Repurposing mebendazole against triple-negative breast cancer CNS metastasis.

PURPOSE: Triple-negative breast cancer (TNBC) often metastasizes to the central nervous system (CNS) and has the highest propensity among breast cancer subtypes to develop leptomeningeal disease (LMD). LMD is a spread of cancer into leptomeningeal space that speeds up the disease progression and severely aggravates the prognosis. LMD has limited treatment options. We sought to test whether the common anti-helminthic drug mebendazole (MBZ) may be effective against murine TNBC LMD. METHODS: A small-molecule screen involving TNBC cell lines identified benzimidazoles as potential therapeutic agents for further study. In vitro migration assays were used to evaluate cell migration capacity and the effect of MBZ. For in vivo testing, CNS metastasis was introduced into BALB/c athymic nude mice through internal carotid artery injections of brain-tropic MDA-MB-231-BR or MCF7-BR cells. Tumor growth and spread was monitored by bioluminescence imaging and immunohistochemistry. MBZ was given orally at 50 and 100 mg/kg doses. MBZ bioavailability was assayed by mass spectrometry. RESULTS: Bioinformatic analysis and migration assays revealed higher migratory capacity of TNBC compared to other breast cancer subtypes. MBZ effectively slowed down migration of TNBC cell line MDA-MB-231 and its brain tropic derivative MDA-MB-231-BR. In animal studies, MBZ reduced leptomeningeal spread, and extended survival in brain metastasis model produced by MDA-MB-231-BR cells. MBZ did not have an effect in the non-migratory MCF7-BR model. CONCLUSIONS: We demonstrated that MBZ is a safe and effective oral agent in an animal model of TNBC CNS metastasis. Our findings are concordant with previous efforts involving MBZ and CNS pathology and support the drug's potential utility to slow down leptomeningeal spread.

Repurposing mebendazole against triple-negative breast cancer leptomeningeal disease.

Purpose: Triple-negative breast cancer (TNBC) is an aggressive subtype that often metastasizes to the brain. Leptomeningeal disease (LMD), a devastating brain metastasis common in TNBC, has limited treatment options. We sought to test whether the common anti-helminthic drug mebendazole (MBZ) may be effective against murine TNBC LMD. Methods: A small-molecule screen involving TNBC cell lines identified benzimidazoles as potential therapeutic agents for further study. In vitro migration assays were used to evaluate cell migration capacity and the effect of MBZ. For in vivo testing, LMD was introduced into BALB/c athymic nude mice through internal carotid artery injections of brain-tropic MDA-MB-231-BR or MCF7-BR cells. Tumor growth and spread was monitored by bioluminescence imaging. MBZ was given orally at 50 and 100 mg/kg doses. MBZ bioavailability was assayed by mass spectrometry. Results: Bioinformatic analysis and migration assays revealed higher migratory capacity of TNBC compared to other breast cancer subtypes. MBZ effectively slowed down migration of TNBC cell line MDA-MB-231 and its brain tropic derivative MDA-MB-231-BR. In animal studies, MBZ reduced tumor growth and extended survival in the LMD model produced by MDA-MB-231-BR cells. MBZ did not have an effect in the non-migratory MCF7-BR model. Conclusions: We demonstrated that MBZ is a safe and effective oral agent in an animal model of TNBC LMD. Our findings are concordant with previous efforts involving MBZ and central nervous system pathology and further support the drug's potential utility as an alternative therapeutic for TNBC LMD.

Regeneration of neuromuscular synapses after acute and chronic denervation by inhibiting the gerozyme 15-prostaglandin dehydrogenase.

To date, there are no approved treatments for the diminished strength and paralysis that result from the loss of peripheral nerve function due to trauma, heritable neuromuscular diseases, or aging. Here, we showed that denervation resulting from transection of the sciatic nerve triggered a marked increase in the prostaglandin-degrading enzyme 15-hydroxyprostaglandin dehydrogenase (15-PGDH) in skeletal muscle in mice, providing evidence that injury drives early expression of this aging-associated enzyme or gerozyme. Treating mice with a small-molecule inhibitor of 15-PGDH promoted regeneration of motor axons and formation of neuromuscular synapses leading to an acceleration in recovery of force after an acute nerve crush injury. In aged mice with chronic denervation of muscles, treatment with the 15-PGDH inhibitor increased motor neuron viability and restored neuromuscular junctions and function. These presynaptic changes synergized with previously reported muscle tissue remodeling to result in a marked increase in the strength of aged muscles. We further found that 15-PGDH aggregates defined the target fibers that are histopathologic hallmarks of human neurogenic myopathies, suggesting that the gerozyme may be involved in their etiology. Our data suggest that inhibition of 15-PGDH may constitute a therapeutic strategy to physiologically boost prostaglandin E2, restore neuromuscular connectivity, and promote recovery of strength after acute or chronic denervation due to injury, disease, or aging.

Identification of Widespread Antibiotic Exposure in Patients With Cholera Correlates With Clinically Relevant Microbiota Changes.

BACKGROUND: A first step to combating antimicrobial resistance in enteric pathogens is to establish an objective assessment of antibiotic exposure. Our goal was to develop and evaluate a liquid chromatography-ion trap mass spectrometry (LC/MS) method to determine antibiotic exposure in patients with cholera. METHODS: A priority list for targeted LC/MS was generated from medication-vendor surveys in Bangladesh. A study of patients with and those without cholera was conducted to collect and analyze paired urine and stool samples. RESULTS: Among 845 patients, 11% (90) were Vibrio cholerae positive; among these 90 patients, analysis of stool specimens revealed ≥1 antibiotic in 86% and ≥2 antibiotics in 52%. Among 44 patients with cholera and paired urine and stool specimens, ≥1 antibiotic was detected in 98% and ≥2 antibiotics were detected in 84%, despite 55% self-reporting medication use. Compared with LC/MS, a low-cost antimicrobial detection bioassay lacked a sufficient negative predictive value (10%; 95% confidence interval, 6%-16%). Detection of guideline-recommended antibiotics in stool specimens did (for azithromycin; P = .040) and did not (for ciprofloxacin) correlate with V. cholerae suppression. A nonrecommended antibiotic (metronidazole) was associated with decreases in anaerobes (ie, Prevotella organisms; P < .001). CONCLUSION: These findings suggest that there may be no true negative control group when attempting to account for antibiotic exposure in settings like those in this study.

Vitamin D supplementation decreases serum 27-hydroxycholesterol in a pilot breast cancer trial.

PURPOSE: 27-hydroxycholesterol (27HC), an endogenous selective estrogen receptor modulator (SERM), drives the growth of estrogen receptor-positive (ER+) breast cancer. 1,25-dihydroxyvitamin D (1,25(OH)2D), the active metabolite of vitamin D, is known to inhibit expression of CYP27B1, which is very similar in structure and function to CYP27A1, the synthesizing enzyme of 27HC. Therefore, we hypothesized that 1,25(OH)2D may also inhibit expression of CYP27A1, thereby reducing 27HC concentrations in the blood and tissues that express CYP27A1, including breast cancer tissue. METHODS: 27HC, 25-hydroxyvitamin D (25OHD), and 1,25(OH)2D were measured in sera from 29 breast cancer patients before and after supplementation with low-dose (400 IU/day) or high-dose (10,000 IU/day) vitamin D in the interval between biopsy and surgery. RESULTS: A significant increase (p = 4.3E-5) in 25OHD and a decrease (p = 1.7E-1) in 27HC was observed in high-dose versus low-dose vitamin D subjects. Excluding two statistical outliers, 25OHD and 27HC levels were inversely correlated (p = 7.0E-3). CONCLUSIONS: Vitamin D supplementation can decrease circulating 27HC of breast cancer patients, likely by CYP27A1 inhibition. This suggests a new and additional modality by which vitamin D can inhibit ER+ breast cancer growth, though a larger study is needed for verification.

Reduced carboxylesterase 1 is associated with endothelial injury in methamphetamine-induced pulmonary arterial hypertension.

Pulmonary arterial hypertension is a complication of methamphetamine use (METH-PAH), but the pathogenic mechanisms are unknown. Given that cytochrome P450 2D6 (CYP2D6) and carboxylesterase 1 (CES1) are involved in metabolism of METH and other amphetamine-like compounds, we postulated that loss of function variants could contribute to METH-PAH. Although no difference in CYP2D6 expression was seen by lung immunofluorescence, CES1 expression was significantly reduced in endothelium of METH-PAH microvessels. Mass spectrometry analysis showed that healthy pulmonary microvascular endothelial cells (PMVECs) have the capacity to both internalize and metabolize METH. Furthermore, whole exome sequencing data from 18 METH-PAH patients revealed that 94.4% of METH-PAH patients were heterozygous carriers of a single nucleotide variant (SNV; rs115629050) predicted to reduce CES1 activity. PMVECs transfected with this CES1 variant demonstrated significantly higher rates of METH-induced apoptosis. METH exposure results in increased formation of reactive oxygen species (ROS) and a compensatory autophagy response. Compared with healthy cells, CES1-deficient PMVECs lack a robust autophagy response despite higher ROS, which correlates with increased apoptosis. We propose that reduced CES1 expression/activity could promote development of METH-PAH by increasing PMVEC apoptosis and small vessel loss.

Inhibition of Mycobacterium tuberculosis strains H37Rv and MDR MS-115 by a new set of C5 modified pyrimidine nucleosides.

Two sets of pyrimidine nucleoside derivatives bearing extended alkyloxymethyl or alkyltriazolidomethyl substituents at position 5 of the nucleobase were synthesized and evaluated as potential antituberculosis agents. The impact of modifications at 3'- and 5'-positions of the carbohydrate moiety on the antimycobacterial activity and cytotoxicity was studied. The highest effect was shown for 5-dodecyloxymethyl-2'-deoxyuridine, 5-decyltriazolidomethyl-2'-deoxyuridine, and 5-dodecyltriazolidomethyl-2'-deoxycytidine. They effectively inhibited the growth of two Mycobacterium tuberculosis strains in vitro, laboratory H37Rv (MIC99=20, 10, and 20µg/mL, respectively) and clinical MDR MS-115 resistant to five top antituberculosis drugs (МIC99=50, 10, and 10µg/mL, respectively).

The synthesis and antituberculosis activity of 5'-nor carbocyclic uracil derivatives.

A series of new carbocyclic uracil derivatives were synthesized and evaluated as potential antituberculosis agents. Racemic 1-[4'-hydroxy-2'-cyclopenten-1'-yl]-5-tetradecynyluracil completely inhibited the growth of Mycobacterium tuberculosis H37Rv in vitro at a concentration of 10 µg/ml. Individual (+) and (-) isomers of the above uracil derivative were isolated and showed the same level of activity against two strains of Mycobacterium tuberculosis: laboratory sensitive (H37Rv) and clinical resistant to five top antituberculosis drugs (MS-115).

Neural precursor cell-expressed developmentally down-regulated protein 4-2 (Nedd4-2) regulation by 14-3-3 protein binding at canonical serum and glucocorticoid kinase 1 (SGK1) phosphorylation sites.

Regulation of epithelial Na(+) channel (ENaC)-mediated transport in the distal nephron is a critical determinant of blood pressure in humans. Aldosterone via serum and glucocorticoid kinase 1 (SGK1) stimulates ENaC by phosphorylation of the E3 ubiquitin ligase Nedd4-2, which induces interaction with 14-3-3 proteins. However, the mechanisms of SGK1- and 14-3-3-mediated regulation of Nedd4-2 are unclear. There are three canonical SGK1 target sites on Nedd4-2 that overlap phosphorylation-dependent 14-3-3 interaction motifs. Two of these are termed "minor," and one is termed "major," based on weak or strong binding to 14-3-3 proteins, respectively. By mass spectrometry, we found that aldosterone significantly stimulates phosphorylation of a minor, relative to the major, 14-3-3 binding site on Nedd4-2. Phosphorylation-deficient minor site Nedd4-2 mutants bound less 14-3-3 than did wild-type (WT) Nedd4-2, and minor site Nedd4-2 mutations were sufficient to inhibit SGK1 stimulation of ENaC cell surface expression. As measured by pulse-chase and cycloheximide chase assays, a major binding site Nedd4-2 mutant had a shorter cellular half-life than WT Nedd4-2, but this property was not dependent on binding to 14-3-3. Additionally, a dimerization-deficient 14-3-3ε mutant failed to bind Nedd4-2. We conclude that whereas phosphorylation at the Nedd4-2 major site is important for interaction with 14-3-3 dimers, minor site phosphorylation by SGK1 may be the relevant molecular switch that stabilizes Nedd4-2 interaction with 14-3-3 and thus promotes ENaC cell surface expression. We also propose that major site phosphorylation promotes cellular Nedd4-2 protein stability, which potentially represents a novel form of regulation for turnover of E3 ubiquitin ligases.

Non-nucleoside inhibitors of HCV polymerase NS5B. Part 3: synthesis and optimization studies of benzothiazine-substituted tetramic acids.

Benzothiazine-substituted tetramic acids were discovered as highly potent non-nucleoside inhibitors of HCV NS5B polymerase. X-ray crystallography studies confirmed the binding mode of these inhibitors with HCV NS5B polymerase. Rational optimization of time dependent inactivation of CYP 3A4 and clearance was accomplished by incorporation of electron-withdrawing groups to the benzothiazine core.

Non-nucleoside inhibitors of HCV polymerase NS5B. Part 4: structure-based design, synthesis, and biological evaluation of benzo[d]isothiazole-1,1-dioxides.

A series of benzo[d]isothiazole-1,1-dioxides were designed and evaluated as inhibitors of HCV polymerase NS5B. Structure-based design led to the incorporation of a high affinity methyl sulfonamide group. Structure-activity relationship (SAR) studies of this series revealed analogues with submicromolar potencies in the HCV replicon assay and moderate pharmacokinetic properties. SAR studies combined with structure based drug design focused on the sulfonamide region led to a novel and potent cyclic analogue.

Non-nucleoside inhibitors of HCV polymerase NS5B. Part 2: Synthesis and structure-activity relationships of benzothiazine-substituted quinolinediones.

A new series of benzothiazine-substituted quinolinediones were evaluated as inhibitors of HCV polymerase NS5B. SAR studies on this series revealed a methyl sulfonamide group as a high affinity feature. Analogues with this group showed submicromolar potencies in the HCV cell based replicon assay. Pharmacokinetic and toxicology studies were also performed on a selected compound (34) to evaluate in vivo properties of this new class of inhibitors of HCV NS5B polymerase.

3,4-Dihydro-2H-benzo[1,4]oxazine derivatives as 5-HT6 receptor antagonists.

A series of novel 3,4-dihydro-2H-benzo[1,4]oxazine derivatives has been designed and synthesized as 5-HT(6) receptor antagonists. Many of the compounds displayed subnanomolar affinities for the 5-HT(6) receptor and good brain penetration in rats. The relationship of structure and lipophilicity to hERG inhibition of this series of compounds is discussed.

Synthesis of novel alkyl triphosphates and their substrate properties toward terminal deoxynucleotidyltransferase.

Novel triphosphate derivatives bearing bulky or small groups at alpha-position attached to the triphosphate residue through linkers of different structures and lengths were synthesized and studied as substrates toward terminal deoxynucleotidyltransferase. The substrate efficacy depends on the structure of substituents, linker length, and nature of metal activator. The replacement of hydrophobic groups by small substituents decreased the substrate efficacy by about 20 times in respect to hydrophobic residues. The dependence on metal activator is the following: Co(2+) > Mn(2+) >> Mg(2+). The model of interaction of alkyl triphosphates with linkers of different lengths bearing TdT active site is presented.

Application of polarity switching in the identification of the metabolites of RO9237.

Polarity switching mass spectrometry is an efficient way to collect structural data on drug metabolites. The value of this approach is illustrated with the in vitro metabolism of RO9237. Metabolites are identified by positive and negative electrospray ionization (ESI) full scan mass spectrometry, MS/MS and MS(3) using unlabelled and (14)C-radiolabelled versions of the drug. Comparison of the relative detectability of these metabolites by +ESI and -ESI shows that neither ESI mode is universal. It is advantageous to screen for metabolites using both positive and negative ionization modes. This is especially true for phase II metabolism which tends to make molecules more polar and often more acidic. Identification of phase II metabolites also benefits greatly from MS(3) experiments because the conjugating groups typically are cleaved in MS/MS and information on the core structure is only obtained in MS(3). A special case of phase II metabolism is the generation of glutathione (GSH) conjugates from reactive metabolites. The detection of GSH conjugates also benefits from generating both positive and negative ESI mass spectral data.

New triphosphate conjugates bearing reporter groups: labeling of DNA fragments for microarray analysis.

A simple and convenient method for incorporation of fluorescent or ligand groups into 3'-termini of DNA fragments is proposed. A set of triphosphoric acid monoesters bearing fluorescent groups or biotin attached to the triphosphate fragment through linkers of different lengths and structures was synthesized. All the compounds were substrates for calf thymus terminal deoxynucleotidyltransferase and were used for incorporation of marker groups into 3'-termini of DNA fragments. The compounds were successfully applied for DNA labeling during post-PCR target preparation for microarray analysis.

Expanding the repertoire of DNA polymerase substrates: template-instructed incorporation of non-nucleoside triphosphate analogues by DNA polymerases beta and lambda.

We have recently shown that neither the base nor the sugar moieties of a nucleotide is an essential feature for its incorporation by DNA polymerases (pols) lambda and beta. Here we present the identification of novel non-nucleoside triphosphate (NNTP) derivatives belonging to three classes: (i) non-substrate-specific inhibitors of DNA pol lambda; (ii) substrate inhibitors which could preferentially be incorporated by either DNA pol lambda wild type or its Y505A mutant and (iii) the substrate inhibitor N-(Biphenylcarbonyl)-4-oxobutyl triphosphate which could be incorporated exclusively by DNA pol beta in a Mg2+-dependent manner, and preferentially pairs with A on the template. This compound represents the first example of a substrate lacking both nucleobase and ribose residue, showing distinct base-pairing properties with normal bases. Therefore, this NNTP analog can be considered as the prototype of an entirely novel class of DNA pol substrates.

Hypophosphoric acid is a unique substrate of pyrophosphorolysis catalyzed by HIV-1 reverse transcriptase.

Pyrophosphate analogues, namely, pyrophosphorous, hypophosphoric, and hypophosphorous acids, were evaluated as inhibitors in elongation reactions and substrates in pyrophosphorolysis reaction catalyzed by HIV-1 reverse transcriptase and DNA polymerase I (the Klenow fragment). The substrate efficacy of hypophosphoric acid in pyrophosphorolysis reaction exceeded that of pyrophosphate for both enzymes by more than ten times. The product of the reaction was a dNTP analogue bearing a hypophosphate in the beta,gamma-position. Pyrophosphorous and hypophosphorous acids were neither inhibitors nor substrates for the enzymes. Kinetic parameters of the pyrophosphorolysis reaction catalyzed by HIV reverse transcriptase in the presence of hypophosphoric acid were evaluated. The dTMP analogue bearing a hypophosphate in the beta,gamma-position was synthesized and its substrate properties in elongation reaction catalyzed by HIV-1 reverse transcriptase were similar to those of natural dTTP. Hypophosphoric acid was capable of removing ddTMP, ddTMP(3'N3), and ddTMP(3'NH2) from the 3'-end of primers with an equal efficacy.

Incorporation of non-nucleoside triphosphate analogues opposite to an abasic site by human DNA polymerases beta and lambda.

A novel class of non-nucleoside triphosphate analogues, bearing hydrophobic groups sterically similar to nucleosides linked to the alpha-phosphate but lacking the chemical functional groups of nucleic acids, were tested against six different DNA polymerases (polymerases). Human polymerases alpha, beta and lambda, and Saccharomyces cerevisiae polymerase IV, were inhibited with different potencies by these analogues. On the contrary, Escherichia coli polymerase I and HIV-1 reverse transcriptase were not. Polymerase beta incorporated these derivatives in a strictly Mn++-dependent manner. On the other hand, polymerase lambda could incorporate some alkyltriphosphate derivatives with both Mg++ and Mn++, but only opposite to an abasic site on the template strand. The active site mutant polymerase lambda Y505A showed an increased ability to incorporate the analogues. These results show for the first time that neither the base nor the sugar moieties of nucleotides are required for incorporation by family X DNA polymerases.

Pre-clinical and clinical evaluation of solution and soft gelatin capsule formulations for a BCS class 3 compound with atypical physicochemical properties.

R1481 is a sub-type selective muscarinic receptor antagonist with the potential treatment of overactive bladder. R1481 presents two challenges for drug development. The first is the viscous semi-solid nature of the active pharmaceutical ingredient (API). The second challenge is the poor oral bioavailability of this water soluble, metabolically stable compound due to low intestinal permeability, and the P-glycoprotein (P-gp) efflux mechanism. Vitamin E TPGS is reported by others to enhance bioavailability by increasing the solubility of active compounds and by inhibiting P-gp in the intestine. In this report, compatibility of R1481 in Capmul MCM-based formulations with and without vitamin E TPGS is summarized. Review of accelerated stability studies of oral formulations led to the identification of a soft gelatin capsule formulation using neat Capmul MCM as an acceptable formulation for Phase 1 clinical studies. Soft gelatin capsules (5 mg strength) were manufactured with and without the addition of vitamin E TPGS. Clinical data show that vitamin E TPGS does not improve systemic exposure of R1481 in humans.