A highly potent and selective DGAT-1 inhibitor was identified and used in rodent models of obesity and postprandial chylomicron excursion to validate DGAT-1 inhibition as a novel approach for the ...treatment of metabolic diseases. Specifically, compound 4a conferred weight loss and a reduction in liver triglycerides when dosed chronically in DIO mice and depleted serum triglycerides following a lipid challenge in a dose-dependent manner, thus, reproducing major phenotypical characteristics of DGAT-1−/− mice.
BACKGROUND:Drug-coated balloons (DCBs) are a predominant revascularization therapy for symptomatic femoropopliteal artery disease. Because of the differences in excipients, paclitaxel dose, and ...coating morphologies, varying clinical outcomes have been observed with different DCBs. We report the results of 2 studies investigating the pharmacokinetic and clinical outcomes of a new DCB to treat femoropopliteal disease.
METHODS:In the ILLUMENATE Pivotal Study (Prospective, Randomized, Single-Blind, U.S. Multi-Center Study to Evaluate Treatment of Obstructive Superficial Femoral Artery or Popliteal Lesions With A Novel Paclitaxel-Coated Percutaneous Angioplasty Balloon), 300 symptomatic patients (Rutherford class 2–4) were randomly assigned to DCB (n=200) or standard angioplasty (percutaneous transluminal angioplasty PTA) (n=100). The primary safety end point was freedom from device- and procedure-related death through 30 days, and freedom from target limb major amputation and clinically driven target lesion revascularization through 12 months. The primary effectiveness end point was primary patency through 12 months. In the ILLUMENATE PK study (Pharmacokinetic Study of the Stellarex Drug-Coated Angioplasty Balloon), paclitaxel plasma concentrations were measured after last DCB deployment and at prespecified times (at 1, 4, 24 hours and at 7 and 14 days postprocedure) until no longer detectable.
RESULTS:In the ILLUMENATE Pivotal Study, baseline characteristics were similar between groups50% had diabetes mellitus, 41% were women, mean lesion length was 8.3 cm, and 44% were severely calcified. The primary safety end point was met (92.1% for DCB versus 83.2% for PTA, P=0.025 for superiority) and the primary patency rate was significantly higher with DCB (76.3% for DCB versus 57.6% for PTA, P=0.003). Primary patency per Kaplan-Meier estimates at day 365 was 82.3% for DCB versus 70.9% for PTA (P=0.002). The rate of clinically driven target lesion revascularization was significantly lower in the DCB cohort (7.9% versus 16.8%, P=0.023). Improvements in ankle-brachial index, Rutherford class, and quality of life were comparable, but the PTA cohort required twice as many revascularizations. Pharmacokinetic outcomes showed that all patients had detectable paclitaxel levels after DCB deployment that declined within the first hour (54.4±116.9 ng/mL to 1.4±1.0 ng/mL).
CONCLUSIONS:The data demonstrate superior safety and effectiveness of the Stellarex DCB in comparison with PTA, and plasma levels of paclitaxel fall to low levels within 1 hour.
CLINICAL TRIAL REGISTRATION:URLhttp://clinicaltrials.gov. Unique identifiersNCT01858428 and NCT01912937.
We present the 5-year results of a prospective regulatory study of the INCRAFT device, a low-profile endovascular stent graft system for repair of abdominal aortic aneurysms.
This was an open-label ...prospective nonrandomized single-arm study enrolling in centers in the United States and Japan. The primary effectiveness outcome was successful aneurysm treatment and the primary safety outcome was the incidence of major adverse events at 30 days after the procedure. Major long-term outcomes were mortality, reintervention, adverse limb outcomes, and suprarenal stent fracture.
One hundred and ninety patients (mean age, 73.8 ± 7.6 years; 90% male; 69% white and 30% Asian) were enrolled from 32 centers throughout the United States and Japan. Minimal access vessel size was less than 7 mm on both sides in 43.9% of the study cohort. Thirty-day major adverse events occurred in 3.2% of patients (6/190). Periprocedural technical success was 94.1% (176/187). Successful aneurysm treatment was 100% at 30 days and 87.9% at 1 year. Two patients required open conversion for thromboembolic complications, 3 developed new type I or III endoleaks, and 7 experienced graft or limb occlusion. Freedom from graft occlusion was 96 ± 2% at 1 year and 94 ± 2% at 5 years. Freedom from stent fracture was 97 ± 1% at 1 year and 87 ± 3% at 5 years. Freedom from aneurysm-related mortality was 99 ± 1% at 1 and 5 years.
This study demonstrates good efficacy and safety and a very low rate of aneurysm related deaths with the INCRAFT device in a population with a high proportion of challenging anatomy.
Ghrelin, a 28 amino acid, octanoylated peptide, is an endogenous ligand for the growth hormone secretagogue receptor (GHS-R). In addition to various endocrine functions, including stimulation of GH ...release, ghrelin has been characterized as an important regulator of energy homeostasis. Ghrelin administration has been shown to increase adiposity in rodents and stimulate food intake in humans. Studies suggest that these orexigenic effects are mediated primarily through GHS-R expression in hypothalamic and pituitary neuronal pathways. In this context, GHS-R has been recognized as a potential target for the treatment of GH deficiency and body weight disorders. Cell lines provide convenient in vitro systems to identify and characterize potential pharmacophores and to analyze GHS-R functional activity. While recombinant cell lines that overexpress GHS-R have served as effective research tools for these studies, such cell lines may differ in signaling response to ghrelin compared with hypothalamic or pituitary cells expressing GHS-R. We show here that a cell line derived from a rat anterior pituitary adenoma, RC-4B/C, expresses endogenous GHS-R as judged by reverse transcriptase-PCR. In a Ca2+mobilization assay, RC-4B/C cells demonstrate a dose-dependent increase in intracellular Ca2+ on stimulation with rat ghrelin and a related peptide agonist, hexarelin (EC50, 1.0 nM and 1.7 nM respectively), but are unresponsive to treatment with inactive des-octanoyl rat ghrelin. A subclone, RC-4B/C.40, with a more robust and stable ghrelin response, was isolated from the parental population of cells to allow further analysis of GHS-R signal transduction. Using pertussis toxin and the phospholipase C inhibitor U-73122, we show that ghrelin signals through the Gq pathway in the RC-4B/C.40 cells. We also demonstrate that the ghrelin-induced rise of intracellular Ca2+ in RC-4B/C.40 cells involves initial Ca2+release from intracellular stores followed by a sustained elevation that occurs via influx of extracellular Ca2+ through ion channels. In addition, unlike observations reported in recombinant cell systems, the RC-4B/C.40 cells do not exhibit a high level of GHS-R constitutive activity as determined in a phosphatidylinositol hydrolysis assay. Overall, the data presented here suggest that the RC-4B/C parental and RC-4B/C.40 cells provide novel in vitro systems for the characterization of GHS-R pharmacophores and ghrelin signaling.
A critical early event in the inflammatory cascade is the induced expression of cell adhesion molecules on the lumenal surface of vascular endothelial cells. These adhesion molecules include ...E-selectin, ICAM-1, and VCAM-1, which serve to recruit circulating leukocytes to the site of the inflammation. These adhesive interactions allow the leukocytes to firmly adhere to and cross the vascular endothelium and migrate to the site of tissue injury. Pharmaceutical agents which would prevent the induced expression of one or more of the cell adhesion molecules on the endothelium might be expected to provide a novel mechanism to attenuate the inflammatory responses associated with chronic inflammatory diseases. A thieno2,3-dpyrimidine, A-155918, was identified from a whole-cell high-throughput assay for compounds which inhibited the tumor necrosis factor-α (TNFα)-induced expression of E-selectin, ICAM-1, or VCAM-1 on human vascular endothelial cells. Traditional medicinal chemistry methods were applied to this low-micromolar inhibitor, resulting in the 2,4-disubstituted thieno2,3-cpyridine A-205804, a potent and selective lead inhibitor of E-selectin and ICAM-1 expression (IC50 = 20 and 25 nM, respectively). The relative position of the nitrogen atom in the thienopyridine isomer was shown to be critical for activity, as was a small amide 2-substituent.
The elevated expression of cell adhesion molecules (CAMs) on the lumenal surface of vascular endothelial cells is a critical early event in the complex inflammatory process. The adhesive interactions ...of these CAMs that include E-selectin, ICAM-1, and VCAM-1 with their counter-receptors on leukocytes, such as integrins of the αLβ2 family, result in migration of the leukocytes to the site of inflammation and cause tissue injury. Pharmaceutical agents that could suppress the induced expression of one or more of these cell adhesion molecules would provide a novel mechanism to attenuate the inflammatory responses associated with chronic inflammatory diseases. A-205804 (1), a potent and selective inhibitor of the induced expression of E-selectin and ICAM-1 over VCAM-1, was further modified with emphasis at the C-4 and C-2 positions to identify a more potent drug candidate with a good pharmacokinetic profile and physical properties. Replacement of the C-4 sulfur linkage in 1 with an oxygen atom eliminated one of the two major metabolites for this lead molecule. The para-position of the 4-phenoxy group of the thieno2,3-cpyridine lead is found to be very critical for a higher in vitro potency and selectivity of E-selectin and ICAM-1 over VCAM-1 expression. This position is presumably close to the solvent-accessible region of the target protein−inhibitor complex. An attempt to install a water-solubilizing group at the para-position of the phenoxy group to increase the aqueous solubility of this lead series through various linkages failed to provide an ideal inhibitor. Only small substituents such as fluorine are tolerated at the meta- and ortho-positions of the 4-phenoxy to retain a good in vitro potency. Bromo, trifluoromethyl, pyrazol-1-yl, and imidazol-1-yl are among the better substituents at the para-position. With fine-tuning at the C-2 position we discovered a series of very potent (IC50 < 5 nM for ICAM-1) and selective (>200-fold vs VCAM-1) inhibitors with a good pharmacokinetic profile. Demonstrated efficacy in a rat rheumatoid arthritis model and in a mice asthma model with selected compounds is also reported.
A series of potent and highly CNS penetrable melanin-concentrating hormone inhibitors is described.
Prior SAR studies on 2-amino-8-alkoxyquinoline MCHr1 antagonists demonstrated that compounds with ...acyclic amide-containing sidechains displayed exceptional binding and functional potency, but negligible CNS penetration. Related analogs with acyclic benzylamine-containing sidechains showed greatly improved CNS exposure, but suffered in functional potency. In this report, we demonstrate that cyclization of these benzylic amine sidechains affords compounds that combine the best elements of potency and CNS penetration among this class of antagonists. This is exemplified by compound
21, which has sub-nanomolar MCHr1 binding affinity, good functional potency, and excellent CNS exposure over 24
h.
Melanin-concentrating hormone (MCH), an orexigenic neuropeptide in mammals, activates a G-protein coupled receptor, MCHR1. It is expected that antagonists of MCHR1 function will prove therapeutically ...useful as anti-obesity agents. Intracellular signaling by MCHR1 has been investigated primarily using non-neural cell lines expressing the recombinant receptor, in which MCHR1 has been shown to couple to Gα
i/o and Gα
q G-proteins. While these cell lines have been widely utilized to discover and optimize small molecule antagonists, it is unknown whether the intracellular signaling pathways in these cells accurately reflect those in neurons. Thus, we sought to develop a neurally derived cell line endogenously expressing MCHR1. IMR32, a human neuroblastoma cell line, has been shown to express MCHR1 mRNA; however, we were unable to detect either MCH-binding or MCH-stimulated Ca
++-mobilization in these cells. Following transfection of IMR32 cells with a plasmid encoding human Gα
16 G-protein, we isolated a cell line, I3.4.2, which responded to MCH in Ca
++-mobilization assays. We found that the expression level of MCHR1 mRNA in I3.4.2 cells was 2000-fold higher than in the parent cell line. Using
125IMCH saturation-binding to I3.4.2 cell membranes, we estimated the
B
max as 0.72
pmol/mg protein and the
K
d as 0.35
nM. We report that Ca
++-mobilization in I3.4.2 cells was insensitive to pertussis toxin (Ptx) treatment, indicating that signaling was via Gα
q G-proteins. Furthermore, negative results in cAMP accumulation assays confirmed the lack of signaling via the Gα
i/o G-proteins. Our results suggest that the I3.4.2 cell line may be useful for characterization of MCHR1 activity in a neural-derived cell line.
Abstract
Introduction
Immunotherapy offers the potential for durable responses in a growing list of cancer indications.1 One approach towards stimulating the immune system is to target the ...heterotrimeric interleukin2 receptor, IL2R. Binding of ligand to the β subunit leads to expansion of tumor killing memory effector T cells, whereas engagement of the α subunit expands undesirable regulatory T cells, promoting immune suppression in the tumor.2 NKTR-214 uses polymer technology to engineer a cytokine that has reduced affinity for the IL2Rα subunit, diminishing the expansion of regulatory T cells in the tumor, while maintaining affinity for the β subunit expands memory effector T cells. The selective immunological profile in the tumor tips the balance towards tumor killing and results in superior efficacy and tolerability in a resistant mouse melanoma model.
Methods
The affinity of NKTR-214 to IL2Rα and IL2Rβ was measured directly by SPR and compared to that of clinically available IL2 (Aldesleukin). C57BL/6 mice bearing subcutaneous B16F10 melanoma were treated with NKTR-214 (IV, Q9dx3), Aldesleukin (BIDx5, 2 cycles) or vehicle. Immune cell populations in the tumor microenvironment were characterized by flow cytometry. Anti-tumor efficacy was measured by monitoring tumor volumes over time.
Data
The affinity of the activated form of NKTR-214 to IL2Rα was reduced by ∼8x compared to clinically available Aldesleukin. Mice receiving NKTR-214 showed an increase of ∼8-fold in CD8 memory effector T cells in the tumor compared to Aldesleukin .Conversely, mice receiving NKTR-214 showed a ∼6-fold decrease of CD4 regulatory T cells in the tumor compared to Aldesleukin. Improved survival and substantial tumor growth delay were observed in the NKTR-214 treated mice as compared to Aldesleukin, despite using 10-fold fewer moles of protein. The mice in the NKTR-214 treated group were not moribund, did not shiver, and showed an 84% decrease in mortality as compared to the Aldesleukin treated group.
Conclusions
Engineering a cytokine through polymer modification leads to a differential IL2 receptor binding profile and an improved tumor infiltrating T cell profile. The modification increases CD8 memory effector T cells and decreases suppressive regulatory T cells in the tumor compared to a clinically available IL2 (Aldesleukin). The reduced level of immune suppression in the tumor may explain the substantially improved therapeutic index observed for NKTR-214, despite using 10-fold fewer moles of protein, and infrequent dosing (Q9Dx3). The drug was well tolerated by the mice, with an 84% decrease in mortality compared to Aldesleukin. The results demonstrate that polymer engineering of a cytokine can have a profound differentiating effect on its in vitro and in vivo behavior.
1L. Calabro, Semin Oncol 2010 37 460-467
2O. Boyman, J. Sprent Nature Reviews Immunology 2012 12 180
Citation Format: Deborah Charych, Steven Lee, Murali Addepalli, Thomas Chang, Peter Kirk, Marina Konakova, Laurie Vanderveen, Rupesh Kanhere, Dennis Fry, Cherie Ali, Stephen Harrison, Seema Kantak, Stephen Doberstein. Tipping the balance in the tumor microenvironment: An engineered cytokine (NKTR-214) with altered IL2 receptor binding selectivity and improved efficacy. abstract. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 482. doi:10.1158/1538-7445.AM2013-482
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