amphotericin B (AMB; MIC ≥ 2.0 mg/liter), AMB-5FC (0.25/1.0 mg/liter) yielded 100% inhibition. Six C. auris isolates resistant to three echinocandins (anidulafungin [AFG], MIC ≥ 4.0 mg/liter; caspofungin [CAS], MIC ≥ 2.0 mg/liter; and micafungin [MFG], MIC ≥ 4.0 mg/liter) were 100% inhibited by AFG-5FC and CAS-5FC (0.0078/1 mg/liter) and MFG-5FC (0.12/1 mg/liter). None of the combinations were effective for C. auris 18-1 and 18-13 (fluconazole [FLC] > 256 mg/liter, 5FC > 32 mg/liter) except MFG-5FC (0.1/0.06 mg/liter). Thirteen isolates with a high voriconazole (VRC) MIC (>2 mg/liter) were 100% inhibited by the VRC-5FC (0.015/1 mg/liter). The simplified two-drug combination susceptibility test format would permit laboratories to provide clinicians and public health experts with additional data to manage multidrug-resistant C. auris.

scholarly journals Comparison of a SYBR Green I-Based Assay with a Histidine-Rich Protein II Enzyme-Linked Immunosorbent Assay for In Vitro Antimalarial Drug Efficacy Testing and Application to Clinical Isolates

2007 ◽  
Vol 51 (4) ◽  
pp. 1172-1178 ◽  
Author(s):  
David J. Bacon ◽  
Christine Latour ◽  
Carmen Lucas ◽  
Olga Colina ◽  
Pascal Ringwald ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Immunosorbent Assay ◽  
Sybr Green ◽  
Sybr Green I ◽  
Clinical Isolates ◽  
Enzyme Linked Immunosorbent Assay ◽  
Complete Medium ◽  
Phenol Red ◽  
Reference Strains

ABSTRACT In vitro drug susceptibility testing with the malaria parasite has been used to assess the antimalarial activities of new compounds and to monitor drug resistance in field isolates. We investigated the validity of a SYBR green I fluorescent-based assay under various culture conditions and compared the assay results to those of previously published histidine-rich protein II (HRPII) enzyme-linked immunosorbent assay (ELISA) methods. Reference strains of Plasmodium falciparum were cultured in vitro by using standard conditions in complete medium with and without phenol red before they were dispensed into 96-well plates predosed with chloroquine, mefloquine, or quinine. Following incubation, the culture supernatants were divided and the 50% inhibitory concentrations (IC50s) were determined by using a SYBR green I-based method and the HRPII capture ELISA method. There were no significant differences in IC50 values when phenol red was included in the medium. The IC50s and the IC90s of the antimalarials tested by both methods were similar or identical for each of the reference strains. Fresh clinical isolates of P. falciparum collected from imported cases of malaria in Lyon, France, were tested for in vitro resistance to chloroquine and mefloquine by using the validated SYBR green I and HRPII ELISA methods. The SYBR green I-based method was able to calculate IC50 and IC90 values similar or identical to those calculated by the HRPII assay with fresh clinical samples without removal of white blood cells. The SYBR green I-based method for determination of drug sensitivity levels produced results comparable to those produced by other methods, showing that this method can be used routinely to conduct surveillance for drug resistance in P. falciparum with fresh or cultured parasites.

scholarly journals Rapid optimization of drug combinations for the optimal angiostatic treatment of cancer

Angiogenesis ◽  
2015 ◽  
Vol 18 (3) ◽  
pp. 233-244 ◽  
Author(s):  
Andrea Weiss ◽  
Xianting Ding ◽  
Judy R. van Beijnum ◽  
Ieong Wong ◽  
Tse J. Wong ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Drug Combination ◽  
Search Algorithm ◽  
Drug Combinations ◽  
System Control ◽  
Single Drug ◽  
Drug Concentrations

Abstract Drug combinations can improve angiostatic cancer treatment efficacy and enable the reduction of side effects and drug resistance. Combining drugs is non-trivial due to the high number of possibilities. We applied a feedback system control (FSC) technique with a population-based stochastic search algorithm to navigate through the large parametric space of nine angiostatic drugs at four concentrations to identify optimal low-dose drug combinations. This implied an iterative approach of in vitro testing of endothelial cell viability and algorithm-based analysis. The optimal synergistic drug combination, containing erlotinib, BEZ-235 and RAPTA-C, was reached in a small number of iterations. Final drug combinations showed enhanced endothelial cell specificity and synergistically inhibited proliferation (p < 0.001), but not migration of endothelial cells, and forced enhanced numbers of endothelial cells to undergo apoptosis (p < 0.01). Successful translation of this drug combination was achieved in two preclinical in vivo tumor models. Tumor growth was inhibited synergistically and significantly (p < 0.05 and p < 0.01, respectively) using reduced drug doses as compared to optimal single-drug concentrations. At the applied conditions, single-drug monotherapies had no or negligible activity in these models. We suggest that FSC can be used for rapid identification of effective, reduced dose, multi-drug combinations for the treatment of cancer and other diseases.

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