Sepantronium bromide (YM155) enhances response of human B-cell non-Hodgkin lymphoma to rituximab.

In the treatment of B-cell non-Hodgkin lymphoma (B-NHL) rituximab improves long-term survival in combination with conventional chemotherapy. However, because the majority of patients with B-NHL eventually relapse, the development of more effective therapies is needed. Here, we evaluated the antitumor effects of a combination treatment involving sepantronium bromide (YM155), a first-in-class survivin suppressant, and rituximab in B-NHL xenograft mice models. To determine the efficacy of the combination treatment, YM155- and rituximab-treated B-NHL cell xenografted mice were monitored for tumor size and survival and subjected to 2'-deoxy-2'-(18)F-fluoro-D-glucose ((18)F-FDG) and 3'-(18)F-fluoro-3'-deoxythymidine ((18)F-FLT) positron emission tomography (PET) imaging. In addition, the cell proliferation status of excised tumors was examined by Ki-67 immunohistochemistry. In DB, WSU-DLCL-2, and Mino xenograft-bearing mice, the combination treatment of YM155 and rituximab induced significant tumor growth inhibition and tumor regression compared with either single agent. On day 3 after the initiation of treatment a significant decrease in both (18)F-FDG and (18)F-FLT tumor uptake from pretreatment levels was observed in combination treatment groups. The Ki-67 proliferation index was significantly decreased on day 3 in the xenograft models treated with combination treatment, suggesting that the combination of YM155 and rituximab reduced cell proliferation and glucose metabolism. Furthermore, compared with monotherapy, combination treatment prolonged survival times of severe combined immunodeficient mice with disseminated WSU-FSCCL and Jeko B-NHL tumors. Our findings demonstrate that YM155 and rituximab combination treatment enhances antitumor activity in B-NHL xenografts, and (18)F-FLT and (18)F-FDG PET imaging may allow the early functional evaluation of treatment responses in patients with B-NHL.

Inverse agonist histamine H3 receptor PET tracers labelled with carbon-11 or fluorine-18.

Two histamine H3 receptor (H3R) inverse agonist PET tracers have been synthesized and characterized in preclinical studies. Each tracer has high affinity for the histamine H3 receptor, has suitable lipophilicity, and neither is a substrate for the P-glycoprotein efflux pump. A common phenolic precursor was used to synthesize each tracer with high specific activity and radiochemical purity by an alkylation reaction using either [(11)C]MeI or [(18)F]FCD(2)Br. Autoradiographic studies in rhesus monkey and human brain slices showed that each tracer had a widespread distribution with high binding densities in frontal cortex, globus pallidus and striatum, and lower uptake in cerebellum. The specificity of this expression pattern was demonstrated by the blockade of the autoradiographic signal by either the H3R agonist R-alpha-methylhistamine or a histamine H3R inverse agonist. In vivo PET imaging studies in rhesus monkey showed rapid uptake of each tracer into the brain with the same distribution seen in the autoradiographic studies. Each tracer could be blocked by pretreatment with a histamine H3R inverse agonist giving a good specific signal. Comparison of the in vitro metabolism of each compound showed slower metabolism in human liver microsomes than in rhesus monkey liver microsomes, with each compound having a similar clearance rate in humans. The in vivo metabolism of 1b in rhesus monkey showed that at 60 min, approximately 35% of the circulating counts were due to the parent. These tracers are very promising candidates as clinical PET tracers to both study the histamine H3R system and measure receptor occupancy of H3R therapeutic compounds.

Discovery of substituted 2,4,4-triarylimidazoline derivatives as potent and selective neuropeptide Y Y5 receptor antagonists.

Novel imidazoline derivatives were discovered to be potent neuropeptide Y Y5 receptor antagonists. High-throughput screening of Merck sample collections against the human Y5 receptor resulted in the identification of 2,4,4-triphenylimidazoline (1), which had an IC(50) of 54nM. Subsequent optimization led to the identification of several potent derivatives.

Synthesis and evaluation of a spiro-isobenzofuranone class of histamine H3 receptor inverse agonists.

Spiro-isobenzofuranones 1a and 1b were discovered as potent, selective, and brain-penetrable non-imidazole H3 receptor inverse agonists. Our corporate sample collection was screened to identify 2a as a lead. Recognizing the right-hand portion of 2a as an essential pharmacophore, an extensive screen of the left-hand piperidine portion was carried out to yield the potent spiro-derivatives 2t-x. Spiro-isobenzofuranone 2x, the most potent among the derivatives, was converted to the corresponding amide 1a, which possessed dramatically improved H3 activity (IC(50)=0.72 nM; more than 20-fold improvement over 2x). Further elaboration led to the identification of 1b, a 5-methoxy derivative with an IC(50) of 0.54 nM. Our studies demonstrated that derivatives 1a and 1b to be potent, selective, and brain-penetrable H3 inverse agonists.

Characterization of neuropeptide Y (NPY) Y5 receptor-mediated obesity in mice: chronic intracerebroventricular infusion of D-Trp(34)NPY.

To clarify the role of the neuropeptide Y (NPY) Y5 receptor subtype in energy homeostasis, the effect of the intracerebroventricular infusion of a selective Y5 agonist, D-Trp(34)NPY, was investigated in C57BL/6J mice. Intracerebroventricular infusion of D-Trp(34)NPY (5 and 10 microg/d) produced hyperphagia and body weight gain, accompanied by increased adipose tissue weight, hypercholesterolemia, hyperinsulinemia, and hyperleptinemia. Oral administration of a selective Y5 antagonist at a dose of 100 mg/kg twice a day completely suppressed all of these D-Trp(34)NPY-induced changes, indicating that chronic activation of the Y5 receptor produces hyperphagia and obesity. In addition, D-Trp(34)NPY still resulted in an increase in adipose tissue weight accompanied by hyperleptinemia and hypercholesterolemia, although D-Trp(34)NPY-induced food intake was restricted by pair-feeding. Under the pair-fed condition, D-Trp(34)NPY decreased hormone-sensitive lipase activity in white adipose tissue and uncoupling protein-1 mRNA expression in brown adipose tissue. These findings indicate that Y5-mediated obesity may involve metabolic changes, such as decreased lipolysis and thermogenesis, as well as hyperphagia. Therefore, the Y5 receptor can play a key role in regulating energy homeostasis.

Pre-clinical validation of orthotopically-implanted pulmonary tumor by imaging with 18F-fluorothymidine-positron emission tomography/computed tomography.

The development of positron-emission tomography (PET) and X-ray computed tomography (CT) imaging has improved the detection of tumor burden and, in turn, pre-clinical drug development and clinical treatment. In pre-clinical drug development, clinically-relevant murine cancer models, such as orthotopic models of lung cancer, have provided an accurate representation of tumor burden in humans. However, evidence demonstrating the capability of imaging-guided evaluation of these clinically-relevant models is limited. Here, we combined (18)F-fluorothymidine (FLT)-PET/CT imaging and a murine model of human non-small cell lung cancer (NSCLC) to improve the accuracy of anticancer drug evaluation in pre-clinical studies. We found that FLT-PET/CT imaging enabled the progression of pulmonary tumors to be longitudinally monitored rather than FDG-PET/CT. Furthermore, in an efficacy study of a standard treatment of docetaxel in a murine lung cancer model, FLT-PET imaging detected the anticancer response earlier than volumetric analysis by CT imaging. We, thus, observed a relationship between the alteration of FLT signals and Ki-67 index in the pulmonary tumor during the period of chemotherapy. These results indicate that the combination of FLT-PET/CT imaging and an orthotopic NSCLC model is an effective strategy for evaluating clinical efficacy and potential of an anticancer agent during pre-clinical development.

Discovery of tetrasubstituted imidazolines as potent and selective neuropeptide Y Y5 receptor antagonists: reduced human ether-a-go-go related gene potassium channel binding affinity and potent antiobesity effect.

A series of novel imidazoline derivatives was synthesized and evaluated as neuropeptide Y (NPY) Y5 receptor antagonists. Optimization of previously reported imidazoline leads, 1a and 1b, was attempted by introduction of substituents at the 5-position on the imidazoline ring and modification of the bis(4-fluorphenyl) moiety. A number of potent derivatives without human ether-a-go-go related gene potassium channel (hERG) activity were identified. Selected compounds, including 2a, were shown to have excellent brain and CSF permeability. Compound 2a displayed a suitable pharmacokinetic profile for chronic in vivo studies and potently inhibited D-Trp(34)NPY-induced acute food intake in rats. Oral administration of 2a resulted in a potent reduction of body weight in a diet-induced obese mouse model.

(9S)-9-(2-hydroxy-4,4-dimethyl-6-oxo-1-cyclohexen-1-yl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-xanthen-1-one, a selective and orally active neuropeptide Y Y5 receptor antagonist.

(9S)-9-(2-Hydroxy-4,4-dimethyl-6-oxo-1-cyclohexen-1-yl)-3,3-dimethyl-2,3,4,9-tetrahydro-1H-xanthen-1-one ((S)-1) was identified as a selective and orally active neuropeptide Y Y5 receptor antagonist. The structure-activity relationship for this structural class was investigated and showed that limited substitution on the phenyl ring was tolerated and that modification of the 4,4-dimethyl group of the cyclohexenone and the 3,3-dimethyl group of the xanthenone parts slightly improved potency. The plasma concentration-time profile after oral administration of (S)-1 in Sprague-Dawley (SD) rats showed significant in vivo racemization of (S)-1 and that (S)-1 is cleared much more quickly than (R)-1. The duration of (S)-1 in SD rats after oral administration of (RS)-1 racemate was twice as long as that following oral administration of (S)-1. The C max values of (S)-1 after administration of (S)-1 and (RS)-1 were comparable, and the brain to plasma ratio for (S)-1 was 0.34 in SD rats. In our acute D-Trp (34)NPY-induced food intake model, both (S)-1 and (RS)-1 showed potent and dose-dependent efficacy. Therefore, the use of (RS)-1 is suitable for studies that require sustained plasma exposure of (S)-1.

A pair-feeding study reveals that a Y5 antagonist causes weight loss in diet-induced obese mice by modulating food intake and energy expenditure.

Neuropeptide Y (NPY) is thought to have a significant role in the physiological control of energy homeostasis. We recently reported that an NPY Y5 antagonist inhibits body weight gain in diet-induced obese (DIO) mice, with a moderate reduction in food intake. To clarify the mechanism of the antiobesity effects of the Y5 antagonist, we conducted a pair-feeding study in DIO mice. The Y5 antagonist at 100 mg/kg produced a moderate feeding suppression leading to an 18% decrease in body weight, without altering body temperature. In contrast, the pair-fed group showed only a transient weight reduction and a reduced body temperature, thus indicating that the Y5 antagonist stimulates thermogenesis. The Y5 antagonist-treated mice showed an up-regulation of uncoupling protein mRNA in brown adipose tissue (BAT) and white adipose tissue (WAT), suggesting that both BAT and WAT contribute to energy expenditure. Thus, the Y5 antagonist induces its antiobesity effects by acting on both energy intake and expenditure.

A neuropeptide Y Y5 antagonist selectively ameliorates body weight gain and associated parameters in diet-induced obese mice.

Neuropeptide Y (NPY) is thought to have a major role in the physiological control of energy homeostasis. Among five NPY receptors described, the NPY Y5 receptor (Y5R) is a prime candidate to mediate some of the effects of NPY on energy homeostasis, although its role in physiologically relevant rodent obesity models remains poorly defined. We examined the effect of a potent and highly selective Y5R antagonist in rodent obesity and dietary models. The Y5R antagonist selectively ameliorated diet-induced obesity (DIO) in rodents by suppressing body weight gain and adiposity while improving the DIO-associated hyperinsulinemia. The compound did not affect the body weight of lean mice fed a regular diet or genetically obese leptin receptor-deficient mice or rats, despite similarly high brain Y5R receptor occupancy. The Y5R antagonist acts in a mechanism-based manner, as the compound did not affect DIO of Y5R-deficient mice. These results indicate that Y5R is involved in the regulation and development of DIO and suggest utility for Y5R antagonists in the treatment of obesity.

Synthesis and evaluation of substituted 4-alkoxy-2-aminopyridines as novel neuropeptide Y1 receptor antagonists.

A series of substituted 4-alkoxy-2-aminopyridines 2, which were formally derived from neuropeptide Y1 antagonist 1 by replacing the morpholino portion with alkoxy groups, were synthesized and evaluated as neuropeptide Y Y1 receptor antagonists. Primary structure-activity relationships and identification of potent 4-alkoxy derivatives are described.