Novel Bile Acid-Dependent Mechanisms of Hepatotoxicity Associated with Tyrosine Kinase Inhibitors.

Drug-induced liver injury (DILI) is the leading cause of acute liver failure and a major concern in drug development. Altered bile acid homeostasis via inhibition of the bile salt export pump (BSEP) is one mechanism of DILI. Dasatinib, pazopanib, and sorafenib are tyrosine kinase inhibitors (TKIs) that competitively inhibit BSEP and increase serum biomarkers for hepatotoxicity in ∼25-50% of patients. However, the mechanism(s) of hepatotoxicity beyond competitive inhibition of BSEP are poorly understood. This study examined mechanisms of TKI-mediated hepatotoxicity associated with altered bile acid homeostasis. Dasatinib, pazopanib, and sorafenib showed bile acid-dependent toxicity at clinically relevant concentrations, based on the C-DILI assay using sandwich-cultured human hepatocytes (SCHH). Among several bile acid-relevant genes, cytochrome P450 (CYP) 7A1 mRNA was specifically upregulated by 6.2- to 7.8-fold (dasatinib) and 5.7- to 9.3-fold (pazopanib), compared with control, within 8 hours. This was consistent with increased total bile acid concentrations in culture medium up to 2.3-fold, and in SCHH up to 1.4-fold, compared with control, within 24 hours. Additionally, protein abundance of sodium taurocholate co-transporting polypeptide (NTCP) was increased up to 2.0-fold by these three TKIs. The increase in NTCP protein abundance correlated with increased function; dasatinib and pazopanib increased hepatocyte uptake clearance (CLuptake) of taurocholic acid, a probe bile acid substrate, up to 1.4-fold. In conclusion, upregulation of CYP7A1 and NTCP in SCHH constitute novel mechanisms of TKI-associated hepatotoxicity. SIGNIFICANCE STATEMENT: Understanding the mechanisms of hepatotoxicity associated with tyrosine kinase inhibitors (TKIs) is fundamental to development of effective and safe intervention therapies for various cancers. Data generated in sandwich-cultured human hepatocytes, an in vitro model of drug-induced hepatotoxicity, revealed that TKIs upregulate bile acid synthesis and alter bile acid uptake and excretion. These findings provide novel insights into additional mechanisms of bile acid-mediated drug-induced liver injury, an adverse effect that limits the use and effectiveness of TKI treatment in some cancer patients.

Protective effects of taxifolin on pazopanib-induced liver toxicity: an experimental rat model.

Pazopanib is a tyrosine kinase inhibitor that is generally used for the treatment of metastatic renal cell cancer and advanced soft tissue sarcoma. It can cause various degrees of hepatotoxicity. Our study aimed to investigate the effect of taxifolin on pazopanib-induced liver toxicity. A total of 18 rats were divided into three groups: the pazopanib (PP), pazopanib plus taxifolin (TPP), and control (C) group. Taxifolin was administered to the TPP (n=6) group with a dose of 50 mg/kg. Distilled water was orally admnistered to the C (n=6) and PP (n=6) groups as a solvent. Subsequently, pazopanib 200 mg/kg was administered to the TPP and PP groups via the stomach. This procedure was repeated once a day for four weeks. Then, all rats were sacrificed, and their livers were removed. Malondialdehyde (MDA), total glutathione (tGSH), total oxidant status (TOS), and total antioxidant status (TAS) levels were evaluated. MDA and TOS levels were higher in the PP group compared with the levels of the other parameters (P<0.001). tGSH and TAS levels were lower in the PP group than in the TPP and C groups (P<0.001), and the aspartate aminotransferase (AST), alanine aminotransferase (ALT), and lactate dehydrogenase (LDH) levels were higher. Furthermore, liver tissue damage, including hemorrhage, hydropic degeneration, and necrosis was observed in the PP group. Administration of taxifolin before pazopanib significantly improved degenerative changes. Our study demonstrated that the administration of taxifolin is significantly effective in preventing pazopanib-induced hepatotoxicity in rats.

Clinicopathological and immunohistochemical evaluation of lonidamine-entrapped lipid-polymer hybrid nanoparticles in treatment of benign prostatic hyperplasia: An experimental rat model.

Benign prostatic hyperplasia (BPH) is a progressive proliferative disease, the incidence of which is constantly increasing due to aging of population. In this research, a hexokinase-II enzyme inhibiting agent, lonidamine - the use of which is limited in BPH treatment due to high hepatic toxicity observed after three months of treatment - was selected as an active agent, based on its mechanism of action in treating BPH. The aim of this study was to evaluate in vivo therapeutic efficacy and hepatic toxicity of lipid-polymer hybrid nanoparticles of lonidamine in a rat BPH model created in rat prostates. After local injections of hybrid nanoparticles of lonidamine were administered to the rat prostates, hyperplasic structures of prostates were evaluated in terms of prostatic index values, immunohistochemical evaluations, and histopathological findings. Liver blood enzyme values were also determined to specify hepatic toxicity. Apoptosis was evaluated by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) reaction and histopathological methods to determine intravital degenerative destruction in liver. Through this study, lonidamine-loaded hybrid nanoparticles were found to reduce the hepatic toxicity and increase therapeutic efficiency of lonidamine. Therefore, lonidamine-entrapped hybrid nanoparticles may provide a promising, and very safe, drug delivery strategy in the treatment of BPH.

Synthetic Cannabinoids JWH-122 and THJ-2201 Disrupt Endocannabinoid-Regulated Mitochondrial Function and Activate Apoptotic Pathways as a Primary Mechanism of In Vitro Nephrotoxicity at In Vivo Relevant Concentrations.

The widespread recreational use of synthetic cannabinoids (SCBs) represents a major public health issue, as reports of intoxications and deaths following SCB use rapidly mount up. Specifically, a direct link between SCB use and acute kidney injury (AKI) has been established, although the pathophysiologic mechanisms remain undefined. Here we assessed the in vitro nephrotoxicity of 3 commonly detected and structurally distinct SCBs-AB-FUBINACA, JWH-122, and THJ-2201-in human proximal tubule cells (HK-2), to ascertain potential similarities and/or differences regarding their nephrotoxicity signatures. We showed that 2 of the 3 SCBs tested, namely JWH-122 and THJ-2201, at in vivo relevant concentrations (1 nM-1 µM), triggered apoptotic cell death pathways, mainly through a shared mechanism involving the deregulation of mitochondrial function (ie, with mitochondrial membrane hyperpolarization and increased intracellular ATP levels), as the primary molecular signature of nephrotoxicity mechanism. Noteworthy, no SCB affected cell viability (MTT reduction, lactate dehydrogenase release, Neutral Red inclusion). Use of the cannabinoid receptor (CBR) antagonists SR141716A and SR144528, as well as HEK293T cells, which do not express CBRs, confirmed the involvement of these receptors in SCB-mediated mitochondrial membrane hyperpolarization but not on other events, suggesting an off-target action regulating SCB-induced kidney cell death. Our results further strengthen the relevance of the endocannabinoid system in maintaining mitochondrial function in kidney cells, as we demonstrate that HK-2 incubation with CBR antagonists or inhibitors of endocannabinoid biosynthesis (ie, methyl arachydonyl fluorophosphonate, tetrahydrolipstatin) alone produced deleterious effects similar to those now reported for SCBs. Overall, SCB-induced nephrotoxicity seems to be mainly regulated at the mitochondrial level, but the specific mechanisms involved require further clarification.

Novel 4-(3-phenylpropionamido), 4-(2-phenoxyacetamido) and 4-(cinnamamido) substituted benzamides bearing the pyrazole or indazole nucleus: synthesis, biological evaluation and mechanism of action.

Based on some common structural features of known compounds interfering with p53 pathways and our previously synthesized benzamides, we synthesized new ethyl 5-(4-substituted benzamido)-1-phenyl-1H-pyrazole-4-carboxylates 26a-c, ethyl 5-(4-substituted benzamido)-1-(pyridin-2-yl)-1H-pyrazole-4-carboxylates 27a-c and N-(1H-indazol-6-yl)-4-substituted benzamides 31a,b bearing in the 4 position of the benzamido moiety the 2-phenylpropanamido or 2-phenoxyacetamido or cinnamamido groups. A preliminary test to evaluate the antiproliferative activity against human lung carcinoma H292 cells highlighted how compound 26c showed the best activity. This last was therefore selected for further studies with the aim to find the mechanism of action. Compound 26c induces intrinsic apoptotic pathway by activating p53 and is also able to activate TRAIL-inducing death pathway by promoting increase of DR4 and DR5 death receptors, downregulation of c-FLIPL and caspase-8 activation.

Inflammation and oxidative stress are key mediators in AKB48-induced neurotoxicity in vitro.

Synthetic cannabinoids were introduced into market in early 2000s; since these "legal highs" are dramatically popular among youth, it becomes a deadly problem. Synthetic cannabinoids have high affinity to cannabinoid receptors; leading to various clinical symptoms. AKB48 (Apinaca) has been classified as a third-generation synthetic cannabinoid for the first time in 2014. The toxicity profile of AKB48 is unclear due to little information that mainly obtained from clinical and forensic cases; however, it is believed to be similar with other psychoactive substances. Thus, we aimed to investigate the possible toxicity mechanisms of AKB48 in SH-SY5Y (human bone marrow neuroblastoma) cell line. IC50 value of AKB48 was calculated as 160.91 µM by MTT assay. AKB48 treatment enhanced (≥1.2-fold) the fluorescence intensity indicating increased reactive oxygen species production; however, glutathione levels did not changed in the range of 25-200 µM exposure concentrations. Cannabinoid type-1 receptor (CB1) expression was increased ≥15-fold in the range of 25-50 µM of AKB48, while cannabinoid type-2 receptor (CB2) did not expressed in SH-SY5Y cells. Interleukin-6 (IL-6) and tumor necrosis factor-α (TNF- α) were up-regulated with a dose-dependent manner, and the profiles were almost identical; however, mitogen-activated protein kinase 8 (MAPK 8) was only upregulated with 25 µM of AKB48 and nuclear factor kappa B (NF-ĸB) did not change. Our results should raise the concerns about the safety associated with synthetic cannabinoids uses.

Design, synthesis and evaluate of novel dual FGFR1 and HDAC inhibitors bearing an indazole scaffold.

Both histone deacetylase (HDAC) and fibroblast growth factor receptor (FGFR) are important targets for cancer therapy. Although combining dual HDAC pharmacophore with tyrosine kinase inhibitors (TKIs) had achieved a successful progress, dual HDAC/FGFR1 inhibitors haven't been reported yet. Herein, we designed a series of hybrids bearing 1H-indazol-3-amine and benzohydroxamic acids scaffold with scaffold hopping and molecular hybridization strategies. Among them, compound 7j showed the most potent inhibitory activity against HDAC6 with IC50 of 34 nM and exhibited the great inhibitory activities against a human breast cancer cell line MCF-7 with IC50 of 9 µM in vitro. Meanwhile, the compound also exhibited moderate FGFR1 inhibitory activities. This study provides new tool compounds for further exploration of dual HDAC/FGFR1 inhibition.

Quantifying the relationship between inhibition of VEGF receptor 2, drug-induced blood pressure elevation and hypertension.

BACKGROUND AND PURPOSE: Several anti-angiogenic cancer drugs that inhibit VEGF receptor (VEGFR) signalling for efficacy are associated with a 15-60% incidence of hypertension. Tyrosine kinase inhibitors (TKIs) that have off-target activity at VEGFR-2 may also cause blood pressure elevation as an undesirable side effect. Therefore, the ability to translate VEGFR-2 off-target potency into blood pressure elevation would be useful in development of novel TKIs. Here, we have sought to quantify the relationship between VEGFR-2 inhibition and blood pressure elevation for a range of kinase inhibitors. EXPERIMENTAL APPROACH: Porcine aortic endothelial cells overexpressing VEGFR-2 (PAE) were used to determine IC50 for VEGFR-2 phosphorylation. These IC50 values were compared with published reports of exposure attained during clinical use and the corresponding incidence of all-grade hypertension. Unbound average plasma concentration (Cav,u ) was selected to be the most appropriate pharmacokinetic parameter. The pharmacokinetic-pharmacodynamic (PKPD) relationship for blood pressure elevation was investigated for selected kinase inhibitors, using data derived either from clinical papers or from rat telemetry experiments. KEY RESULTS: All-grade hypertension was predominantly observed when the Cav,u was >0.1-fold of the VEGFR-2 (PAE) IC50 . Furthermore, based on the PKPD analysis, an exposure-dependent blood pressure elevation >1 mmHg was observed only when the Cav,u was >0.1-fold of the VEGFR-2 (PAE) IC50 . CONCLUSIONS AND IMPLICATIONS: Taken together, these data show that the risk of blood pressure elevation is proportional to the amount of VEGFR-2 inhibition, and a margin of >10-fold between VEGFR-2 IC50 and Cav,u appears to confer a minimal risk of hypertension.

Axitinib in the treatment of renal cell carcinoma: design, development, and place in therapy.

Since 2005, the approved first-line treatment of metastatic renal cell carcinoma consists in tyrosine kinase inhibitors (TKIs) targeting the vascular endothelial growth factor receptors (VEGFRs). Axitinib is an oral second-generation TKI and a potent VEGFR inhibitor with a half maximal inhibitory concentration for the VEGF family receptors 10-fold lower than other TKIs. Axitinib activity in renal cell carcinoma (RCC) patients has been studied in various settings and particularly as second-line treatment. In this setting, axitinib with clinically based dose escalation compared to sorafenib has demonstrated an improvement in progression-free survival in a randomized Phase III trial leading to US Food and Drug Administration approval. In the first-line setting, axitinib failed to demonstrate improved efficacy over sorafenib, but the field of RCC treatment is rapidly changing with novel TKIs as cabozantinib or the emergence of check point inhibitors as nivolumab and the place of axitinib in therapy is therefore challenged. In this review, we focus on axitinib pharmacological and clinical properties in RCC patients and discuss its place in the treatment of patients with RCC.

Use of early phenotypic in vivo markers to assess human relevance of an unusual rodent non-genotoxic carcinogen in vitro.

Foci of altered hepatocytes (FAH) are considered putative, pre-neoplastic lesions that can occur spontaneously in aging rodents, but can also be induced by chemicals or drugs. Progression of FAH to hepatocellular neoplasms has been reported repeatedly but increases in foci in rodents do not necessarily lead to tumors in carcinogenicity studies and the relevance for humans often remains unclear. Here we present the case of RG3487, a molecule which induced FAH and, later on, tumors in rats. Because the molecule was negative in genotoxicity assays it was classified as a non-genotoxic carcinogen. In order to assess the potential for liver tumor formation in humans, we analyzed treatment-induced changes in vivo to establish a possible mode of action (MoA). In vivo and in vitro gene expression analysis revealed that nuclear receptor signaling was unlikely to be the relevant MoA and no other known mechanism could be established. We therefore took an approach comparing phenotypic markers, including mRNA changes, proliferation and glycogen accumulation, in vitro using cells of different species to assess the human relevance of this finding. Since the alterations observed in rats were not seen in the liver of mice or dogs in vivo, we could validate the relevance of the cell models chosen by use of hepatocytes from these species in vitro. This ultimately allowed for a cross-species comparison, which suggested that the formation of FAH and liver tumors was rat specific and unlikely to translate to human. Our work showed that phenotypic species comparison in vitro is a useful approach for assessment of the human relevance of pre-clinical findings where no known mechanism can be established.

The Typical Metabolic Modifiers Conferring Improvement in Cancer Resistance.

BACKGROUND: Cancer metabolic reprogramming rekindles enthusiasm for the research of metabolic regulation in cancer drug resistance. A growing number of metabolic modifiers combined with cancer drugs obtain the expected efficacy in in vitro or in vivo studies, also in clinical trial studies, indicating a good potential of enhancing efficacy and reducing resistance. Hence, a comprehensive review on the attenuations of metabolic modifiers in cancer drug resistance is necessary for rational drug design and clinical cancer drug research. METHODS: Cancer drug resistance and cancer metabolic reprogramming were used as the key words to collect publications with reference value in bibliographic databases. Specifically, the focused question is the advances of metabolic modifiers on cancer resistance improvement. Figures and tables were applied to analyze the interventions in accordance with the inclusion criteria. RESULTS: This review summarized the advances of metabolic modifiers combined with cancer drugs in in vitro, in vivo and clinical trial studies, especially for cancer resistance improvement. The relationship between metabolic regulation and cancer resistance was elaborated, and the potential metabolic modifiers were embraced. Metabolic targets were also visualized in categorization in 4 figures and expatiated in 4 tables. Three typical metabolic modifiers, namely lonidamine, 2-DG and 3-BrPA, conferring attenuation to cancer resistance were elucidated systematically. CONCLUSION: Metabolic regulation is an intervention with targeted perturbation in a modest manner and reflects homeostasis balance. When combined with cancer drugs, the metabolic modifiers always show exciting potential with practical significance, enhancing activity or exerting synergism.

Antiangiogenic and Toxic Effects of Genistein, Usnic Acid, and Their Copper Complexes in Zebrafish Embryos at Different Developmental Stages.

Angiogenesis plays a major role in the normal embryonic development and in diseases such as cancer. Drugs that control angiogenesis are an alternative way to tackle this disease. The polyphenols usnic acid (3), genistein (5), and daidzein (6) were tested for antiangiogenic and unwanted effects in zebrafish embryos whose blood vessel system resembles that of mammals. The established tyrosine kinase inhibitors axitinib (1) and tyrphostin AG490 (2) were included for comparison. All compounds except 6 caused distinct antiangiogenic effects such as a concentration-dependent reduction of intersegmental vessels, dorsal longitudinal anastomotic vessels, subintestinal veins and secondary sprouts. As side effects, pericardial oedema and the impairment of blood flow were observed. Usnic acid (3), genistein (5) and Cu(II)-genisteinate (7) gave rise to a curvature of the spine. Compounds 5 and 7 also induced cell death in the head of the embryos at higher doses. All effects were more pronounced when the compounds had been applied at an early stage (24 hpf) rather than at 48 hpf. The copper complexes 4 and 7 showed a stronger antiangiogenic effect than the free ligands 3 and 5. The genistein complex 7 was antiangiogenic at doses so low that side effects were tolerable, and thus it may be a potential anticancer drug candidate.

Discovery of Indazoles as Potent, Orally Active Dual Neurokinin 1 Receptor Antagonists and Serotonin Transporter Inhibitors for the Treatment of Depression.

Combination studies of neurokinin 1 (NK1) receptor antagonists and serotonin-selective reuptake inhibitors (SSRIs) have shown promise in preclinical models of depression. Such a combination may offer important advantages over the current standard of care. Herein we describe the discovery and optimization of an indazole-based chemotype to provide a series of potent dual NK1 receptor antagonists/serotonin transporter (SERT) inhibitors to overcome issues of ion channel blockade. This effort culminated in the identification of compound 9, an analogue that demonstrated favorable oral bioavailability, excellent brain uptake, and robust in vivo efficacy in a validated depression model. Over the course of this work, a novel heterocycle-directed asymmetric hydrogenation was developed to facilitate installation of the key stereogenic center.

Mechanism of antineoplastic activity of lonidamine.

Lonidamine (LND) was initially introduced as an antispermatogenic agent. It was later found to have anticancer activity sensitizing tumors to chemo-, radio-, and photodynamic-therapy and hyperthermia. Although the mechanism of action remained unclear, LND treatment has been known to target metabolic pathways in cancer cells. It has been reported to alter the bioenergetics of tumor cells by inhibiting glycolysis and mitochondrial respiration, while indirect evidence suggested that it also inhibited l-lactic acid efflux from cells mediated by members of the proton-linked monocarboxylate transporter (MCT) family and also pyruvate uptake into the mitochondria by the mitochondrial pyruvate carrier (MPC). Recent studies have demonstrated that LND potently inhibits MPC activity in isolated rat liver mitochondria (Ki 2.5µM) and cooperatively inhibits l-lactate transport by MCT1, MCT2 and MCT4 expressed in Xenopus laevis oocytes with K0.5 and Hill coefficient values of 36-40µM and 1.65-1.85, respectively. In rat heart mitochondria LND inhibited the MPC with similar potency and uncoupled oxidation of pyruvate was inhibited more effectively (IC50~7µM) than other substrates including glutamate (IC50~20µM). LND inhibits the succinate-ubiquinone reductase activity of respiratory Complex II without fully blocking succinate dehydrogenase activity. LND also induces cellular reactive oxygen species through Complex II and has been reported to promote cell death by suppression of the pentose phosphate pathway, which resulted in inhibition of NADPH and glutathione generation. We conclude that MPC inhibition is the most sensitive anti-tumour target for LND, with additional inhibitory effects on MCT-mediated l-lactic acid efflux, Complex II and glutamine/glutamate oxidation.

Short-term treatment with VEGF receptor inhibitors induces retinopathy of prematurity-like abnormal vascular growth in neonatal rats.

Retinal arterial tortuosity and venous dilation are hallmarks of plus disease, which is a severe form of retinopathy of prematurity (ROP). In this study, we examined whether short-term interruption of vascular endothelial growth factor (VEGF) signals leads to the formation of severe ROP-like abnormal retinal blood vessels. Neonatal rats were treated subcutaneously with the VEGF receptor (VEGFR) tyrosine kinase inhibitors, KRN633 (1, 5, or 10 mg/kg) or axitinib (10 mg/kg), on postnatal day (P) 7 and P8. The retinal vasculatures were examined on P9, P14, or P21 in retinal whole-mounts stained with an endothelial cell marker. Prevention of vascular growth and regression of some preformed capillaries were observed on P9 in retinas of rats treated with KRN633. However, on P14 and P21, density of capillaries, tortuosity index of arterioles, and diameter of veins significantly increased in KRN633-treated rats, compared to vehicle (0.5% methylcellulose)-treated animals. Similar observations were made with axitinib-treated rats. Expressions of VEGF and VEGFR-2 were enhanced on P14 in KRN633-treated rat retinas. The second round of KRN633 treatment on P11 and P12 completely blocked abnormal retinal vascular growth on P14, but thereafter induced ROP-like abnormal retinal blood vessels by P21. These results suggest that an interruption of normal retinal vascular development in neonatal rats as a result of short-term VEGFR inhibition causes severe ROP-like abnormal retinal vascular growth in a VEGF-dependent manner. Rats treated postnatally with VEGFR inhibitors could serve as an animal model for studying the mechanisms underlying the development of plus disease.

Antiangiogenic therapy inhibits venous thrombus resolution.

OBJECTIVE: Venous thromboembolism is a common complication in patients with cancer, resulting in significant morbidity and mortality. Clinical studies suggest that the incidence of venous thromboembolic events increased after treatment of these patients with antiangiogenic agents. Thrombi resolve through a process of remodeling, involving the formation of microvascular channels within the thrombus. Our aim was to determine whether inhibiting angiogenesis affects venous thrombus resolution. APPROACH AND RESULTS: Thrombus was induced in the inferior vena cava of mice. These mice were treated with axitinib (50 mg/kg per day), 2-methoxyestradiol (2ME, 150 mg/kg per day), or vehicle control. Thrombus size, recanalization, neovascularization, inflammatory cell content, and collagen content were assessed after axitinib (days 3, 10, 17) and 2ME (day 10 only) treatment (n=6/group). Axitinib treatment resulted in reduced thrombus resolution (P<0.002) and vein recanalization (P<0.001) compared with vehicle-treated controls. This was associated with inhibition of organization as seen through reduced thrombus neovascularization (P<0.0001) and collagen (P<0.0001) content, as well as reduced macrophage accumulation in the thrombus (P<0.001). Treatment with a second antiangiogenic agent, 2ME, mirrored these findings, with a similar order of magnitude of effect of treatment over vehicle control in all of the parameters measured, with the exception of neutrophil content, which was significantly reduced after 2ME treatment but not affected by axitinib. CONCLUSIONS: Antiangiogenic therapy (using axitinib and 2ME) inhibits the resolution of venous thrombi, which could lead to persistent venous obstruction and the possibility of thrombus extension. This potential prolongation of venous occlusion by antiangiogenic agents should therefore be taken into consideration in trials of these agents and when managing the complications of venous thromboembolic events in patients with cancer.

The Davis-Beirut Reaction: a novel entry into 2H-indazoles and indazolones. Recent biological activity of indazoles.

A novel, easy method for the syntheses of richly diversified 2H-indazoles and indazolones, called the Davis-Beirut reaction, and other recent 2H-indazole synthetic routes are briefly reviewed. An update on the biological activity of indazoles is also surveyed.

Biological and chemical study of fused tri- and tetracyclic indazoles and analogues with important antiparasitic activity.

A series of fused tri- and tetracyclic indazoles and analogues compounds (NID) with potential antiparasitic effects were studied using voltamperometric and spectroscopic techniques. Nitroanion radicals generated by cyclic voltammetry were characterized by electron spin resonance spectroscopy (ESR) and their spectral lines were explained and analyzed using simulated spectra. In addition, we examined the interaction between radical species generated from nitroindazole derivatives and glutathione (GSH). Biological assays such as activity against Trypanosoma cruzi and cytotoxicity against macrophages were carried out. Finally, spin trapping and molecular modeling studies were also done in order to elucidate the potentials action mechanisms involved in the trypanocidal activity.

Synthesis of substituted 3-amino-N-phenyl-1H-indazole-1-carboxamides endowed with antiproliferative activity.

Several new N-phenyl-1H-indazole-1-carboxamides 1c-h and 4l,m were prepared by reacting phenyl isocyanate derivatives 3a,b with 3-amino-1H-indazole derivatives 2c,e,g or 1H-indazole 2l respectively. Chemical transformations of compounds 1a,b and 1g,h gave 3-acetamido-N-phenyl-1H-indazole-1-carboxamide derivatives 5a,b, and 3,5-diamino-N-phenyl-1H-indazole-1-carboxamide derivatives 4i,j respectively. Finally, 3,5-diacetamido-N-phenyl-1H-indazole-1-carboxamide derivatives 6a,b were prepared by acetylation of 4i,j. Some of synthesized compounds were evaluated for their in vitro antiproliferative activity against the full NCI tumor cell lines panel derived from nine clinically isolated cancer types (leukemia, non-small cell lung, colon, CNS, melanoma, ovarian, renal, prostate and breast). Compound 1c, the most active of the series, was able to inhibit cell growth showing GI(50) values in the 0.041-33.6 µM range, mean GI(50) 1.90 µM, being very effective against colon and melanoma cell lines. Cell cycle analysis in K562 cells showed that 1c causes a marked increase of cells in G0-G1 phase. Moreover, it increases the ratio between hypophosphorylated pRb and total pRb.

Inhibition of Akt inhibits growth of glioblastoma and glioblastoma stem-like cells.

A commonly activated signaling cascade in many human malignancies, including glioblastoma multiforme, is the Akt pathway. This pathway can be activated via numerous upstream alterations including genomic amplification of epidermal growth factor receptor, PTEN deletion, or PIK3CA mutations. In this study, we screened phosphatidylinositol 3-kinase/Akt small-molecule inhibitors in an isogenic cell culture system with an activated Akt pathway secondary to a PIK3CA mutation. One small molecule, A-443654, showed the greatest selective inhibition of cells with the mutant phenotype. Based on these findings, this inhibitor was screened in vitro against a panel of glioblastoma multiforme cell lines. All cell lines tested were sensitive to A-443654 with a mean IC(50) of approximately 150 nmol/L. An analogue of A-443654, methylated at a region that blocks Akt binding, was on average 36-fold less active. Caspase assays and dual flow cytometric analysis showed an apoptotic mechanism of cell death. A-443654 was further tested in a rat intracranial model of glioblastoma multiforme. Animals treated intracranially with polymers containing A-443654 had significantly extended survival compared with control animals; animals survived 79% and 43% longer than controls when A-443654-containing polymers were implanted simultaneously or in a delayed fashion, respectively. This small molecule also inhibited glioblastoma multiforme stem-like cells with similar efficacy compared with traditionally cultured glioblastoma multiforme cell lines. These results suggest that local delivery of an Akt small-molecule inhibitor is effective against experimental intracranial glioma, with no observed resistance to glioblastoma multiforme cells grown in stem cell conditions.