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PDF file, 156K, Species and target specificity of IMC-3G3 and 1E10.

In lung cancer, platelet-derived growth factor receptor α (PDGFRα) is expressed frequently by tumor-associated stromal cells and by cancer cells in a subset of tumors. We sought to determine the effect of targeting stromal PDGFRα in preclinical lung tumor xenograft models (human tumor, mouse stroma). Effects of anti-human (IMC-3G3) and anti-mouse (1E10) PDGFRα monoclonal antibodies (mAb) on proliferation and PDGFRα signaling were evaluated in lung cancer cell lines and mouse fibroblasts. Therapy studies were conducted using established PDGFRα-positive H1703 cells and PDGFRα-negative Calu-6, H1993, and A549 subcutaneous tumors in immunocompromised mice treated with vehicle, anti-PDGFRα mAbs, chemotherapy, or combination therapy. Tumors were analyzed for growth and levels of growth factors. IMC-3G3 inhibited PDGFRα activation and the growth of H1703 cells in vitro and tumor growth in vivo, but had no effect on PDGFRα-negative cell lines or mouse fibroblasts. 1E10 inhibited growth and PDGFRα activation of mouse fibroblasts, but had no effect on human cancer cell lines in vitro. In vivo, 1E10-targeted inhibition of murine PDGFRα reduced tumor growth as single-agent therapy in Calu-6 cells and enhanced the effect of chemotherapy in xenografts derived from A549 cells. We also identified that low expression cancer cell expression of VEGF-A and elevated expression of PDGF-AA were associated with response to stromal PDGFRα targeting. We conclude that stromal PDGFRα inhibition represents a means for enhancing control of lung cancer growth in some cases, independent of tumor cell PDGFRα expression. Mol Cancer Ther; 11(11); 2473–82. ©2012 AACR.

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The programmed cell death protein 1 receptor (PD-1) and programmed death ligand 1 (PD-L1) coinhibitory pathway suppresses T-cell–mediated immunity. We hypothesized that cotargeting of PD-1 and PD-L1 with a bispecific antibody molecule could provide an alternative therapeutic approach, with enhanced antitumor activity, compared with monospecific PD-1 and PD-L1 antibodies. Here, we describe LY3434172, a bispecific IgG1 mAb with ablated Fc immune effector function that targets both human PD-1 and PD-L1. LY3434172 fully inhibited the major inhibitory receptor–ligand interactions in the PD-1 pathway. LY3434172 enhanced functional activation of T cells in vitro compared with the parent anti–PD-1 and anti–PD-L1 antibody combination or respective monotherapies. In mouse tumor models reconstituted with human immune cells, LY3434172 therapy induced dramatic and potent antitumor activity compared with each parent antibody or their combination. Collectively, these results demonstrated the enhanced immunomodulatory (immune blockade) properties of LY3434172, which improved antitumor immune response in preclinical studies, thus supporting its evaluation as a novel bispecific cancer immunotherapy.

Following the discovery of small molecule acyl piperazine ROMK inhibitors, the acyl octahydropyrazino[2,1-c][1,4]oxazine series was identified. This series displays improved ROMK/hERG selectivity, and as a consequence, the resulting ROMK inhibitors do not evoke QTc prolongation in an in vivo cardiovascular dog model. Further efforts in this series led to the discovery of analogs with improved pharmacokinetic profiles. This new series also retained comparable ROMK potency compared to earlier leads.

ROMK, the renal outer medullary potassium channel, is involved in potassium recycling at the thick ascending loop of Henle and potassium secretion at the cortical collecting duct in the kidney nephron. Because of this dual site of action, selective inhibitors of ROMK are expected to represent a new class of diuretics/natriuretics with superior efficacy and reduced urinary loss of potassium compared to standard-of-care loop and thiazide diuretics. Following our earlier work, this communication will detail subsequent medicinal chemistry endeavors to further improve lead selectivity against the hERG channel and preclinical pharmacokinetic properties. Pharmacological assessment of highlighted inhibitors will be described, including pharmacodynamic studies in both an acute rat diuresis/natriuresis model and a subchronic blood pressure model in spontaneous hypertensive rats. These proof-of-biology studies established for the first time that the human and rodent genetics accurately predict the in vivo pharmacology of ROMK inhibitors and supported identification of the first small molecule ROMK inhibitor clinical candidate, MK-7145.

A new subseries of ROMK inhibitors exemplified by 28 has been developed from the initial screening hit 1. The excellent selectivity for ROMK inhibition over related ion channels and pharmacokinetic properties across preclinical species support further preclinical evaluation of 28 as a new mechanism diuretic. Robust pharmacodynamic effects in both SD rats and dogs have been demonstrated.

A sub-class of distinct small molecule ROMK inhibitors were developed from the original lead 1. Medicinal chemistry endeavors led to novel ROMK inhibitors with good ROMK functional potency and improved hERG selectivity. Two of the described ROMK inhibitors were characterized for the first in vivo proof-of-concept biology studies, and results from an acute rat diuresis model confirmed the hypothesis that ROMK inhibitors represent new mechanism diuretic and natriuretic agents.

In lung cancer, platelet-derived growth factor receptor α (PDGFRα) is expressed frequently by tumor-associated stromal cells and by cancer cells in a subset of tumors. We sought to determine the effect of targeting stromal PDGFRα in preclinical lung tumor xenograft models (human tumor, mouse stroma). Effects of anti-human (IMC-3G3) and anti-mouse (1E10) PDGFRα monoclonal antibodies (mAb) on proliferation and PDGFRα signaling were evaluated in lung cancer cell lines and mouse fibroblasts. Therapy studies were conducted using established PDGFRα-positive H1703 cells and PDGFRα-negative Calu-6, H1993, and A549 subcutaneous tumors in immunocompromised mice treated with vehicle, anti-PDGFRα mAbs, chemotherapy, or combination therapy. Tumors were analyzed for growth and levels of growth factors. IMC-3G3 inhibited PDGFRα activation and the growth of H1703 cells in vitro and tumor growth in vivo, but had no effect on PDGFRα-negative cell lines or mouse fibroblasts. 1E10 inhibited growth and PDGFRα activation of mouse fibroblasts, but had no effect on human cancer cell lines in vitro. In vivo, 1E10-targeted inhibition of murine PDGFRα reduced tumor growth as single-agent therapy in Calu-6 cells and enhanced the effect of chemotherapy in xenografts derived from A549 cells. We also identified that low expression cancer cell expression of VEGF-A and elevated expression of PDGF-AA were associated with response to stromal PDGFRα targeting. We conclude that stromal PDGFRα inhibition represents a means for enhancing control of lung cancer growth in some cases, independent of tumor cell PDGFRα expression. Mol Cancer Ther; 11(11); 2473–82. ©2012 AACR.

The renal outer medullary potassium (ROMK) channel is a member of the inwardly rectifying family of potassium (Kir) channels. ROMK (Kir1.1) is predominantly expressed in kidney where it plays a major role in the salt reabsorption process. Loss-of-function mutations in the human Kir1.1 channel are associated with antenatal Bartter's syndrome type II, a life-threatening salt and water balance disorder. Heterozygous carriers of Kir1.1 mutations associated with antenatal Bartter's syndrome have reduced blood pressure and a decreased risk of developing hypertension by age 60. These data suggest that Kir1.1 inhibitors could represent novel diuretics for the treatment of hypertension. Because little is known about the molecular pharmacology of Kir1.1 channels, assays that provide a robust, reliable readout of channel activity-while operating in high-capacity mode-are needed. In the present study, we describe high-capacity, 384- and 1,536-well plate, functional thallium flux, and IonWorks electrophysiology assays for the Kir1.1 channel that fulfill these criteria. In addition, 96-well (86)Rb(+) flux assays were established that can operate in the presence of 100% serum, and can provide an indication of the effect of a serum shift on compound potencies. The ability to grow Madin-Darby canine kidney cells expressing Kir1.1 in Transwell supports provides a polarized cell system that can be used to study the mechanism of Kir1.1 inhibition by different agents. All these functional Kir1.1 assays together can play an important role in supporting different aspects of drug development efforts during lead identification and/or optimization.

To compare safety and efficacy of biphasic insulin aspart 70/30 (BIAsp 30) with exenatide in subjects with type 2 diabetes mellitus (T2DM) not achieving glycemic targets with metformin and sulfonylurea in a randomized, open-label, 24-week trial.Subjects (N = 372, T2DM6 months, ageor = 18 andor = 80 years, HbA1cor = 8%, insulin naive not achieving glycaemic targets, receiving metformin and sulfonylurea) were randomized 1: 1: 1 to receive either BIAsp 30 QD (12 U before supper); BIAsp 30 BID (12 U divided equally between pre-breakfast and pre-supper); or exenatide (5 microg BID for 4 weeks and 10 microg BID thereafter). Efficacy (HbA1c, fasting plasma glucose [FPG]) and safety (adverse events and hypoglycemic episodes) were assessed.Glycemic control achieved with both BIAsp 30 BID and BIAsp 30 QD was superior to that with exenatide (BIAsp 30 BID-exenatide: HbA1c difference -0.91% [95% CI: -1.23 to -0.59%] and BIAsp 30 QD-exenatide: difference: -0.67% [95% CI: -0.99 to -0.34%]). At the end of the study, more subjects achieved HbA1c7% andor = 6.5% in the BIAsp 30 BID group than in the exenatide group (HbA1c7%: 37% vs. 20%, p = 0.0060; HbA1cor = 6.5%: 25% vs. 8%, p = 0.0004, respectively). Combined hypoglycemic episodes (major, minor, symptoms only) were reported by 56%, 61%, and 29% of the subjects in the BIAsp 30 QD, BIAsp 30 BID, and exenatide groups, respectively. Weight gain was observed in the BIAsp 30 group (BIAsp 30 QD: 2.85 kg, BIAsp 30 BID: 4.08 kg) and weight loss was observed in the exenatide group (-1.96 kg). Nausea or vomiting was responsible for discontinuation of seven subjects in the exenatide group and one subject in the BIAsp 30 BID group.Significantly more T2DM patients (poorly controlled with combination metformin/sulfonylurea) achieved glycemic goals when treated with BIAsp 30 than with exenatide. The high baseline HbA1c values (approximately 10.2%) and the long duration of diabetes (approximately 9 years) suggests that some subjects may have been in an advanced stage of their diabetes and may not have had sufficient beta-cell function for a GLP-1 mimetic to be effective. The insulin-treated groups had more minor hypoglycemic events and weight gain but less gastrointestinal side-effects. In summary, BIAsp 30 was more efficacious in helping patients with high baseline HbA1c achieve glycemic goals.www.clinicaltrials.gov, NCT00097877.

Following the discovery of small molecule acyl piperazine ROMK inhibitors and their initial preclinical validation as a novel diuretic agent, our group set out to discover new ROMK inhibitors with reduced risk for QT effects, suitable for further pharmacological experiments in additional species. Several strategies for decreasing hERG affinity while maintaining ROMK inhibition were investigated and are described herein. The most promising candidate, derived from the newly discovered 4-N-heteroaryl acetyl series, improved functional hERG/ROMK ratio by >10× over the previous lead. In vivo evaluation demonstrated comparable diuretic effects in rat with no detectable QT effects at the doses evaluated in an in vivo dog model.

Inward rectifier potassium channels (Kir) play critical roles in cell physiology. Despite representing the simplest tetrameric potassium channel structures, the pharmacology of this channel family remains largely undeveloped. In this respect, tertiapin (TPN), a 21 amino acid peptide isolated from bee venom, has been reported to inhibit Kir1.1 and Kir3.1/3.4 channels with high affinity by binding to the M1-M2 linker region of these channels. The features of the peptide-channel interaction have been explored electrophysiologically, and these studies have identified ways by which to alter the composition of the peptide without affecting its biological activity. In the present study, the TPN derivative, TPN-Y1/K12/Q13, has been synthesized and radiolabeled to high specific activity with (125)I. TPN-Y1/K12/Q13 and mono-iodo-TPN-Y1/K12/Q13 ([(127)I]TPN-Y1/K12/Q13) inhibit with high affinity rat but not human Kir1.1 channels stably expressed in HEK293 cells. [(125)I]TPN-Y1/K12/Q13 binds in a saturable, time-dependent, and reversible manner to HEK293 cells expressing rat Kir1.1, as well as to membranes derived from these cells, and the pharmacology of the binding reaction is consistent with peptide binding to Kir1.1 channels. Studies using chimeric channels indicate that the differences in TPN sensitivity between rat and human Kir1.1 channels are due to the presence of two nonconserved residues within the M1-M2 linker region. When these results are taken together, they demonstrate that [(125)I]TPN-Y1/K12/Q13 represents the first high specific activity radioligand for studying rat Kir1.1 channels and suggest its utility for identifying other Kir channel modulators.

The renal outer medullary potassium (ROMK) channel, which is located at the apical membrane of epithelial cells lining the thick ascending loop of Henle and cortical collecting duct, plays an important role in kidney physiology by regulating salt reabsorption. Loss-of-function mutations in the human ROMK channel are associated with antenatal type II Bartter's syndrome, an autosomal recessive life-threatening salt-wasting disorder with mild hypokalemia. Similar observations have been reported from studies with ROMK knockout mice and rats. It is noteworthy that heterozygous carriers of Kir1.1 mutations associated with antenatal Bartter's syndrome have reduced blood pressure and a decreased risk of developing hypertension by age 60. Although selective ROMK inhibitors would be expected to represent a new class of diuretics, this hypothesis has not been pharmacologically tested. Compound A [5-(2-(4-(2-(4-(1H-tetrazol-1-yl)phenyl)acetyl)piperazin-1-yl)ethyl)isobenzofuran-1(3H)-one)], a potent ROMK inhibitor with appropriate selectivity and characteristics for in vivo testing, has been identified. Compound A accesses the channel through the cytoplasmic side and binds to residues lining the pore within the transmembrane region below the selectivity filter. In normotensive rats and dogs, short-term oral administration of compound A caused concentration-dependent diuresis and natriuresis that were comparable to hydrochlorothiazide. Unlike hydrochlorothiazide, however, compound A did not cause any significant urinary potassium losses or changes in plasma electrolyte levels. These data indicate that pharmacologic inhibition of ROMK has the potential for affording diuretic/natriuretic efficacy similar to that of clinically used diuretics but without the dose-limiting hypokalemia associated with the use of loop and thiazide-like diuretics.

The renal outer medullary potassium channel (ROMK or Kir1.1) is a putative drug target for a novel class of diuretics that could be used for the treatment of hypertension and edematous states such as heart failure. An internal high-throughput screening campaign identified 1,4-bis(4-nitrophenethyl)piperazine (5) as a potent ROMK inhibitor. It is worth noting that this compound was identified as a minor impurity in a screening hit that was responsible for all of the initially observed ROMK activity. Structure–activity studies resulted in analogues with improved rat pharmacokinetic properties and selectivity over the hERG channel, providing tool compounds that can be used for in vivo pharmacological assessment. The featured ROMK inhibitors were also selective against other members of the inward rectifier family of potassium channels.

Hypoglycemia and wide glucose excursions continue to be major obstacles to achieving target HbA(1c) values and the associated reductions in long-term complications (and economic costs) in people with insulin-treated diabetes. In this study we evaluated the accuracy, safety, and clinical effectiveness of a continuous glucose-sensing device.A total of 91 insulin-requiring patients with type 1 (n = 75) and type 2 (n = 16) diabetes were enrolled in this multicenter randomized study. Subjects wore a transcutaneous, 3-day, continuous glucose-sensing system for three consecutive 72-h periods. Subjects were randomly assigned (1:1 ratio) to either a control group (continuous glucose data not provided) or a display group (continuous glucose data not provided during period 1 but displayed during periods 2 and 3). During periods 2 and 3, patients in the display group had real-time access to sensor glucose values, could review glucose trends over the preceding 1, 3, and 9 h, and were provided with high (or = 200 mg/dl) and low (or = 80 mg/dl) alerts and a low (or = 55 mg/dl) alarm. Sensors were inserted by patients, and both groups used (or wore) the system during daily activities. Device accuracy was assessed by comparing continuous glucose values to paired self-monitoring of blood glucose (SMBG) meter readings. Clinical effectiveness was evaluated by analyzing between-group (control vs. display, periods 2 and 3) and within-group (display, period 1 vs. period 3) differences in time spent in high, low, and target (81-140 mg/dl) glucose zones.When prospective, real-time sensor values were compared with SMBG values, 95.4% of 6,767 paired glucose values fell within Clarke error grid A and B zones. Pearson's correlation coefficient was 0.88, and mean and median absolute relative differences were 21.2 and 15.9%, respectively. No systematic bias was detected at any of the prespecified glucose levels (50, 80, 100, 150, and 200 mg/dl). When compared with control subjects, the display group spent 21% less time as hypoglycemic (55 mg/dl), 23% less time as hyperglycemic (or = 240 mg/dl), and 26% more time in the target (81-140 mg/dl) glucose range (P0.001 for each comparison). Nocturnal (10:00 p.m. to 6:00 a.m.) hypoglycemia, as assessed at two thresholds, was also reduced by 38% (55 mg/dl; P0.001) and 33% (55-80 mg/dl; P0.001) in the display group compared with control subjects.We conclude that real-time continuous glucose monitoring for periods up to 72 h is accurate and safe in insulin-requiring subjects with type 1 and type 2 diabetes. This study demonstrates that availability of real-time, continuously measured glucose levels can significantly improve glycemic excursions by reducing exposure to hyperglycemia without increasing the risk of hypoglycemia, which may reduce long-term diabetes complications and their associated economic costs.

Macrophage stimulating 1-receptor (RON) is a member of the c-Met receptor tyrosine kinase family. RON is normally expressed on macrophages and epithelial cells. However, it is overexpressed and activated in a large number of human tumors. The fully human anti-RON antibody, IMC-RON8, blocks the RON ligand, macrophage-stimulating protein (MSP), from binding to RON and has been demonstrated to have antitumor activity against human colon, lung, and pancreatic xenografts in mice. Overexpression of RON correlates with a worse clinical outcome for patients in at least two human cancer indications, namely breast and bladder. Given this correlation, we investigated the effect of IMC-RON8 in several in vitro and in vivo systems with the RON-positive breast and bladder cancer cell lines JIMT-1 and BFTC-905, respectively. Both of these were found from a screen of breast and bladder cancer cell lines designed to identify those that are RON positive and responsive to MSP through activation of the MAP Kinase signaling pathway. IMC-RON8 inhibited the phosphorylation of MAP Kinase in response to 5nM MSP stimulation for JIMT-1 and BFTC-905. IMC-RON8 also inhibited the MSP-induced cellular migration of JIMT-1 in a wound healing assay, completely preventing progression towards closing of the wound. As measured by flow cytometry, IMC-RON8 at 33.3nM induced the internalization of 38% and 31% cell-surface RON expressed on JIMT-1 and BFTC-905 cells, respectively, following 24 hours of treatment at 37°C. To investigate IMC-RON8-induced RON downmodulation, clonal Hela cells stably expressing a RON-GFP protein were generated. These cells were treated with MSP, a RON agonist antibody (RON2), or IMC-RON8 and the level of RON-GFP was then measured by confocal microscopy in a live-cell time lapse experiment. IMC-RON8 treatment actively induced receptor interalization and caused RON-GFP degradation (reduction of the GFP signal) relative to IgG controls, although to a lesser extent than MSP and RON2. In an in vivo JIMT-1 xenograft model, IMC-RON8 significantly inhibited tumor growth with a %T/C value of 59 when administered at 60 mg/kg twice a week. Combination of IMC-RON8 with the chemotherapeutic agent, docetaxel at 12 mg/kg once per week, significantly improved the anti-tumor effects compared to either monotherapy with a %T/C value of 20. In an in vivo BFTC-905 xenograft model, IMC-RON8 significantly inhibited tumor growth with a %T/C value of 58 when administered at 60 mg/kg twice a week. BFTC-905 tumors removed after the last IMC-RON8 dose (6 total doses over a 3 week study period), showed a decrease in the total level of RON receptor present in tumors relative to controls (p-value of Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C54.

Type 2 diabetes exerts a huge toll on both morbidity and mortality, despite an expanding range of antiglycemic drugs and epidemiological evidence highlighting the benefits of effective glycemic control. Incretin-based agents offer important benefits, including a meal-dependent mode of action that may protect against hypoglycemia, and weight loss—in contrast to other antihyperglycemic drugs that cause weight gain. There are now two glucagon-like peptide-1 (GLP-1) receptor agonists and three dipeptidyl peptidase-4 (DPP-4) inhibitors approved for the management of type 2 diabetes in the US. Clinical trials have established the efficacy of incretin-based agents in controlling fasting and post-prandial blood glucose levels as well as glycosylated hemoglobin (HbA1c), both as monotherapy (including as first-line pharmacological treatment) and in combination with other antihyperglycemic treatments. GLP-1 receptor agonists and DPP-4 inhibitors have different mechanisms of action, which may explain their inconsistent efficacy results in direct comparator trials; for example, liraglutide has better efficacy than sitagliptin. However, GLP-1 receptor agonists can cause transient nausea in some patients. There is also evidence of different effects of individual agents within the same class; for example, liraglutide has shown superior efficacy to exenatide when added to metformin and/or sulfonylurea. Linagliptin is not cleared through renal mechanisms, unlike sitagliptin and saxagliptin. Isolated cases of pancreatitis led to concerns about a putative link with incretin-based therapies. However, the data currently available do not support a mechanistic or epidemiological link, although there does appear to be an increased risk of pancreatitis in people with diabetes that is independent of incretin-based treatment. Ongoing studies aim to extend our longer-term understanding of these agents, and hence, allow us to develop an optimal approach to patient management.

1. The effects of naloxone (8 mg) on the pressor and plasma catecholamine response to a standard cold-pressor test have been evaluated in six normal male subjects. Plasma catecholamines were estimated by high performance liquid chromatography coupled to electrochemical detection. 2. Cold stimulation induced significant elevations in plasma noradrenaline and adrenaline to reach mean peak levels 61% and 108% above their respective basal levels (P < 0.05). Systolic blood pressure increased by 23 ± 6.5 mmHg (P < 0.001), and heart rate increased by 7.5 ± 2.5 beats/min (P < 0.001). 3. Naloxone pretreatment significantly enhanced the plasma adrenaline response to the cold stimulus by 98% (P < 0.01) with concomitant changes in peak systolic blood pressure (peak increment 31 ± 6 mmHg) and pulse rate (12.5 ± 3.5 beats/min) responses (both P < 0.05). The mean plasma noradrenaline response to cold also increased after naloxone, but this failed to achieve significance. 4. Endogenous opioids are likely to be involved in the sympathoadrenal response to a mild acute stress in man.