Preclinical Studies Evaluating Subacute Toxicity and Therapeutic Efficacy of LQB-118 in Experimental Visceral Leishmaniasis.

Visceral leishmaniasis (VL) is the most severe form of leishmaniasis and is the second major cause of death by parasites, after malaria. The arsenal of drugs against leishmaniasis is small, and each has a disadvantage in terms of toxicity, efficacy, price, or treatment regimen. Our group has focused on studying new drug candidates as alternatives to current treatments. The pterocarpanquinone LQB-118 was designed and synthesized based on molecular hybridization, and it exhibited antiprotozoal and anti-leukemic cell line activities. Our previous work demonstrated that LQB-118 was an effective treatment for experimental cutaneous leishmaniasis. In this study, we observed that treatment with 10 mg/kg of body weight/day LQB-118 orally inhibited the development of hepatosplenomegaly with a 99% reduction in parasite load. An in vivo toxicological analysis showed no change in the clinical, biochemical, or hematological parameters. Histologically, all of the analyzed organs were normal, with the exception of the liver, where focal points of necrosis with leukocytic infiltration were observed at treatment doses 5 times higher than the therapeutic dose; however, these changes were not accompanied by an increase in transaminases. Our findings indicate that LQB-118 is effective at treating different clinical forms of leishmaniasis and presents no relevant signs of toxicity at therapeutic doses; thus, this framework is demonstrated suitable for developing promising drug candidates for the oral treatment of leishmaniasis.

LQB-118, an orally active pterocarpanquinone, induces selective oxidative stress and apoptosis in Leishmania amazonensis.

OBJECTIVES: The pterocarpanquinone LQB-118, previously demonstrated to be effective in vivo via oral delivery, was investigated for its mechanism in selective parasite killing. METHODS: Oxidative stress in Leishmania amazonensis was analysed by evaluating reactive oxygen species (ROS) production (2',7'-dichlorodihydrofluorescein diacetate) and the loss of mitochondrial membrane potential (ΔΨm) using rhodamine, JC-1 and MitoCapture. Ultrastructural analysis was performed using transmission electron microscopy (TEM). DNA fragmentation was evaluated using terminal deoxyribonucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL). RESULTS: Treatment with LQB-118 induced ROS production in the promastigotes of L. amazonensis in a concentration-dependent manner for the first 4 h and was sustained for 24 h. TEM analysis revealed several alterations typical of apoptosis. Promastigotes presented a reduction of ΔΨm after 24 h of incubation with 2.5 µM (18.7%), 5 µM (63.7%) or 10 µM (70.7%) LQB-118. A sub-G0/G1 cell cycle phenotype was observed in 21%-83% of the promastigotes incubated with 1.25-10 µM LQB-118. Concentration-dependent DNA fragmentation was observed in promastigotes treated with 2.5-10 µM LQB-118, and selective DNA fragmentation was observed in intracellular amastigotes after 72 h with 2.5 µM treatment. CONCLUSIONS: Our results suggest that LQB-118 selectively induces ROS-triggered and mitochondria-dependent apoptosis in this parasite.

Effects of anti-Leishmania compounds in the behavior of the sand fly vector Lutzomyia longipalpis.

BACKGROUND: Leishmaniasis is an infectious parasitic disease caused by pathogens of the genus Leishmania transmitted through the bite of adult female sand flies. To reduce case numbers, it is necessary to combine different control approaches, especially those aimed at the sand fly vectors. Innovative forms of control with the use of attractive sugar baits explored the fact that adult sand flies need to feed on sugars of plant origin. Leishmania parasites develop in the gut of sand flies, interacting with the sugars in the diet of adults. Recent studies have shown that sugar baits containing plant-derived compounds can reduce sand fly survival, the number of parasites per gut, and the percentage of infected sand flies. Several synthetic compounds produced from naphthoquinones and pterocarpans have anti-parasitic activity on Leishmania amazonensis and/or Leishmania infantum in cell culture. This work aimed to assess the inclusion of these compounds in sugar baits for blocking transmission, targeting the development of the Leishmania parasite inside the sand fly vector. RESULTS: We evaluated the attractant or repellent properties of these compounds, as well as of the reference compound N,N'-diethyl-m-toluamide (DEET), in sugar baits. We also observed changes in feeding preference caused by these compounds, looking for anti-feeding or stimulation of ingestion. Pterocarpanquinone L4 and pentamidine showed attractant and repellent properties, respectively. CONCLUSION: Based on the effects in feeding preference and intake volume, pterocarpanquinone L6, and the pyrazole-derived compound P8 were chosen as the most promising compounds for the future development of anti-Leishmania sugar baits. © 2022 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Novel Ru(II)-bipyridine/phenanthroline-lapachol complexes as potential anti-cancer agents.

For the first time, we herein report on the syntheses of two new Ru(II)/bipyridine/phenanthroline complexes containing lapachol as ligand: complex (1), [Ru (bipy)2(Lap)]PF6 and complex (2), [Ru(Lap)(phen)2]PF6, where bipy = 2,2'-bipyridine and ph en = 1,10-phenanthroline; Lap = lapachol (2-hydroxy-3-(3-methylbut-2-en-1- yl)naphthalene-1,4-dione). The complexes were synthesized and characterized by elemental analyses, molar conductivity, mass spectrometry, ultraviolet-visible and infrared spectroscopies, nuclear magnetic resonance (1H, 13C), and single crystal X-ray diffraction, for complex (2). In addition, in vitro cytotoxicity was tested against six cancer cells: A549 (lung carcinoma); DU-145 (human prostate carcinoma); HepG2 (human hepatocellular carcinoma), PC-3 (human prostate adenocarcinoma); MDA-MB-231 (human breast adenocarcinoma); Caco-2 (human colorectal adenocarcinoma), and against two non-cancer cells, FGH (human gingival normal fibroblasts) and PNT-2 (prostate epithelial cells). Complex (1) was slightly more toxic and selective than complex (2) for all cell lines, except against the A549 cells, where (2) was more potent than complex (1). The complexes induced an increase in the reactive oxygen species, and the co-treatment with N-acetyl-L-cysteine remarkably suppressed the ROS generation and prevented the reduction of cell viability, suggesting that the cytotoxicity of the complexes is related to the ROS-mediated pathway. Further studies indicated that the complexes may bind to DNA via minor groove interaction. Our studies also revealed that free Lap induces gene mutations in Salmonella Typhimurium, nevertheless, the complexes demonstrated the absence of genotoxicity by the Ames test. The present study provides a relevant contribution to understanding the anti-cancer potential and genetic toxicological events of new ruthenium complexes containing the lapachol molecule as a ligand.

Effectiveness of the local or oral delivery of the novel naphthopterocarpanquinone LQB-118 against cutaneous leishmaniasis.

OBJECTIVES: This paper describes the antileishmanial properties of LQB-118, a new compound designed by molecular hybridization, orally active in Leishmania amazonensis-infected BALB/c mice. METHODS: In vitro antileishmanial activity was determined in L. amazonensis-infected macrophages. For in vivo studies, LQB-118 was administered intralesionally (15 µg/kg/day, five times a week), intraperitoneally (4.5 mg/kg/day, five times a week) or orally (4.5 mg/kg/day, five times a week) to L. amazonensis-infected BALB/c mice throughout experiments lasting 85 or 105 days. At the end of the experiments, serum levels of alanine aminotransferase, aspartate aminotransferase and creatinine were measured as toxicological parameters. RESULTS: LQB-118 was active against intracellular amastigotes of L. amazonensis [50% inhibitory concentration (IC(50)) 1.4 µM] and significantly less so against macrophages (IC(50) 18.5 µM). LQB-118 administered intralesionally, intraperitoneally or orally was found to control both lesion and parasite growth in L. amazonensis-infected BALB/c mice, without altering serological markers of toxicity. CONCLUSIONS: These results demonstrate that the molecular hybridization of a naphthoquinone core to pterocarpan yielded a novel antileishmanial compound that was locally and orally active in an experimental cutaneous leishmaniasis model.

The pterocarpanquinone LQB­118 compound induces apoptosis of cytarabine­resistant acute myeloid leukemia cells.

Acute myeloid leukemia (AML) is a complex hematological disorder characterized by blockage of differentiation and high proliferation rates of myeloid progenitors. Anthracycline and cytarabine­based therapy has remained the standard treatment for AML over the last four decades. Although this treatment strategy has increased survival rates, patients often develop resistance to these drugs. Despite efforts to understand the mechanisms underlying cytarabine resistance, there have been few advances in the field. The present study developed an in vitro AML cell line model resistant to cytarabine (HL­60R), and identified chromosomal aberrations by karyotype evaluation and potential molecular mechanisms underlying chemoresistance. Cytarabine decreased cell viability, as determined by MTT assay, and induced cell death and cell cycle arrest in the parental HL­60 cell line, as revealed by Annexin V/propidium iodide (PI) staining and PI DNA incorporation, respectively, whereas no change was observed in the HL­60R cell line. In addition, the HL­60R cell line exhibited a higher tumorigenic capacity in vivo compared with the parental cell line. Notably, no reduction in tumor volume was detected in mice treated with cytarabine and inoculated with HL­60R cells. In addition, western blotting revealed that the protein expression levels of Bcl­2, X­linked inhibitor of apoptosis protein (XIAP) and c­Myc were upregulated in HL­60R cells compared with those in HL­60 cells, along with predominant nuclear localization of the p50 and p65 subunits of NF­κB in HL­60R cells. Furthermore, the antitumor effect of LQB­118 pterocarpanquinone was investigated; this compound induced apoptosis, a reduction in cell viability and a decrease in XIAP expression in cytarabine­resistant cells. Taken together, these data indicated that acquired cytarabine resistance in AML was a multifactorial process, involving chromosomal aberrations, and differential expression of apoptosis and cell proliferation signaling pathways. Furthermore, LQB­118 could be a potential alternative therapeutic approach to treat cytarabine­resistant leukemia cells.

LQB­118 compound inhibits migration and induces cell death in glioblastoma cells.

Glioblastoma (GBM) is the most frequent malignant brain tumor. It represents the most aggressive astrocytoma with an overall survival of 14 months. Despite improvements in surgery techniques, radio­ and chemotherapy, most patients present treatment resistance, recurrence and disease progression. Therefore, development of effective alternative therapies is essential to overcome treatment failure. The purpose of the study was to evaluate the antitumoral activity of the synthetic compound LQB­118, in vitro. Monolayer and three­dimensional (3D) cell culture systems of human­derived GBM cell lines were used to evaluate the effect of LQB­118 on cell viability, cell death and migration. LQB­118 reduced cell viability as determined by MTT and trypan blue exclusion assays and promoted apoptosis in monolayer cell lines with an intrinsic temozolomide (TMZ)­resistance profile. In 3D culture models, LQB­118 reduced cell viability as evaluated by APH assay and inhibited cell migration while the TMZ resistance profile was maintained. Moreover, LQB­118 reduced p38 and AKT expression and phosphorylation, whereas it reduced only the phosphorylated ERK1/2 form. LQB­118 reduced p38 and NRF2 expression, an axis that is associated with TMZ resistance, revealing a mechanism to overcome resistance. LQB­118 also demonstrated an additional effect when combined with ionizing radiation and cisplatin. In conclusion, the present data demonstrated that LQB­118 maintained its effectiveness in a 3D cell conformation, which shares more similarities with the tumor mass. LQB­118 is a promising agent for GBM treatment as monotherapy and associated with radiotherapy or cisplatin. Its effect is associated with inhibition of GBM­related survival signaling pathways.

The pterocarpanquinone LQB 118 inhibits inflammation triggered by zymosan in vivo and in vitro.

LQB 118, a hydride molecule, has been described as an antineoplastic and antiparasitic drug. Recently, LQB118 was also shown to display anti-inflammatory properties using an LPS-induced lung inflammation model. However, LQB 118 effects on the inflammatory response induced by zymosan has not been demonstrated. In this study, swiss mice were LQB 118 intraperitoneally (i.p.) treated and zymosan was used to induce peritoneal inflammation. Peritoneal fluid was collected and used for cell counting and proinflammatory cytokines quantification (IL-1ß, IL-6, and TNF-α) by immunoenzymatic assay (ELISA). For in vitro studies, peritoneal macrophages zymosan-stimulated were used. Results demonstrated that LQB 118 treatment reduced polymorphonuclear cell migration and TNF-α, IL-1ß, and IL-6 levels in the peritoneal cavity. In macrophages, LQB 118 treatment display no cytotoxic effect and is also able to reduce cytokines levels. To investigate LQB 118 putative mechanism of action, TLR2, CD69, and P-p38 MAPK expression were evaluated. LQB 118 treatment reduced CD69 expression and p38 phosphorylation induced by zymosan. Furthermore, LQB 118 was able to negatively modulate TLR2 expression in the presence of inflammatory stimulus. Thus, our study provide new evidences for the mechanisms related to the anti-inflammatory effect of LQB 118 in vivo and in vitro.

Second-generation pterocarpanquinones: synthesis and antileishmanial activity.

BACKGROUND: Despite the development of new therapies for leishmaniasis, among the 200 countries or territories reporting to the WHO, 87 were identified as endemic for Tegumentary Leishmaniasis and 75 as endemic for Visceral Leishmaniasis. The identification of antileishmanial drug candidates is essential to fill the drug discovery pipeline for leishmaniasis. In the hit molecule LQB-118 selected, the first generation of pterocarpanquinones was effective and safe against experimental visceral and cutaneous leishmaniasis via oral delivery. In this paper, we report the synthesis and antileishmanial activity of the second generation of pterocarpanoquinones. METHODS: The second generation of pterocarpanquinones 2a-f was prepared through a palladium-catalyzed oxyarylation of dihydronaphtalen and chromens with iodolawsone, easily prepared by iodination of lawsone. The spectrum of antileishmanial activity was evaluated in promastigotes and intracellular amastigotes of L. amazonensis, L. braziliensis, and L. infantum. Toxicity was assessed in peritoneal macrophages and selective index calculated by CC50/IC50. Oxidative stress was measured by intracellular ROS levels and mitochondrial membrane potential in treated cells. RESULTS: In this work, we answered two pertinent questions about the structure of the first-generation pterocarpanquinones: the configuration and positions of rings B (pyran) and C (furan) and the presence of oxygen in the B ring. When rings B and C are exchanged, we noted an improvement of the activity against promastigotes and amastigotes of L. amazonensis and promastigotes of L. infantum. As to the oxygen in ring B of the new generation, we observed that the oxygenated compound 2b is approximately twice as active against L. braziliensis promastigotes than its deoxy derivative 2a. Another modification that improved the activity was the addition of the methylenedioxy group. A variation in the susceptibility among species was evident in the clinically relevant form of the parasite, the intracellular amastigote. L. amazonensis was the species most susceptible to novel derivatives, whilst L. infantum was resistant to most of them. The pterocarpanoquinones (2b and 2c) that possess the oxygen atom in ring B showed induction of increased ROS production. CONCLUSIONS: The data presented indicate that the pterocarpanoquinones are promising compounds for the development of new leishmanicidal agents.

Further evidence that naphthoquinone inhibits Toxoplasma gondii growth in vitro.

Toxoplasmosis is a widely disseminated disease caused by Toxoplasma gondii, an intracellular protozoan parasite. Standard treatment causes many side effects, such as depletion of bone marrow cells, skin rashes and gastrointestinal implications. Therefore, it is necessary to find chemotherapeutic alternatives for the treatment of this disease. It was shown that a naphthoquinone derivative compound is active against T. gondii, RH strain, with an IC50 around 2.5 µM. Here, three different naphthoquinone derivative compounds with activity against leukemia cells and breast carcinoma cell were tested against T. gondii (RH strain) infected LLC-MK2 cell line. All the compounds were able to inhibit parasite growth in vitro, but one of them showed an IC50 activity below 1 µM after 48 h of treatment. The compounds showed low toxicity to the host cell. In addition, these compounds were able to induce tachyzoite-bradyzoite conversion confirmed by morphological changes, Dolichus biflorus lectin cyst wall labeling and characterization of amylopectin granules in the parasites by electron microscopy analysis using the Thierry technique. Furthermore, the compounds induced alterations on the ultrastructure of the parasite. Taken together, our results point to the naphthoquinone derivative (LQB 151) as a potential compound for the development of new drugs for the treatment of toxoplasmosis.

A new type of pterocarpanquinone that affects Toxoplasma gondii tachyzoites in vitro.

Toxoplasma gondii, the agent of Toxoplasmosis, is an obligate intracellular protozoan able to infect a wide range of vertebrate cells, including nonprofessional and professional phagocytes. Therefore, drugs must have intracellular activities in order to control this parasite. The most common therapy for Toxoplasmosis is the combination of sulfadiazine and pyrimethamine. This treatment is associated with adverse reactions, thus, the development of new drugs is necessary. In previous studies, naphthoquinone derivatives showed anti-cancer activity functioning as agents capable of acting on groups of DNA, preventing cancer cells duplication. These derivatives also display anti-parasitic activity against Plasmodium falciparum and Leishmania amazonensis. The derivative pterocarpanquinone tested in this work resulted from the molecular hybridization between pterocarpans and naphtoquinone that presents anti-tumoral and anti-parasitic activities of lapachol. The aim of this work was to determine if this derivative is able to change T. gondii growth within LLC-MK2 cells. The drug did not arrest host cell growth, but was able to decrease the infection index of T. gondii with an IC(50) of 2.5 µM. Scanning and transmission electron microscopy analysis showed morphological changes of parasites including membrane damage. The parasite that survived tended to encyst as seen by Dolichos biflorus lectin staining and Bag-1 expression. These results suggest that pterocarpanquinones are drugs potentially important for the killing and encystment of T. gondii.