Can Power Motion Imaging Mode Combined with Contrast Agent Assess Myocardial Contraction and Perfusion? In Vitro and In Vivo Studies

Power motion imaging (PMI) is a new Doppler imaging technology that can enhance contrast agent visualization. We hypothesized that PMI combined with Albunex injection (CPMI) could provide new insights into myocardial contractility and perfusion. In a first step, PMI signal was studied with an in vitro phantom. In a second step, PMI signal was studied in 10 rabbits at different workloads. In a third step, 10 rabbits were studied before and after ischemia produced by coronary ligation and finally during reperfusion. During the latter protocol, epicardial echocardiography analyzed by PMI and fundamental mode was performed before and after Albunex injection. PMI signal was well correlated with phantom velocity. PMI signal and myocardial wall thickening were well correlated, particularly in the septal area. On the coronary occlusion model, ischemia was associated with a significant decrease in PMI and CPMI signals, whereas reperfusion was associated with a significant decrease in PMI signals only, indicating stunning. We conclude that PMI combined with CPMI is a powerful tool to assess myocardial contractility and perfusion.     

Preclinical In Vitro and In Vivo Characterization of Synaptic Vesicle 2A–Targeting Compounds Amenable to F-18 Labeling as Potential PET Radioligands for Imaging of Synapse Integrity

Molecular Imaging and Biology - Current synaptic vesicle 2A (SV2A) positron emission tomography (PET) imaging agents include the nanomolar affinity probes [11C]UCB-J and [18F]UCB-H derived from the...     

Validation of an Engineered Cell Model for In Vitro and In Vivo HIF-1α Evaluation by Different Imaging Modalities

Purpose

The aim of this study was to characterize a cell-based model for the molecular study of hypoxia-inducible factor (HIF)-1α activity, in the context of hypoxia, by means of different imaging techniques.

Procedures

Engineered U251-HRE glioma cells were used to analyze the molecular mechanisms underlying HIF-1α activity in vitro in relation to luciferase expression. The same cells were orthotopically implanted in mice to evaluate tumor progression and hypoxia induction by bioluminescence imaging, fluorescence imaging, positron emission tomography (PET), and magnetic resonance imaging (MRI).

Results

In vitro analyses highlighted the relationship between HIF-1α and luciferase activity in hypoxic conditions and after pharmacological treatments in U251-HRE cells. Through in vivo studies, it was possible to assess hypoxia establishment in relation to tumor growth by optical imaging, PET and MRI.

Conclusions

The findings of this study indicate that the U251-HRE orthotopic murine model can be used to reliably evaluate processes modulating HIF-1α activity, using both molecular and preclinical non-invasive imaging techniques.     

In Vivo Multiparametric Ultrasound Imaging of Structural and Functional Tumor Modifications during Therapy

Longitudinal imaging techniques are needed that can meaningfully probe the tumor microenvironment and its spatial heterogeneity. Contrast-enhanced ultrasound, shear wave elastography and quantitative ultrasound are ultrasound-based techniques that provide information on the vascular function and micro-/macroscopic tissue structure. Modifications of the tumor microenvironment induced by cytotoxic and anti-angiogenic molecules in ectopic murine Lewis lung carcinoma tumors were monitored. The most heterogenous structures were found in tumors treated with anti-angiogenic drug that simultaneously accumulated the highest levels of necrosis and fibrosis. The anti-angiogenic group presented the highest number of correlations between parameters related to vascular function and those related to the micro-/macrostructure of the tumor microenvironment. Results suggest how patterns of multiparametric ultrasound modifications can be related to provide a more insightful marker of changes occurring within tumors during therapy.     

Evaluation of Two Optical Probes for Imaging the Integrin αvβ6− In Vitro and In Vivo in Tumor-Bearing Mice

Purpose

The purpose of this study was to develop and evaluate two α_vβ₆-targeted fluorescent imaging agents. The integrin subtype α_vβ₆ is significantly upregulated in a wide range of epithelial derived cancers, plays a key role in invasion and metastasis, and expression is often located at the invasive edge of tumors. α_vβ₆-targeted fluorescent imaging agents have the potential to guide surgical resection leading to improved patient outcomes. Both imaging agents were based on the bi-PEGylated peptide NH₂-PEG₂₈-A20FMDV2-K16R-PEG₂₈ (1), a peptide that has high affinity and selectivity for the integrin α_vβ₆: (a) 5-FAM-X-PEG₂₈-A20FMDV2-K16R-PEG₂₈ (2), and (b) IRDye800-PEG₂₈-A20FMDV2-K16R-PEG₂₈ (3).

Procedures

Peptides were synthesized using solid-phase peptide synthesis and standard Fmoc chemistry. Affinity for α_vβ₆ was evaluated by ELISA. In vitro binding, internalization, and localization of 2 was monitored using confocal microscopy in DX3puroβ₆ (α_vβ₆+) and DX3puro (α_vβ₆?) cells. The in vivo imaging and ex vivo biodistribution of 3 was evaluated in three preclinical mouse models, DX3puroβ₆/DX3puro and BxPC-3 (α_vβ₆+) tumor xenografts and a BxPC-3 orthotopic pancreatic tumor model.

Results

Peptides were obtained in >?99% purity. IC₅₀ values were 28 nM (2) and 39 nM (3). Rapid α_vβ₆-selective binding and internalization of 2 was observed. Fluorescent intensity (FLI) measurements extracted from the in vivo images and ex vivo biodistribution confirmed uptake and retention of 3 in the α_vβ₆ positive subcutaneous and orthotopic tumors, with negligible uptake in the α_vβ₆-negative tumor. Blocking studies with a known α_vβ₆-targeting peptide demonstrated α_vβ₆-specific binding of 3.

Conclusion

Two fluorescence imaging agents were developed. The α_vβ₆-specific uptake, internalization, and endosomal localization of the fluorescence agent 2 demonstrates potential for targeted therapy. The selective uptake and retention of 3 in the α_vβ₆-positive tumors enabled clear delineation of the tumors and surgical resection indicating 3 has the potential to be utilized during image-guided surgery.

     

Development and Screening of Contrast Agents for In Vivo Imaging of Parkinson’s Disease

Purpose

The goal was to identify molecular imaging probes that would enter the brain, selectively bind to Parkinson’s disease (PD) pathology, and be detectable with one or more imaging modalities.

Procedure

A library of organic compounds was screened for the ability to bind hallmark pathology in human Parkinson’s and Alzheimer’s disease tissue, alpha-synuclein oligomers and inclusions in two cell culture models, and alpha-synuclein aggregates in cortical neurons of a transgenic mouse model. Finally, compounds were tested for blood–brain barrier permeability using intravital microscopy.

Results

Several lead compounds were identified that bound the human PD pathology, and some showed selectivity over Alzheimer’s pathology. The cell culture models and transgenic mouse models that exhibit alpha-synuclein aggregation did not prove predictive for ligand binding. The compounds had favorable physicochemical properties, and several were brain permeable.

Conclusions

Future experiments will focus on more extensive evaluation of the lead compounds as PET ligands for clinical imaging of PD pathology.     

Comparison of the Adsorption Capacities of an Activated-Charcoal–Yogurt Mixture Versus Activated-Charcoal–Water Slurry In Vivo and In Vitro

Background. An activated charcoal–yogurt mixture was evaluated in vivo to determine the effect on the gastrointestinal absorption of paracetamol, as compared to activated-charcoal–water slurry. The potential advantage of the activated-charcoal–yogurt mixture is a better palatability and general acceptance by the patients without loss of efficacy. In addition, paracetamol adsorption studies were carried out in vitro to calculate the maximum adsorption capacity of paracetamol to activated-charcoal–yogurt mixture. Methods. In vivo: A randomized crossover study on 15 adult volunteers, using paracetamol 50 mg/kg as a simulated overdose. Each study day volunteers were given a standard meal 1 h before paracetamol, then 50 g activated charcoal 1 h later in either of two preparations: standard water slurry or mixed with 400 mL yogurt. Paracetamol serum concentrations were measured using HPLC. The areas under the concentration-time curve (AUC) of the two preparations were compared and used to estimate the efficacy of each preparation. The palatability of both preparations was evaluated using a visual-analogue scale where the volunteers were asked to evaluate the appearance, smell, flavor, texture, ability to swallow, and overall impression of the mixtures. The time spent to consume the activated charcoal was also registered. In vitro: Activated charcoal, simulated gastric (pH 1.2) or intestinal (pH 7.2) fluid, and paracetamol were mixed with yogurt followed by 1 h incubation. The maximum adsorption capacity of paracetamol to activated charcoal was calculated using Langmuir's adsorption isotherm. Paracetamol concentration was analyzed using HPLC. Results. In vivo there was no significant difference (p > 0.05) in the AUC of paracetamol between the two activated-charcoal preparations. Geometric mean values and 95% CI for the AUCs were (in mg/l · min): 6307 (4932–8065) for the activated charcoal–water slurry and 6525 (5111–8330) for the activated charcoal–yogurt mixture. The palatability study showed significant difference (p < 0.05) only in duration of administration, in favor of the activated charcoal-water slurry. In vitro the maximum adsorption capacity of activated charcoal with added yogurt was 544 mg paracetamol/g activated charcoal (pH 1.2), and 569 mg paracetamol/g activated charcoal (pH 7.2). Conclusion. The two activated-charcoal preparations showed equal (NS) absorption reduction of paracetamol in vivo. Mixing activated charcoal with yogurt rather than water prolonged the ingestion time, but did not improve the palatability in adults. The presence of yogurt reduced the adsorption capacity in vitro by 9–13% (p < 0.05) compared to control without yogurt (previous study with the same setup).     

Correction to: Evaluation of Two Optical Probes for Imaging the Integrin αvβ6− In Vitro and In Vivo in Tumor-Bearing Mice

Molecular Imaging and Biology - This article was updated to correct the axes in Figures&nbsp;4e and 5d.     

Synthesis of a Novel l-Methyl-Methionine–ICG-Der-02 Fluorescent Probe for In Vivo Near Infrared Imaging of Tumors

Purpose

A novel near infrared fluorescent probe, l-methyl-methionine (Met)?CICG-Der-02, was synthesized and characterized for in vivo imaging of tumors and early diagnosis of cancers.

Method

Met was conjugated with ICG-Der-02 dye through the amide bond function by ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride/N-hydroxysuccinimide catalysis chemistry. Met?CICG-Der-02 probe uptake was evaluated on PC3, MDA-MB-231, and human embryonic lung fibroblast cell lines. The dynamics of Met?CICG-Der-02 was investigated in athymic nude mice prior to evaluation of the probe targeting capability in prostate and breast cancer models.

Results

Met?CICG-Der-02 was successfully synthesized. Cell experiments demonstrated excellent cellular uptake of Met?CICG-Der-02 on cancer cell lines without cytotoxicity. Optical imaging showed a distinguishable fluorescence signal in the tumor area at 2?h while maximal tumor-to-normal tissue contrast ratio was at 12?h Met?CICG-Der-02 post-injection. Additionally, dynamic study of the probe indicated intestinal and liver?Ckidney clearance pathways.

Conclusion

Met?CICG-Der-02 probe is a promising optical imaging agent for tumor diagnosis, especially in their early stage.     

In Vitro and In Vivo Efficacy of β-Lactams against Replicating and Slowly Growing/Nonreplicating Mycobacterium tuberculosis

Beta-lactams, in combination with beta-lactamase inhibitors, are reported to have activity against Mycobacterium tuberculosis bacteria growing in broth, as well as inside the human macrophage. We tested representative beta-lactams belonging to 3 different classes for activity against replicating M. tuberculosis in broth and nonreplicating M. tuberculosis under hypoxia, as well as against streptomycin-starved M. tuberculosis strain 18b (ss18b) in the presence or absence of clavulanate. Most of the combinations showed bactericidal activity against replicating M. tuberculosis, with up to 200-fold improvement in potency in the presence of clavulanate. None of the combinations, including those containing meropenem, imipenem, and faropenem, killed M. tuberculosis under hypoxia. However, faropenem- and meropenem-containing combinations killed strain ss18b moderately. We tested the bactericidal activities of meropenem-clavulanate and amoxicillin-clavulanate combinations in the acute and chronic aerosol infection models of tuberculosis in BALB/c mice. Based on pharmacokinetic/pharmacodynamic indexes reported for beta-lactams against other bacterial pathogens, a cumulative percentage of a 24-h period that the drug concentration exceeds the MIC under steady-state pharmacokinetic conditions (%T_MIC) of 20 to 40% was achieved in mice using a suitable dosing regimen. Both combinations showed marginal reduction in lung CFU compared to the late controls in the acute model, whereas both were inactive in the chronic model.     

In Vitro and In Vivo Antifungal Activity of Amphotericin B Lipid Complex: Are Phospholipases Important?

Amphotericin B lipid complex for injection (ABLC) is a suspension of amphotericin B complexed with the lipids l-α-dimyristoylphosphatidylcholine (DMPC) and l-α-dimyristoylphosphatidylglycerol. ABLC is less toxic than amphotericin B deoxycholate (AmB-d), while it maintains the antifungal activity of AmB-d. Active amphotericin B can be released from ABLC by exogenously added (snake venom, bacteria, or Candida-derived) phospholipases or by phospholipases derived from activated mammalian vascular tissue (rat arteries). Such extracellular phospholipases are capable of hydrolyzing the major lipid in ABLC. Mutants of C. albicans that were resistant to ABLC but not AmB-d in vitro were deficient in extracellular phospholipase activity, as measured on egg yolk agar or as measured by their ability to hydrolyze DMPC in ABLC. ABLC was nevertheless effective in the treatment of experimental murine infections produced by these mutants. Isolates of Aspergillus species, apparently resistant to ABLC in vitro (but susceptible to AmB-d), were also susceptible to ABLC in vivo. We suggest that routine in vitro susceptibility tests with ABLC itself as the test material may not accurately predict the in vivo activity of ABLC and that the enhanced therapeutic index of ABLC relative to that of AmB-d in vivo may be due, in part, to the selective release of active amphotericin B from the complex at sites of fungal infection through the action of fungal or host cell-derived phospholipases.Amphotericin B has been the agent of choice for the treatment of serious fungal infections for more than 35 years. However, administration of the most common preparation of amphotericin B (a sodium deoxycholate colloidal suspension) is associated with severe, dose-limiting acute and chronic toxicities, particularly nephrotoxicity.Amphotericin B lipid complex for injection (ABLC) is a suspension of amphotericin B complexed with the lipids l-α-dimyristoylphosphatidylcholine (DMPC) and l-α-dimyristoylphosphatidylglycerol (DMPG) (11). The safety and efficacy of ABLC have been extensively evaluated in laboratory (4–6, 14) and clinical (7, 24, 25) studies. Those studies have shown that ABLC is, in general, markedly less toxic than amphotericin B deoxycholate (AmB-d) and has antifungal activity at least comparable to and sometimes enhanced over that of AmB-d.Complexation with lipids appears to stabilize amphotericin B in a self-associated state so that it is not available to interact with cellular membranes (the presumed major site of its antifungal activity and its mammalian toxicity) (12, 17). It was previously demonstrated that ABLC is more than 1,000-fold less hemolytic to erythrocytes in vitro than AmB-d and that active (hemolytic) amphotericin B can be released from ABLC by a heat-labile, extracellular fungal product (lipase) (17). It has also been demonstrated that in vitro the MICs of ABLC for certain phospholipase-deficient non-Candida albicans Candida species and phospholipase-deficient mutants of C. albicans are higher than those of AmB-d (13). In the present study we further evaluated the role of phospholipases in the in vitro and in vivo antifungal activity of ABLC.     

Soluble Domain 1 of Platelet–Endothelial Cell Adhesion Molecule (PECAM) Is Sufficient to Block Transendothelial Migration In Vitro and In Vivo

The inflammatory response involves sequential adhesive interactions between cell adhesion molecules of leukocytes and the endothelium. Unlike the several adhesive steps that precede it, transendothelial migration (diapedesis), the step in which leukocytes migrate between apposed endothelial cells, appears to involve primarily one adhesion molecule, platelet–endothelial cell adhesion molecule (PECAM, CD31). Therefore, we have focused on PECAM as a target for antiinflammatory therapy. We demonstrate that soluble chimeras made of the entire extracellular portion of PECAM, or of only the first immunoglobulin domain of PECAM, fused to the Fc portion of IgG, block diapedesis in vitro and in vivo. Furthermore, the truncated form of the PECAM-IgG chimera does not bind stably to its cellular ligand. This raises the possibility of selective anti-PECAM therapies that would not have the untoward opsonic or cell-activating properties of antibodies directed against PECAM.The emigration of leukocytes from the bloodstream into a site of inflammation involves a series of interactions between cell adhesion molecules (CAMs)¹ on the leukocyte and the venular endothelium. This phenomenon has been dissected into discrete steps of rolling, activation, tight adhesion, transmigration, and migration across the basement membrane (1–4). If a relevant leukocyte or endothelial CAM is inhibited, leukocytes do not proceed to the next step. During transendothelial migration (TEM), the leukocytes squeeze between tightly apposed endothelial cells. This process involves the function of platelet–endothelial cell adhesion molecule (PECAM, CD31), a member of the immunoglobulin gene superfamily, which is expressed on the surfaces of monocytes (Mo), granulocytes, NK cells, some T cell subsets, and concentrated at the borders between endothelial cells (5–7).In the presence of appropriate anti-PECAM mAbs, leukocytes can bind tightly to endothelial monolayers and migrate to the endothelial junctions, but they do not proceed through the junctions (8, 9). This process is reversible since diapedesis resumes shortly after removing the blocking mAb (8). Transmigration appears to involve homophilic interaction of PECAM on the leukocyte with PECAM on the endothelial cell. Blocking the PECAM on either cell is sufficient to maximally block TEM in vitro; blocking PECAM on both cells has no additional effect (8).Depending on the leukocyte type and the inflammatory stimulus, more than one CAM can participate in each of the steps of rolling, activation, and tight adhesion (1, 10). Thus, it is difficult to block inflammation using individual reagents directed at the particular molecules involved in these steps. In contrast, PECAM mediates a common final step in emigration for many leukocyte types activated by a variety of stimuli. In addition, PECAM has no other known function in vivo. Most of the other CAMs important in emigration of leukocytes have other roles in the immune system (1, 11), the blockade of which could lead to untoward consequences.Therefore, PECAM is an attractive target molecule for antiinflammatory therapy. In fact, mAbs (12) and polyclonal antibodies (13–15) against PECAM block acute inflammation in response to a variety of stimuli. However, xenogeneic mAb has the potential to opsonize leukocytes, leading to leukopenia, as well as to stimulate production of neutralizing antibodies by the host, making it a poor agent for chronic therapy. Moreover, engagement of CAMs by high affinity mAbs can activate cells, especially leukocytes. Ligation of leukocyte PECAM by a variety of mAbs can trigger an adhesion cascade resulting in the upregulation of leukocyte integrin binding activity on T cells (16), PMN, Mo (17), and NK cells (9, 18).To avoid these potential problems, we fused portions of the extracellular region of autologous PECAM to the human Fc chain. These soluble chimeras competitively inhibit TEM in vitro and in vivo. A chimera containing only PECAM domain 1, which is incapable of binding stably to cellular PECAM, blocks emigration of both PMN and monocytes into the inflamed peritoneal cavity. This is the first demonstration that a portion of a CAM with no stable binding activity itself can block inflammation in vivo.     

Echo Decorrelation Imaging of Rabbit Liver and VX2 Tumor during In Vivo Ultrasound Ablation

In open surgical procedures, image-ablate ultrasound arrays performed thermal ablation and imaging on rabbit liver lobes with implanted VX2 tumor. Treatments included unfocused (bulk ultrasound ablation, N = 10) and focused (high-intensity focused ultrasound ablation, N = 13) exposure conditions. Echo decorrelation and integrated backscatter images were formed from pulse-echo data recorded during rest periods after each therapy pulse. Echo decorrelation images were corrected for artifacts using decorrelation measured prior to ablation. Ablation prediction performance was assessed using receiver operating characteristic curves. Results revealed significantly increased echo decorrelation and integrated backscatter in both ablated liver and ablated tumor relative to unablated tissue, with larger differences observed in liver than in tumor. For receiver operating characteristic curves computed from all ablation exposures, both echo decorrelation and integrated backscatter predicted liver and tumor ablation with statistically significant success, and echo decorrelation was significantly better as a predictor of liver ablation. These results indicate echo decorrelation imaging is a successful predictor of local thermal ablation in both normal liver and tumor tissue, with potential for real-time therapy monitoring.     

Development of a Fluorinated Analogue of Erlotinib for PET Imaging of EGFR Mutation–Positive NSCLC

Molecular Imaging and Biology - Positron emission tomography (PET) using [11C]erlotinib identifies non-small cell lung carcinoma (NSCLC) tumors with activating mutations in the epidermal growth...     

2-D Left Ventricular Flow Estimation by Combining Speckle Tracking With Navier–Stokes-Based Regularization: An In Silico,In Vitro and In Vivo Study

Despite the availability of multiple ultrasound approaches to left ventricular (LV) flow characterization in two dimensions, this technique remains in its childhood and further developments seem warranted. This article describes a new methodology for tracking the 2-D LV flow field based on ultrasound data. Hereto, a standard speckle tracking algorithm was modified by using a dynamic kernel embedding Navier–Stokes-based regularization in an iterative manner. The performance of the proposed approach was first quantified in synthetic ultrasound data based on a computational fluid dynamics model of LV flow. Next, an experimental flow phantom setup mimicking the normal human heart was used for experimental validation by employing simultaneous optical particle image velocimetry as a standard reference technique. Finally, the applicability of the approach was tested in a clinical setting. On the basis of the simulated data, pointwise evaluation of the estimated velocity vectors correlated well (mean r = 0.84) with the computational fluid dynamics measurement. During the filling period of the left ventricle, the properties of the main vortex obtained from the proposed method were also measured, and their correlations with the reference measurement were also calculated (radius, r = 0.96; circulation, r = 0.85; weighted center, r = 0.81). In vitro results at 60 bpm during one cardiac cycle confirmed that the algorithm properly measures typical characteristics of the vortex (radius, r = 0.60; circulation, r = 0.81; weighted center, r = 0.92). Preliminary qualitative results on clinical data revealed physiologic flow fields.     

Evaluation of Cu-64 and Ga-68 Radiolabeled Glucagon-Like Peptide-1 Receptor Agonists as PET Tracers for Pancreatic β cell Imaging

Purpose

Copper-64 (Cu-64) and Galium-68 (Ga-68) radiolabeled DO3A and NODA conjugates of exendin-4 were used for preclinical imaging of pancreatic β cells via targeting of glucagon-like peptide-1 receptor (GLP-1R).

Procedures

DO3A-VS- and NODA-VS-tagged Cys⁴⁰exendin-4 (DO3A-VS-Cys⁴⁰-exendin-4 and NODA-VS-Cys⁴⁰-exendin-4, respectively) were labeled with Cu-64 and Ga-68 using standard techniques. Biodistribution and dynamic positron emission tomography (PET) were carried out in normal Sprague–Dawley (SD) rats. Ex vivo autoradiography imaging was conducted with freshly frozen pancreatic thin sections.

Results

DO3A-VS- and NODA-VS-Cys⁴⁰-exendin-4 analogues were labeled with Cu-64 and Ga-68 to a specific activity of 518.7?±?3.7 Ci/mmol (19.19?±?0.14 TBq/mmol) and radiochemical yield above 98 %. Biodistribution data demonstrated pancreatic uptake of 0.11?±?0.02 %ID/g for [⁶⁴Cu]DO3A-VS-, 0.14?±?0.02 %ID/g for [⁶⁴Cu]NODA-VS-, 0.11?±?0.03 for [⁶⁸Ga]DO3A-VS-, and 0.26?±?0.03 for [⁶⁸Ga]NODA-VS-Cys⁴⁰-exendin-4. Excess exendin-4 and exendin-(9–39)-amide displaced all four Cu-64 and Ga-68 labeled exendin-4 derivatives in blocking studies.

Conclusions

[⁶⁴Cu]/[⁶⁸Ga]DO3A-VS-Cys⁴⁰- and [⁶⁴Cu]/[⁶⁸Ga]NODA-VS-Cys⁴⁰-exendin-4 can be used as PET imaging agents specific for GLP-1R expressed on β cells. Here, we report the first evidence of pancreatic uptake visualized with exendin-4 derivative in a rat animal model via in vivo dynamic PET imaging.

     

In Vitro and In Vivo Studies of the Antiparasitic Activity of Sterol 14α-Demethylase (CYP51) Inhibitor VNI against Drug-Resistant Strains of Trypanosoma cruzi

Chagas disease affects more than 10 million people worldwide, and yet, as it has historically been known as a disease of the poor, it remains highly neglected. Two currently available drugs exhibit severe toxicity and low effectiveness, especially in the chronic phase, while new drug discovery has been halted for years as a result of a lack of interest from pharmaceutical companies. Although attempts to repurpose the antifungal drugs posaconazole and ravuconazole (inhibitors of fungal sterol 14α-demethylase [CYP51]) are finally in progress, development of cheaper and more efficient, preferably Trypanosoma cruzi-specific, chemotherapies would be highly advantageous. We have recently reported that the experimental T. cruzi CYP51 inhibitor VNI cures with 100% survival and 100% parasitological clearance both acute and chronic murine infections with the Tulahuen strain of T. cruzi. In this work, we further explored the potential of VNI by assaying nitro-derivative-resistant T. cruzi strains, Y and Colombiana, in highly stringent protocols of acute infection. The data show high antiparasitic efficacy of VNI and its derivative (VNI/VNF) against both forms of T. cruzi that are relevant for mammalian host infection (bloodstream and amastigotes), with the in vivo potency, at 25 mg/kg twice a day (b.i.d.), similar to that of benznidazole (100 mg/kg/day). Transmission electron microscopy and reverse mutation tests were performed to explore cellular ultrastructural and mutagenic aspects of VNI, respectively. No mutagenic potential could be seen by the Ames test at up to 3.5 μM, and the main ultrastructural damage induced by VNI in T. cruzi was related to Golgi apparatus and endoplasmic reticulum organization, with membrane blebs presenting an autophagic phenotype. Thus, these preliminary studies confirm VNI as a very promising trypanocidal drug candidate for Chagas disease therapy.