In Vitro and In Vivo Activities of 2-Aminopyrazines and 2-Aminopyridines in Experimental Models of Human African Trypanosomiasis

ABSTRACT The success of nifurtimox-eflornithine combination therapy (NECT) for the treatment of human African trypanosomiasis (HAT) has renewed interest in the potential of nitro drugs as chemotherapeutics. In order to study the implications of the more widespread use of nitro drugs against these parasites, we examined the in vivo and in vitro resistance potentials of nifurtimox and fexinidazole and its metabolites. Following selection in vitro by exposure to increasing concentrations of nifurtimox, Trypanosoma brucei brucei nifurtimox-resistant clones designated NfxR1 and NfxR2 were generated. Both cell lines were found to be 8-fold less sensitive to nifurtimox than parental cells and demonstrated cross-resistance to a number of other nitro drugs, most notably the clinical trial candidate fexinidazole (∼27-fold more resistant than parental cells). Studies of mice confirmed that the generation of nifurtimox resistance in these parasites did not compromise virulence, and NfxR1 remained resistant to both nifurtimox and fexinidazole in vivo. In the case of fexinidazole, drug metabolism and pharmacokinetic studies indicate that the parent drug is rapidly metabolized to the sulfoxide and sulfone form of this compound. These metabolites retained trypanocidal activity but were less effective in nifurtimox-resistant lines. Significantly, trypanosomes selected for resistance to fexinidazole were 10-fold more resistant to nifurtimox than parental cells. This reciprocal cross-resistance has important implications for the therapeutic use of nifurtimox in a clinical setting and highlights a potential danger in the use of fexinidazole as a monotherapy.

Download Full-text

Inhibition of Isoleucyl-tRNA Synthetase as a Potential Treatment for Human African Trypanosomiasis

Journal of Biological Chemistry ◽

10.1074/jbc.m112.447441 ◽

2013 ◽

Vol 288 (20) ◽

pp. 14256-14263 ◽

Cited By ~ 11

Author(s):

Igor Cestari ◽

Kenneth Stuart

Keyword(s):

Structure Prediction ◽

Mammalian Cells ◽

Human African Trypanosomiasis ◽

Competitive Inhibitor ◽

Trna Synthetase ◽

New Drugs ◽

African Trypanosomiasis ◽

Approved Drugs

Trypanosoma brucei sp. causes human African trypanosomiasis (HAT; African sleeping sickness). The parasites initially proliferate in the hemolymphatic system and then invade the central nervous system, which is lethal if not treated. New drugs are needed for HAT because the approved drugs are few, toxic, and difficult to administer, and drug resistance is spreading. We showed by RNAi knockdown that T. brucei isoleucyl-tRNA synthetase is essential for the parasites in vitro and in vivo in a mouse model of infection. By structure prediction and experimental analysis, we also identified small molecules that inhibit recombinant isoleucyl-tRNA synthetase and that are lethal to the parasites in vitro and highly selective compared with mammalian cells. One of these molecules acts as a competitive inhibitor of the enzyme and cures mice of the infection. Because members of this class of molecules are known to cross the blood-brain barrier in humans and to be tolerated, they may be attractive as leading candidates for drug development for HAT.

Download Full-text

Pharmacokinetics, Trypanosoma brucei gambiense Efficacy, and Time of Drug Action of DB829, a Preclinical Candidate for Treatment of Second-Stage Human African Trypanosomiasis

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00398-13 ◽

2013 ◽

Vol 57 (11) ◽

pp. 5330-5343 ◽

Cited By ~ 20

Author(s):

Tanja Wenzler ◽

Sihyung Yang ◽

Olivier Braissant ◽

David W. Boykin ◽

Reto Brun ◽

...

Keyword(s):

Single Dose ◽

Trypanosoma Brucei ◽

Human African Trypanosomiasis ◽

Drug Action ◽

African Trypanosomiasis ◽

Content Type ◽

Second Stage ◽

Trypanosoma Brucei Gambiense

ABSTRACTHuman African trypanosomiasis (HAT, also called sleeping sickness), a neglected tropical disease endemic to sub-Saharan Africa, is caused by the parasitesTrypanosoma brucei gambienseandT. brucei rhodesiense. Current drugs against this disease have significant limitations, including toxicity, increasing resistance, and/or a complicated parenteral treatment regimen. DB829 is a novel aza-diamidine that demonstrated excellent efficacy in mice infected withT. b. rhodesienseorT. b. bruceiparasites. The current study examined the pharmacokinetics,in vitroandin vivoactivity againstT. b. gambiense, and time of drug action of DB829 in comparison to pentamidine. DB829 showed outstandingin vivoefficacy in mice infected with parasites ofT. b. gambiensestrains, despite having higherin vitro50% inhibitory concentrations (IC50s) than againstT. b. rhodesiensestrain STIB900. A single dose of DB829 administered intraperitoneally (5 mg/kg of body weight) cured all mice infected with differentT. b. gambiensestrains. No cross-resistance was observed between DB829 and pentamidine inT. b. gambiensestrains isolated from melarsoprol-refractory patients. Compared to pentamidine, DB829 showed a greater systemic exposure when administered intraperitoneally, partially contributing to its improved efficacy. Isothermal microcalorimetry andin vivotime-to-kill studies revealed that DB829 is a slower-acting trypanocidal compound than pentamidine. A single dose of DB829 (20 mg/kg) administered intraperitoneally clears parasites from mouse blood within 2 to 5 days. In summary, DB829 is a promising preclinical candidate for the treatment of first- and second-stage HAT caused by bothTrypanosoma bruceisubspecies.

Download Full-text

Anti-Trypanosomal Proteasome Inhibitors Cure Hemolymphatic and Meningoencephalic Murine Infection Models of African Trypanosomiasis

Tropical Medicine and Infectious Disease ◽

10.3390/tropicalmed5010028 ◽

2020 ◽

Vol 5 (1) ◽

pp. 28 ◽

Cited By ~ 1

Author(s):

Srinivasa P S Rao ◽

Suresh B Lakshminarayana ◽

Jan Jiricek ◽

Marcel Kaiser ◽

Ryan Ritchie ◽

...

Keyword(s):

Trypanosoma Brucei ◽

Mouse Models ◽

Human African Trypanosomiasis ◽

Proteasome Inhibitors ◽

African Trypanosomiasis ◽

Single Digit ◽

Pharmacological Characterization ◽

Brain Infection

Current anti-trypanosomal therapies suffer from problems of longer treatment duration, toxicity and inadequate efficacy, hence there is a need for safer, more efficacious and ‘easy to use’ oral drugs. Previously, we reported the discovery of the triazolopyrimidine (TP) class as selective kinetoplastid proteasome inhibitors with in vivo efficacy in mouse models of leishmaniasis, Chagas Disease and African trypanosomiasis (HAT). For the treatment of HAT, development compounds need to have excellent penetration to the brain to cure the meningoencephalic stage of the disease. Here we describe detailed biological and pharmacological characterization of triazolopyrimidine compounds in HAT specific assays. The TP class of compounds showed single digit nanomolar potency against Trypanosoma brucei rhodesiense and Trypanosoma brucei gambiense strains. These compounds are trypanocidal with concentration-time dependent kill and achieved relapse-free cure in vitro. Two compounds, GNF6702 and a new analog NITD689, showed favorable in vivo pharmacokinetics and significant brain penetration, which enabled oral dosing. They also achieved complete cure in both hemolymphatic (blood) and meningoencephalic (brain) infection of human African trypanosomiasis mouse models. Mode of action studies on this series confirmed the 20S proteasome as the target in T. brucei. These proteasome inhibitors have the potential for further development into promising new treatment for human African trypanosomiasis.

Download Full-text

Neutralization of a Low Molecular Weight Heparin (LHN-1) and Conventional Heparin by Protamine Sulfate in Rats

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661675 ◽

1986 ◽

Vol 56 (03) ◽

pp. 318-322 ◽

Cited By ~ 18

Author(s):

V Diness ◽

P B Østergaard

Keyword(s):

Molecular Weight ◽

Low Molecular Weight Heparin ◽

Thrombus Formation ◽

Experimental Models ◽

Protamine Sulfate ◽

Low Molecular Weight ◽

Antithrombotic Effect ◽

Higher Doses

SummaryThe neutralization of a low molecular weight heparin (LHN-1) and conventional heparin (CH) by protamine sulfate has been studied in vitro and in vivo. In vitro, the APTT activity of CH was completely neutralized in parallel with the anti-Xa activity. The APTT activity of LHN-1 was almost completely neutralized in a way similar to the APTT activity of CH, whereas the anti-Xa activity of LHN-1 was only partially neutralized.In vivo, CH 3 mg/kg and LHN-1 7.2 mg/kg was given intravenously in rats. The APTT and anti-Xa activities, after neutralization by protamine sulfate in vivo, were similar to the results in vitro. In CH treated rats no haemorrhagic effect in the rat tail bleeding test and no antithrombotic effect in the rat stasis model was found at a protamine sulfate to heparin ratio of about 1, which neutralized APTT and anti-Xa activities. In LHN-1 treated rats the haemorrhagic effect was neutralized when APTT was close to normal whereas higher doses of protamine sulfate were required for neutralization of the antithrombotic effect. This probably reflects the fact that in most experimental models higher doses of heparin are needed to induce bleeding than to prevent thrombus formation. Our results demonstrate that even if complete neutralization of APTT and anti-Xa activities were not seen in LHN-1 treated rats, the in vivo effects of LHN-1 could be neutralized as efficiently as those of conventional heparin. The large fall in blood pressure caused by high doses of protamine sulfate alone was prevented by the prior injection of LHN-1.

Download Full-text

Antimicrobial peptides for the treatment of pulmonary tuberculosis, allies or foes?

Current Pharmaceutical Design ◽

10.2174/1381612824666180327162357 ◽

2018 ◽

Vol 24 (10) ◽

pp. 1138-1147

Author(s):

Bruno Rivas-Santiago ◽

Flor Torres-Juarez

Keyword(s):

Pulmonary Tuberculosis ◽

Antimicrobial Peptides ◽

Experimental Models ◽

New Drugs ◽

World Health ◽

Public Health Issue ◽

Health Organization ◽

Drug Resistant Strains

Tuberculosis is an ancient disease that has become a serious public health issue in recent years, although increasing incidence has been controlled, deaths caused by Mycobacterium tuberculosis have been accentuated due to the emerging of multi-drug resistant strains and the comorbidity with diabetes mellitus and HIV. This situation is threatening the goals of World Health Organization (WHO) to eradicate tuberculosis in 2035. WHO has called for the creation of new drugs as an alternative for the treatment of pulmonary tuberculosis, among the plausible molecules that can be used are the Antimicrobial Peptides (AMPs). These peptides have demonstrated remarkable efficacy to kill mycobacteria in vitro and in vivo in experimental models, nevertheless, these peptides not only have antimicrobial activity but also have a wide variety of functions such as angiogenesis, wound healing, immunomodulation and other well-described roles into the human physiology. Therapeutic strategies for tuberculosis using AMPs must be well thought prior to their clinical use; evaluating comorbidities, family history and risk factors to other diseases, since the wide function of AMPs, they could lead to collateral undesirable effects.

Download Full-text

Epithelial Organotypic Cultures: A Viable Model to Address Mechanisms of Carcinogenesis by Epitheliotropic Viruses

Current Topics in Medicinal Chemistry ◽

10.2174/1568026618666180410125850 ◽

2018 ◽

Vol 18 (4) ◽

pp. 246-255 ◽

Cited By ~ 1

Author(s):

Lara Termini ◽

Enrique Boccardo

Keyword(s):

Three Dimensional ◽

Cell Types ◽

Experimental Models ◽

Biological Research ◽

Specific Gene ◽

Specific Cell ◽

Organotypic Cultures ◽

Skin Physiology

In vitro culture of primary or established cell lines is one of the leading techniques in many areas of basic biological research. The use of pure or highly enriched cultures of specific cell types obtained from different tissues and genetics backgrounds has greatly contributed to our current understanding of normal and pathological cellular processes. Cells in culture are easily propagated generating an almost endless source of material for experimentation. Besides, they can be manipulated to achieve gene silencing, gene overexpression and genome editing turning possible the dissection of specific gene functions and signaling pathways. However, monolayer and suspension cultures of cells do not reproduce the cell type diversity, cell-cell contacts, cell-matrix interactions and differentiation pathways typical of the three-dimensional environment of tissues and organs from where they were originated. Therefore, different experimental animal models have been developed and applied to address these and other complex issues in vivo. However, these systems are costly and time consuming. Most importantly the use of animals in scientific research poses moral and ethical concerns facing a steadily increasing opposition from different sectors of the society. Therefore, there is an urgent need for the development of alternative in vitro experimental models that accurately reproduce the events observed in vivo to reduce the use of animals. Organotypic cultures combine the flexibility of traditional culture systems with the possibility of culturing different cell types in a 3D environment that reproduces both the structure and the physiology of the parental organ. Here we present a summarized description of the use of epithelial organotypic for the study of skin physiology, human papillomavirus biology and associated tumorigenesis.

Download Full-text

Experimental Models of Hepatocellular Carcinoma—A Preclinical Perspective

Cancers ◽

10.3390/cancers13153651 ◽

2021 ◽

Vol 13 (15) ◽

pp. 3651

Author(s):

Alexandru Blidisel ◽

Iasmina Marcovici ◽

Dorina Coricovac ◽

Florin Hut ◽

Cristina Adriana Dehelean ◽

...

Keyword(s):

Hepatocellular Carcinoma ◽

Molecular Mechanisms ◽

Prediction Models ◽

3D Models ◽

Experimental Models ◽

Health Concern ◽

Liver Carcinoma ◽

Tumor Type

Hepatocellular carcinoma (HCC), the most frequent form of primary liver carcinoma, is a heterogenous and complex tumor type with increased incidence, poor prognosis, and high mortality. The actual therapeutic arsenal is narrow and poorly effective, rendering this disease a global health concern. Although considerable progress has been made in terms of understanding the pathogenesis, molecular mechanisms, genetics, and therapeutical approaches, several facets of human HCC remain undiscovered. A valuable and prompt approach to acquire further knowledge about the unrevealed aspects of HCC and novel therapeutic candidates is represented by the application of experimental models. Experimental models (in vivo and in vitro 2D and 3D models) are considered reliable tools to gather data for clinical usability. This review offers an overview of the currently available preclinical models frequently applied for the study of hepatocellular carcinoma in terms of initiation, development, and progression, as well as for the discovery of efficient treatments, highlighting the advantages and the limitations of each model. Furthermore, we also focus on the role played by computational studies (in silico models and artificial intelligence-based prediction models) as promising novel tools in liver cancer research.

Download Full-text

Current Status of Putative Animal Sources of SARS-CoV-2 Infection in Humans: Wildlife, Domestic Animals and Pets

Microorganisms ◽

10.3390/microorganisms9040868 ◽

2021 ◽

Vol 9 (4) ◽

pp. 868

Author(s):

Max Maurin ◽

Florence Fenollar ◽

Oleg Mediannikov ◽

Bernard Davoust ◽

Christian Devaux ◽

...

Keyword(s):

Animal Species ◽

Biological Properties ◽

Experimental Models ◽

Current Data ◽

The Body ◽

Receptor Expression ◽

Current Status ◽

Susceptible Animal

SARS-CoV-2 is currently considered to have emerged from a bat coronavirus reservoir. However, the real natural cycle of this virus remains to be elucidated. Moreover, the COVID-19 pandemic has led to novel opportunities for SARS-CoV-2 transmission between humans and susceptible animal species. In silico and in vitro evaluation of the interactions between the SARS-CoV-2 spike protein and eucaryotic angiotensin-converting enzyme 2 (ACE2) receptor have tentatively predicted susceptibility to SARS-CoV-2 infection of several animal species. Although useful, these data do not always correlate with in vivo data obtained in experimental models or during natural infections. Other host biological properties may intervene such as the body temperature, level of receptor expression, co-receptor, restriction factors, and genetic background. The spread of SARS-CoV-2 also depends on the extent and duration of viral shedding in the infected host as well as population density and behaviour (group living and grooming). Overall, current data indicate that the most at-risk interactions between humans and animals for COVID-19 infection are those involving certain mustelids (such as minks and ferrets), rodents (such as hamsters), lagomorphs (especially rabbits), and felines (including cats). Therefore, special attention should be paid to the risk of SARS-CoV-2 infection associated with pets.

Download Full-text

Ocular Toxoplasmosis: Mechanisms of Retinal Infection and Experimental Models

Parasitologia ◽

10.3390/parasitologia1020007 ◽

2021 ◽

Vol 1 (2) ◽

pp. 50-60

Author(s):

Veronica Rodriguez Fernandez ◽

Giovanni Casini ◽

Fabrizio Bruschi

Keyword(s):

Ex Vivo ◽

Cell Damage ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Treatment Strategies ◽

Experimental Models ◽

Ocular Toxoplasmosis ◽

Cell Infection

Ocular toxoplasmosis (OT) is caused by the parasite Toxoplasma gondii and affects many individuals throughout the world. Infection may occur through congenital or acquired routes. The parasites enter the blood circulation and reach both the retina and the retinal pigment epithelium, where they may cause cell damage and cell death. Different routes of access are used by T. gondii to reach the retina through the retinal endothelium: by transmission inside leukocytes, as free parasites through a paracellular route, or after endothelial cell infection. A main feature of OT is the induction of an important inflammatory state, and the course of infection has been shown to be influenced by the host immunogenetics. On the other hand, there is evidence that the T. gondii phenotype also has an impact on the distribution of the pathology in different areas. Although considerable knowledge has been acquired on OT, a deeper knowledge of its mechanisms is necessary to provide new, more targeted treatment strategies. In particular, in addition to in vitro and in vivo experimental models, organotypic, ex vivo retinal explants may be useful in this direction.

Download Full-text