Synthesis and Initial Biological Evaluation of Boron-Containing Prostate-Specific Membrane Antigen Ligands for Treatment of Prostate Cancer Using Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT) is a therapeutic modality which has been used for the treatment of cancers, including brain and head and neck tumors. For effective treatment via BNCT, efficient and selective delivery of a high boron dose to cancer cells is needed. Prostate-specific membrane antigen (PSMA) is a target for prostate cancer imaging and drug delivery. In this study, we conjugated boronic acid or carborane functional groups to a well-established PSMA inhibitor scaffold to deliver boron to prostate cancer cells and prostate tumor xenograft models. Eight boron-containing PSMA inhibitors were synthesized. All of these compounds showed a strong binding affinity to PSMA in a competition radioligand binding assay (IC50 from 555.7 to 20.3 nM). Three selected compounds 1a, 1d, and 1f were administered to mice, and their in vivo blocking of 68Ga-PSMA-11 uptake was demonstrated through a positron emission tomography (PET) imaging and biodistribution experiment. Biodistribution analysis demonstrated boron uptake of 4-7 µg/g in 22Rv1 prostate xenograft tumors and similar tumor/muscle ratios compared to the ratio for the most commonly used BNCT compound, 4-borono-l-phenylalanine (BPA). Taken together, these data suggest a potential role for PSMA targeted BNCT agents in prostate cancer therapy following suitable optimization.

Effects of Food and Antacids on Pharmacokinetics and Pharmacodynamics of Lesinurad, a Selective Urate Reabsorption Inhibitor.

Two clinical studies were performed in healthy volunteers to investigate food and antacid effects on lesinurad, a novel selective uric acid reabsorption inhibitor approved for treatment of hyperuricemia associated with gout in combination with xanthine oxidase inhibitors. Study 1 evaluated a high-fat, high-calorie meal or high doses of antacids (3000 mg calcium carbonate or 1600 mg magnesium hydroxide/1600 mg aluminum hydroxide) on the pharmacokinetics (PK) and pharmacodynamics (PD) of 400 mg oral lesinurad. Study 2 evaluated low doses of antacids (1250 mg calcium carbonate or 800 mg magnesium hydroxide/800 mg aluminum hydroxide) on the PK and PD of 400 mg lesinurad. Food did not alter the plasma AUC of lesinurad and only reduced its Cmax by 18%. In the fasted conditions, high-dose calcium carbonate reduced the Cmax and AUC of lesinurad by 54% and 38%, respectively, whereas high-dose magnesium hydroxide/aluminum hydroxide reduced Cmax and AUC by 36% and 31%, respectively. Food enhanced the maximum serum urate (sUA)-lowering effect of lesinurad by approximately 20% despite reducing the Cmax of lesinurad. High-dose calcium carbonate decreased the urate-lowering effect approximately 20% in the first 6 hours, whereas high-dose magnesium hydroxide/aluminum hydroxide reduced the effect by 26%. Low-dose calcium carbonate or magnesium hydroxide/aluminum hydroxide in the presence of food did not significantly affect plasma lesinurad Cmax and AUC or the sUA lowering and renal handling of uric acid. In summary, study results suggest food did not meaningfully alter lesinurad PK and PD. High doses of antacids reduced lesinurad AUC up to 40% and reduced the lesinurad uric acid-lowering effect.

A Phase I Study of the Safety, Pharmacokinetics, and Pharmacodynamics of Combination Therapy with Refametinib plus Sorafenib in Patients with Advanced Cancer.

PURPOSE: To assess the safety and tolerability of the small-molecule allosteric MEK inhibitor refametinib combined with sorafenib, in patients with advanced solid malignancies. EXPERIMENTAL DESIGN: This phase I dose-escalation study included an expansion phase at the maximum tolerated dose (MTD). Patients received refametinib/sorafenib twice daily for 28 days, from a dose of refametinib 5 mg plus sorafenib 200 mg to a dose of refametinib 50 mg plus sorafenib 400 mg. Plasma levels of refametinib, refametinib metabolite M17, and sorafenib were measured for pharmacokinetic assessments. Tumors were biopsied at the MTD for analysis of MEK pathway mutations and ERK phosphorylation. RESULTS: Thirty-two patients were enrolled in the dose-escalation cohort. The MTD was refametinib 50 mg twice daily plus sorafenib 400 mg twice daily. The most common treatment-related toxicities were diarrhea and fatigue. Refametinib was readily absorbed following oral administration (plasma half-life of ∼16 hours at the MTD), and pharmacokinetic parameters displayed near-dose proportionality, with less than 2-fold accumulation after multiple dosing. Another 30 patients were enrolled in the MTD cohort; 19 had hepatocellular carcinoma. The combination was associated with significantly reduced ERK phosphorylation in 5 out of 6 patients biopsied, with the greatest reductions in those with KRAS or BRAF mutations. Disease was stabilized in approximately half of patients, and 1 patient with colorectal cancer achieved a partial response at the MTD lasting approximately 1 year. CONCLUSIONS: In this phase I study, refametinib plus sorafenib was well tolerated, with good oral absorption, near-dose proportionality, and target inhibition in a range of tumor types. Clin Cancer Res; 22(10); 2368-76. ©2015 AACR.

The discovery of the benzazepine class of histamine H3 receptor antagonists.

This Letter describes the discovery of a novel series of H3 receptor antagonists. The initial medicinal chemistry strategy focused on deconstructing and simplifying an early screening hit which rapidly led to the discovery of a novel series of H3 receptor antagonists based on the benzazepine core. Employing an H3 driven pharmacodynamic model, the series was then further optimised through to a lead compound that showed robust in vivo functional activity and possessed overall excellent developability properties.

A new in vivo screening paradigm to accelerate antimalarial drug discovery.

The emergence of resistance to available antimalarials requires the urgent development of new medicines. The recent disclosure of several thousand compounds active in vitro against the erythrocyte stage of Plasmodium falciparum has been a major breakthrough, though converting these hits into new medicines challenges current strategies. A new in vivo screening concept was evaluated as a strategy to increase the speed and efficiency of drug discovery projects in malaria. The new in vivo screening concept was developed based on human disease parameters, i.e. parasitemia in the peripheral blood of patients on hospital admission and parasite reduction ratio (PRR), which were allometrically down-scaled into P. berghei-infected mice. Mice with an initial parasitemia (P0) of 1.5% were treated orally for two consecutive days and parasitemia measured 24 h after the second dose. The assay was optimized for detection of compounds able to stop parasite replication (PRR = 1) or induce parasite clearance (PRR >1) with statistical power >99% using only two mice per experimental group. In the P. berghei in vivo screening assay, the PRR of a set of eleven antimalarials with different mechanisms of action correlated with human-equivalent data. Subsequently, 590 compounds from the Tres Cantos Antimalarial Set with activity in vitro against P. falciparum were tested at 50 mg/kg (orally) in an assay format that allowed the evaluation of hundreds of compounds per month. The rate of compounds with detectable efficacy was 11.2% and about one third of active compounds showed in vivo efficacy comparable with the most potent antimalarials used clinically. High-throughput, high-content in vivo screening could rapidly select new compounds, dramatically speeding up the discovery of new antimalarial medicines. A global multilateral collaborative project aimed at screening the significant chemical diversity within the antimalarial in vitro hits described in the literature is a feasible task.

Hyperpolarized [1-13C]dehydroascorbate MR spectroscopy in a murine model of prostate cancer: comparison with 18F-FDG PET.

UNLABELLED: Reduction and oxidation (redox) chemistry is increasingly implicated in cancer pathogenesis. To interrogate the redox status of prostate tumors noninvasively, we developed hyperpolarized [1-(13)C]dehydroascorbate ((13)C-DHA), the oxidized form of vitamin C, as an MR probe. In a model of transgenic adenocarcinoma of the mouse prostate (TRAMP), increased reduction of hyperpolarized (13)C-DHA to vitamin C was observed in tumor, as compared with normal prostate and surrounding benign tissue. We hypothesized that this difference was due to higher concentrations of glutathione and increased transport of hyperpolarized (13)C-DHA via the glucose transporters (GLUT1, GLUT3, and GLUT4) in TRAMP tumor. To test these hypotheses, hyperpolarized (13)C-DHA MR spectroscopy (MRS) and (18)F-FDG PET were applied as complementary technologies in the TRAMP model. METHODS: Late-stage TRAMP tumors (>4 cm(3)) were studied at similar time points (MR studies conducted < 24 h after PET) in fasting mice by (18)F-FDG PET and hyperpolarized (13)C-DHA MR imaging on a small-animal PET/CT scanner and a (1)H/(3)C 3-T MR scanner. PET data were processed using open-source AMIDE software to compare the standardized uptake values of tumor with those of surrounding muscle, and (13)C-DHA MRS data were processed using custom software to compare the metabolite ratios (vitamin C/[vitamin C + (13)C-DHA]). After in vivo studies, the tumor glutathione concentrations were determined using a spectrophotometric assay, and thiol staining was performed using mercury orange. Real-time polymerase chain reaction was used to evaluate the relevant transporters GLUT1, GLUT3, and GLUT4 and vitamin C transporters SVCT1 and SVCT2. GLUT1 was also evaluated by immunohistochemistry. RESULTS: The average metabolite ratio was 0.28 ± 0.02 in TRAMP tumor, versus 0.11 ± 0.02 in surrounding benign tissue (n = 4), representing a 2.5-fold difference. The corresponding tumor-to-nontumor (18)F-FDG uptake ratio was 3.0. The total glutathione was 5.1 ± 0.4 mM in tumor and 1.0 ± 0.2 mM in normal prostate, whereas reduced glutathione was 2.0 ± 0.3 mM and 0.8 ± 0.3 mM, respectively, corresponding to a 2.5-fold difference. In TRAMP tumor, mercury orange staining demonstrated increased thiols. Real-time polymerase chain reaction showed no significant difference in GLUT1 messenger RNA between TRAMP tumor and normal prostate, with immunohistochemistry (anti-GLUT1) also showing comparable staining. CONCLUSION: Both hyperpolarized (13)C-DHA and (18)F-FDG provide similar tumor contrast in the TRAMP model. Our findings suggest that the mechanism of in vivo hyperpolarized (13)C-DHA reduction and the resulting tumor contrast correlates most strongly with glutathione concentration. In the TRAMP model, GLUT1 is not significantly upregulated and is unlikely to account for the contrast obtained using hyperpolarized (13)C-DHA or (18)F-FDG.

Diverse small molecule inhibitors of human apurinic/apyrimidinic endonuclease APE1 identified from a screen of a large public collection.

The major human apurinic/apyrimidinic endonuclease APE1 plays a pivotal role in the repair of base damage via participation in the DNA base excision repair (BER) pathway. Increased activity of APE1, often observed in tumor cells, is thought to contribute to resistance to various anticancer drugs, whereas down-regulation of APE1 sensitizes cells to DNA damaging agents. Thus, inhibiting APE1 repair endonuclease function in cancer cells is considered a promising strategy to overcome therapeutic agent resistance. Despite ongoing efforts, inhibitors of APE1 with adequate drug-like properties have yet to be discovered. Using a kinetic fluorescence assay, we conducted a fully-automated high-throughput screen (HTS) of the NIH Molecular Libraries Small Molecule Repository (MLSMR), as well as additional public collections, with each compound tested as a 7-concentration series in a 4 µL reaction volume. Actives identified from the screen were subjected to a panel of confirmatory and counterscreen tests. Several active molecules were identified that inhibited APE1 in two independent assay formats and exhibited potentiation of the genotoxic effect of methyl methanesulfonate with a concomitant increase in AP sites, a hallmark of intracellular APE1 inhibition; a number of these chemotypes could be good starting points for further medicinal chemistry optimization. To our knowledge, this represents the largest-scale HTS to identify inhibitors of APE1, and provides a key first step in the development of novel agents targeting BER for cancer treatment.

Nucleic acid binding activity of human Cockayne syndrome B protein and identification of Ca(2+) as a novel metal cofactor.

The Cockayne syndrome group B protein (CSB) is a member of the SWI/SNF2 subgroup of Superfamily 2 ATPases/nucleic acid translocases/helicases and is defective in the autosomal recessive segmental progeroid disorder Cockayne syndrome. This study examines the ATP-dependent and the ATP-independent biochemical functions of human CSB. We show that Ca(2+) is a novel metal cofactor of CSB for ATP hydrolysis, mainly through the enhancement of k(cat), and that a variety of biologically relevant model nucleic acid substrates can function to activate CSB ATPase activity with either Mg(2+) or Ca(2+) present. However, CSB lacked detectable ATP-dependent helicase and single- or double-stranded nucleic acid translocase activities in the presence of either divalent metal. CSB was found to support ATP-independent complementary strand annealing of DNA/DNA, DNA/RNA, and RNA/RNA duplexes, with Ca(2+) again promoting optimal activity. CSB formed a stable protein:DNA complex with a 34mer double-stranded DNA in electrophoretic mobility-shift assays, independent of divalent metal or nucleotide (e.g. ATP). Moreover, CSB was able to form a stable complex with a range of nucleic acid substrates, including bubble and "pseudo-triplex" double-stranded DNAs that resemble replication and transcription intermediates, as well as forked duplexes of DNA/DNA, DNA/RNA, and RNA/RNA composition, the latter two of which do not promote CSB ATPase activity. Association of CSB with DNA, independent of ATP binding or hydrolysis, was seemingly sufficient to displace or rearrange a stable pre-bound protein:DNA complex, a property potentially important for its roles in transcription and DNA repair.

Structurally novel histamine H3 receptor antagonists GSK207040 and GSK334429 improve scopolamine-induced memory impairment and capsaicin-induced secondary allodynia in rats.

GSK207040 (5-[(3-cyclobutyl-2,3,4,5-tetrahydro-1H-3-benzazepin-7-yl)oxy]-N-methyl-2-pyrazinecarboxamide) and GSK334429 (1-(1-methylethyl)-4-({1-[6-(trifluoromethyl)-3-pyridinyl]-4-piperidinyl}carbonyl)hexahydro-1H-1,4-diazepine) are novel and selective non-imidazole histamine H(3) receptor antagonists from distinct chemical series with high affinity for human (pK(i)=9.67+/-0.06 and 9.49+/-0.09, respectively) and rat (pK(i)=9.08+/-0.16 and 9.12+/-0.14, respectively) H(3) receptors expressed in cerebral cortex. At the human recombinant H(3) receptor, GSK207040 and GSK334429 were potent functional antagonists (pA(2)=9.26+/-0.04 and 8.84+/-0.04, respectively versus H(3) agonist-induced changes in cAMP) and exhibited inverse agonist properties (pIC(50)=9.20+/-0.36 and 8.59+/-0.04 versus basal GTPgammaS binding). Following oral administration, GSK207040 and GSK334429 potently inhibited cortical ex vivo [(3)H]-R-alpha-methylhistamine binding (ED(50)=0.03 and 0.35 mg/kg, respectively). Functional antagonism of central H(3) receptors was demonstrated by blockade of R-alpha-methylhistamine-induced dipsogenia in rats (ID(50)=0.02 and 0.11 mg/kg p.o. for GSK207040 and GSK334429, respectively). In more pathophysiologically relevant pharmacodynamic models, GSK207040 (0.1, 0.3, 1 and 3mg/kg p.o.) and GSK334429 (0.3, 1 and 3mg/kg p.o.) significantly reversed amnesia induced by the cholinergic antagonist scopolamine in a passive avoidance paradigm. In addition, GSK207040 (0.1, 0.3 and 1mg/kg p.o.) and GSK334429 (3 and 10mg/kg p.o.) significantly reversed capsaicin-induced reductions in paw withdrawal threshold, suggesting for the first time that blockade of H(3) receptors may be able to reduce tactile allodynia. Novel H(3) receptor antagonists such as GSK207040 and GSK334429 may therefore have therapeutic potential not only in dementia but also in neuropathic pain.

SR 141716 (Rimonabant) precipitates withdrawal in marijuana-dependent mice.

Repeated marijuana use is known to lead to physical dependence in humans; however, its dependence liability has yet to be adequately assessed in laboratory animals. The goals of the present study were to: assess whether the CB(1) antagonist SR 141716 (rimonabant) precipitates withdrawal in mice that had been repeatedly exposed to marijuana smoke, and to compare these precipitated withdrawal effects to those elicited following intravenous administration of its chief psychoactive component Delta(9)-tetrahydrocannabinol (Delta(9)-THC). SR 141716 elicited a significant increase in paw tremors in mice that were repeatedly dosed with either marijuana or Delta(9)-THC. Unexpectedly, the blood and brain concentrations of Delta(9)-THC following marijuana exposure were considerably lower than those found following Delta(9)-THC injection when comparing an equivalent magnitude of paw tremors in both conditions. Finally, Delta(9)-THC dose-dependently alleviated SR 141716-induced paw tremors in marijuana-dependent mice, but marijuana itself failed to reverse the precipitated withdrawal effect. It is likely that marijuana exposure generated insufficient Delta(9)-THC brain levels (i.e., 203+/-19 ng/g) to reverse the withdrawal signs compared with the brain levels following intravenous injection (i.e., 1862+/-82 ng/g). These findings taken together indicate that mice exposed repeatedly to marijuana smoke exhibit similar precipitated withdrawal effects as Delta(9)-THC-injected mice.

Ape1 abasic endonuclease activity is regulated by magnesium and potassium concentrations and is robust on alternative DNA structures.

Abasic lesions are common mutagenic or cytotoxic DNA damages. Ape1 is the major human apurinic/apyrimidinic (AP) endonuclease and initiates repair of abasic sites by catalyzing strand cleavage at the lesion. I show here that Ape1 single-stranded (ss) AP site incision activity prefers 0.5 mM or 2 mM MgCl(2) and low concentrations (< or =50 mM) of KCl, whereas its double-stranded (ds) activity favors 10 mM MgCl(2) and 50 mM KCl or 2 mM MgCl(2) and 200 mM KCl. Both activities favor a pH between 7.0 and 7.5, suggesting a common catalytic mechanism. In conditions designed to mimic the intracellular environment (pH 7.2; 100 mM KCl; 1 mM MgCl(2)), Ape1 ssAP site incision activity is either about fivefold more active or approximately 20-fold less efficient than its ds activity, depending on the oligonucleotide employed. Secondary structure predictions suggest a role for the DNA conformational state in determining the effectiveness of Ape1. Ape1 complex stability in the presence of EDTA (non-incising conditions) is significantly weaker for ssDNA than dsDNA, regardless of the AP substrate. Duplexes where the AP site is positioned opposite the 3' terminus of a complementary primer strand are incised with an efficiency similar (less than twofold difference) to that of the ssAP substrate alone. Moreover, Ape1 cleaved AP sites in fork-like and bubble DNA structures with an efficiency that is identical or up to sevenfold higher than ssAP-DNA. The findings here suggest that Ape1 ssAP and dsAP endonuclease activities are regulated by sequence context and the relative concentrations of certain chemical elements in vivo, and that Ape1 incision activity occurs on complex replication, recombination, and/or transcription DNA intermediates.