Illuminating somatostatin analog action at neuroendocrine tumor receptors

Somatostatin analogs for the diagnosis and therapy of neuroendocrine tumors (NETs) have been used in clinical applications for more than two decades. Five somatostatin receptor subtypes have been identified and molecular mechanisms of somatostatin receptor signaling and regulation have been elucidated. These advances increased understanding of the biological role of each somatostatin receptor subtype, their distribution in NETs, as well as agonist-specific regulation of receptor signaling, internalization, and phosphorylation, particularly for the sst2 receptor subtype, which is the primary target of current somatostatin analog therapy for NETs. Various hypotheses exist to explain differences in patient responsiveness to somatostatin analog inhibition of tumor secretion and growth as well as differences in the development of tumor resistance to therapy. In addition, we now have a better understanding of the action of both first generation (octreotide, lanreotide, Octreoscan) and second generation (pasireotide) FDA-approved somatostatin analogs, including the biased agonistic character of some agonists. The increased understanding of somatostatin receptor pharmacology provides new opportunities to design more sophisticated assays to aid the future development of somatostatin analogs with increased efficacy.

First Clinical Experience With Celt ACD(®) : A Femoral Arterial Puncture Closure Device

Introduction This prospective nonrandomized study compared the safety and efficacy of a novel arterial closure device (ACD) in common femoral artery procedures to that of the FDA submitted historical manual pressure control group, who underwent either a diagnostic angiogram (DA) or a percutaneous coronary intervention (PCI) procedure. Methods and Results A total of 55 patients were enrolled in this study of the novel ACD. Of the 55 patients, 39 were enrolled in the DA group and 16 were enrolled in the PCI group. Six patients were excluded. A device was deployed in 49 patients. Time to hemostasis (TTH), time to ambulation (TTA), device function, and device-related vascular complications were measured. In the device group, the TTH for the combined DA and PCI patients was 32 seconds (0.54 ± 0.93 minutes), significantly lower when compared with 16.0 ± 12.2 minutes (P < 0.0001) for the control group. Overall major vascular complication rate did not differ significantly, device group (1/49) and the historical control group (1/217). TTA in the combined PCI and DA device group was 226.4 ± 231.9 at the German site (site ambulation policy). In the Irish site, the average TTA in the PCI group was 187 minutes (n = 8) and 85 minutes (n = 14) in the DA group. Conclusion The Celt ACD® device is safe, effective, and significantly decreases the TTH compared to manual pressure and has a low vascular complications rate. The device may be effective in early ambulation and discharge of patients postcoronary intervention procedures.

Nuclear antisense effects in cyclophilin A pre-mRNA splicing by oligonucleotides: A comparison of tricyclo-DNA with LNA

The nuclear antisense properties of a series of tricyclo(tc)-DNA oligonucleotide 9-15mers, targeted against the 3' and 5' splice sites of exon 4 of cyclophilin A (CyPA) pre-mRNA, were evaluated in HeLa cells and compared with those of corresponding LNA-oligonucleotides. While the 9mers showed no significant antisense effect, the 11-15mers induced exon 4 skipping and exon 3+4 double skipping to about an equal extent upon lipofectamine mediated transfection in a sequence and dose dependent manner, as revealed by a RT-PCR assay. The antisense efficacy of the tc-oligonucleotides was found to be superior to that of the LNA-oligonucleotides in all cases by a factor of at least 4-5. A tc-oligonucleotide 15mer completely abolished CyPA mRNA production at 0.2‘M concentration. The antisense effect was confirmed by western blot analysis which revealed a reduction of CyPA protein to 13% of its normal level. Fluorescence microscopic investigations with a fluorescein labeled tc-15mer revealed a strong propensity for homogeneous nuclear localization of this backbone type after lipofectamine mediated transfection, while the corresponding lna 15mer showed a less clear cellular distribution pattern. Transfection without lipid carrier showed no significant internalization of both tc- and LNA-oligonucleotides. The obtained results confirm the power of tricyclo-DNA for nuclear antisense applications. Morover, CyPA may become an interesting therapeutic target due to its important role in the early steps of the viral replication of HIV-1.

Vitreoretinal Interface Changes in Geographic Atrophy.

PURPOSE Geographic atrophy (GA) is the end-stage manifestation of atrophic age-related macular degeneration (AMD). The disease progresses slowly over time, eventually causing loss of central vision. Its cause and pathomechanism are not fully known. Previous studies have suggested that vitreoretinal traction (VRT) may contribute to the progression of neovascular AMD. The aim of this study was to examine whether an association between changes at the vitreoretinal interface (VRI), in particular traction (VRT), and the characteristics and progression of GA in eyes with dry AMD can be established. DESIGN Clinic-based prospective cohort study. PARTICIPANTS A total of 97 patients (age range, 61-90 years; mean, 78.4 years) with GA secondary to dry AMD were enrolled. Patients exhibiting neovascular signs on fluorescein angiography in either eye were excluded. METHODS The VRI changes were examined using spectral-domain optical coherence tomography (SD-OCT). Characteristics of GA were examined using fundus autofluorescence (FAF) imaging. All imaging was performed using a Spectralis SLO+OCT device (Heidelberg Engineering, Heidelberg, Germany); GA area was measured using the Region Finder (Heidelberg Engineering) software native to the Spectralis platform. MAIN OUTCOME MEASURES Area and increase in area of GA. RESULTS A total of 97 eyes were examined. Vitreoretinal traction was found in 39 eyes (40%). The GA area at baseline was 6.65±5.64 mm(2) in eyes with VRT and 5.73±4.72 mm(2) in eyes with no VRT. The annual rate of progression of GA area progression was 2.99±0.66 mm(2) in eyes with VRT and 1.45±0.67mm(2) in eyes without VRT. Differences between groups in both parameters were statistically significant (n = 97 total number of eyes; P<0.001). Multiple regression analysis confirmed this finding (B = 0.714, P<0.001; F3,93 = 72.542, P<0.001; adjusted R(2) = 0.691) CONCLUSIONS: Our results indicate an association between VRT and an increased rate of progression of GA area in dry AMD. Monitoring VRT may contribute to an improved estimate of the prospective time of visual loss and to a better timing of emerging therapies in dry AMD.

Effect of synthetic agonists of toll-like receptor 9 on canine lymphocyte proliferation and cytokine production in vitro.

Synthetic agonists of TLR9 containing novel DNA structures and R'pG (wherein R=1-(2'-deoxy-beta-d-ribofuranosyl)-2-oxo-7-deaza-8-methyl-purine) motifs, referred to as immune modulatory oligonucleotides (IMOs), have been shown to stimulate T(H)-1-type-immune responses and potently reverse allergen-induced T(H)-2 responses to T(H)-1 responses in vitro and in vivo in mice. In order to investigate the immunomodulatory potential of IMOs in dogs, canine peripheral blood mononuclear cells (PBMC) from healthy dogs were stimulated with three different IMOs and a control IMO, alone or in combination with concanavalin A (ConA). Lipopolysaccharide (LPS) was used as a positive control for B lymphocyte activation. Carboxyfluorescein diacetate succinimidyl ester and phenotype staining was used to tag proliferating T and B lymphocytes (CD5(+) and CD21(+)) by flow cytometry. Real-time PCR and ELISA were processed to assay cytokine production of IFN-gamma, IL-10, TGF-beta, IL-6 and IL-10. Like LPS, IMOs alone induced neither proliferation of CD5(+) T cells nor CD21(+) B cells, but both LPS and IMO had the capacity to co-stimulate ConA and induced proliferation of B cells. In combination with ConA, one of the IMOs (IMO1) also induced proliferation of T cells. IMO1 also significantly enhanced the expression of IFN-gamma on the mRNA and protein level in canine PBMC, whereas expression of IL-10, TGF-beta and IL-4 mRNAs was not induced by any of the IMOs. These results indicate that in canine PBMC from healthy dogs, IMO1 was able to induce a T(H)-1 immune response including T- and B-cell proliferation.

Characterizing New Fluorescent Tools for Studying 5-HT3 Receptor Pharmacology

The pharmacological characterization of ligands depends upon the ability to accurately measure their binding properties. Fluorescence provides an alternative to more traditional approaches such as radioligand binding. Here we describe the binding and spectroscopic properties of eight fluorescent 5-HT3 receptor ligands. These were tested on purified receptors, expressed receptors on live cells, or in vivo. All compounds had nanomolar affinities with fluorescent properties extending from blue to near infra-red emission. A fluorescein-derivative had the highest affinity as measured by fluorescence polarization (FP; 1.14 nM), flow cytometry (FC; 3.23 nM) and radioligand binding (RB; 1.90 nM). Competition binding with unlabeled 5-HT3 receptor agonists (5-HT, mCPBG, quipazine) and antagonists (granisetron, palonosetron, tropisetron) yielded similar affinities in all three assays. When cysteine substitutions were introduced into the 5-HT3 receptor binding site the same changes in binding affinity were seen for both granisetron and the fluorescein-derivative, suggesting that they both adopt orientations that are consistent with co-crystal structures of granisetron with a homologous protein (5HTBP). As expected, in vivo live imaging in anaesthetized mice revealed staining in the abdominal cavity in intestines, but also in salivary glands. The unexpected presence of 5-HT3 receptors in mouse salivary glands was confirmed by Western blots. Overall, these results demonstrate the wide utility of our new high-affinity fluorescently-labeled 5-HT3 receptor probes, ranging from in vitro receptor pharmacology, including FC and FP ligand competition, to live imaging of 5-HT3 expressing tissues.

Treatment of exudative age-related macular degeneration with a designed ankyrin repeat protein that binds vascular endothelial growth factor: a phase I/II study

PURPOSE To evaluate the safety, tolerability and bioactivity of ascending doses of MP0112, a designed ankyrin repeat protein (DARPin) that binds with high affinity to vascular endothelial growth factor-A (VEGF-A), in treatment-naive patients with exudative age-related macular degeneration (AMD). DESIGN Phase I/II, open-label, multicenter, dose-escalation study. METHODS Patients were to receive a single intravitreal injection of MP0112 at doses ranging from 0.04 to 3.6 mg and be monitored for 16 weeks for safety, efficacy, pharmacokinetics, and dose response. RESULTS Altogether, 32 patients received a single injection of MP0112. The maximum tolerated dose was 1.0 mg because of a case of endophthalmitis in the 2.0 mg cohort. Drug-related adverse events were reported by 13 (41%) of 32 patients; they included ocular inflammation in 11 patients (7 mild, 4 moderate in severity). Visual acuity scores were stable or improved compared with baseline for ≥4 weeks following injection; both retinal thickness and fluorescein angiography leakage decreased in a dose-dependent manner. Rescue therapy was administered to 20 (91%) of 22 patients who received 0.04-0.4 mg MP0112 compared with 4 of 10 (40%) patients who received 1.0 or 2.0 mg. Of patients in the higher-dose cohorts who did not require rescue treatment, 83% (5/6) maintained reductions in central retinal thickness through week 16. CONCLUSIONS A single injection of 1.0 or 2.0 mg MP0112 resulted in mean decreases in retinal thickness and leakage area despite ocular inflammation. Larger-scale studies are warranted to confirm these observations.

Novel Nonbiologic Therapies for Ulcerative Colitis

An extensive array of compounds has been studied for the treatment of ulcerative colitis (UC). The most frequently used nonbiologic drugs for the oral and intravenous treatment of ulcerative colitis include 5-aminosalicylate (5-ASA) drugs (mesalamine and derivatives), sulfasalazine, and other azo-bonded molecules of 5-ASA, steroids, calcineurin inhibitors (cyclosporine, tacrolimus, and sirolimus), thiopurines (azathioprine, 6-mercaptopurine), and methotrexate, which are already presented in other sections of this book and are thus not considered in this chapter. The therapies presented in this section should be considered as potential alternatives, mostly for mild-to-moderate ulcerative colitis (UC). They are substances mostly used without FDA indications, such as heparin, nicotine, rosiglitazone, and N-acetylcysteine as well as “natural” compounds suggested to have anti-inflammatory or reparative properties, such as aloe vera, curcumin, short-chain fatty acids, and Bowman-Birk inhibitor

Investigation of retinal morphology alterations using spectral domain optical coherence tomography in a mouse model of retinal branch and central retinal vein occlusion.

Retinal vein occlusion is a leading cause of visual impairment. Experimental models of this condition based on laser photocoagulation of retinal veins have been described and extensively exploited in mammals and larger rodents such as the rat. However, few reports exist on the use of this paradigm in the mouse. The objective of this study was to investigate a model of branch and central retinal vein occlusion in the mouse and characterize in vivo longitudinal retinal morphology alterations using spectral domain optical coherence tomography. Retinal veins were experimentally occluded using laser photocoagulation after intravenous application of Rose Bengal, a photo-activator dye enhancing thrombus formation. Depending on the number of veins occluded, variable amounts of capillary dropout were seen on fluorescein angiography. Vascular endothelial growth factor levels were markedly elevated early and peaked at day one. Retinal thickness measurements with spectral domain optical coherence tomography showed significant swelling (p<0.001) compared to baseline, followed by gradual thinning plateauing two weeks after the experimental intervention (p<0.001). Histological findings at day seven correlated with spectral domain optical coherence tomography imaging. The inner layers were predominantly affected by degeneration with the outer nuclear layer and the photoreceptor outer segments largely preserved. The application of this retinal vein occlusion model in the mouse carries several advantages over its use in other larger species, such as access to a vast range of genetically modified animals. Retinal changes after experimental retinal vein occlusion in this mouse model can be non-invasively quantified by spectral domain optical coherence tomography, and may be used to monitor effects of potential therapeutic interventions.

Porcine extrahepatic vascular endothelial asialoglycoprotein receptor 1 mediates xenogeneic platelet phagocytosis in vitro and in human-to-pig ex vivo xenoperfusion.

BACKGROUND Asialoglycoprotein receptor-1 (ASGR1) mediates capture and phagocytosis of platelets in pig-to-primate liver xenotransplantation. However, thrombocytopenia is also observed in xenotransplantation or xenoperfusion of other porcine organs than liver. We therefore assessed ASGR1 expression as well as ASGR1-mediated xenogeneic platelet phagocytosis in vitro and ex vivo on porcine aortic, femoral arterial, and liver sinusoidal endothelial cells (PAEC/PFAEC/PLSEC). METHODS Porcine forelimbs were perfused with whole, heparinized human or autologous pig blood. Platelets were counted at regular intervals. Pig limb muscle and liver, as well as PAEC/PFAEC/PLSEC, were characterized for ASGR1 expression. In vitro, PAEC cultured on microcarrier beads and incubated with non-anticoagulated human blood were used to study binding of human platelets and platelet-white blood cell aggregation. Carboxyfluorescein diacetate succinimidyl ester-labeled human platelets were exposed to PAEC/PFAEC/PLSEC and analyzed for ASGR1-mediated phagocytosis. RESULTS Human platelet numbers decreased from 102 ± 33 at beginning to 13 ± 6 × 10/μL (P < 0.0001) after 10 minutes of perfusion, whereas no significant decrease of platelets was seen during autologous perfusions (171 ± 26 to 122 ± 95 × 10/μL). The PAEC, PFAEC, and PLSEC all showed similar ASGR1 expression. In vitro, no correlation was found between reduction in platelet count and platelet-white blood cell aggregation. Phagocytosis of human carboxyfluorescein diacetate succinimidyl ester-labeled platelets by PAEC/PFAEC/PLSEC peaked at 15 minutes and was inhibited (P < 0.05 to P < 0.0001) by rabbit anti-ASGR1 antibody and asialofetuin. CONCLUSIONS The ASGR1 expressed on aortic and limb arterial pig vascular endothelium plays a role in binding and phagocytosis of human platelets. Therefore, ASGR1 may represent a novel therapeutic target to overcome thrombocytopenia associated with vascularized pig-to-primate xenotransplantation.

Synthesis of photo-crosslinking probes and their application for the site-selective chemical modification of the 5-HT3 receptor

The 5-HT3 receptor (5-HT3R) is an important ion channel responsible for the transmission of nerve impulses in the CNS and PNS that is activated by the endogenous agonist serotonin (5-hydroxytryptamine, 5-HT). 5-HT3R is the only serotonin receptor belonging to the Cys-loop superfamily of neurotransmitter receptors. Different structural biology approaches can be applied, such as crystallization and x-ray analysis. Nonetheless, characterizing the exact ligand binding site(s) of these dynamic receptors is still challenging. The use of photo-crosslinking probes is an alternative validated approach allowing identification of regions in the protein that are important for the binding of small molecules. We designed our probes based on the core structure of the 5-HT3R antagonist granisetron, a FDA approved drug used for the treatment of chemotherapy-induced nausea and vomiting. We synthesized a small library of photo-crosslinking probes by conjugating diazirines and benzophenones via various linkers to granisetron. We were able to obtain several compounds with diverse linker lengths and different photo-crosslinking moieties that show nanomolar binding affinity for the orthosteric binding site. Furthermore we established a stable h5-HT3R expressing cell line and a purification protocol to yield the receptor in a high purity. Several experiments showed unambiguously that we are able to photo-crosslink our probes with the receptor site-specifically. The functionalised protein was analysed by Western blot and MS-analysis. This yielded the exact covalent modification site, corroborating current ligand binding models derived from mutagenesis and docking studies.

Prognostic significance of foveal capillary drop-out and previous panretinal photocoagulation for diabetic macular oedema treated with ranibizumab

AIMS To investigate the prognostic significance of macular capillary drop-out and previous panretinal laser photocoagulation in diabetic macular oedema treated with intravitreal ranibizumab. METHODS Retrospective observational case series. Treatment-naive patients with diabetic macular oedema that had been treated with intravitreal ranibizumab as per the RESTORE study protocol for at least 12 months were included. Some patients (n=15) had previous panretinal laser photocoagulation. Best-corrected visual acuity and central retina thickness were recorded monthly. The foveal avascular zone and the perifoveal capillaries were quantitatively and qualitatively assessed on fluorescein angiography on two occasions during the observational period. RESULTS From the 46 eyes (46 patients) in this study, 13 (28%) had evidence of perifoveal capillary drop-out. Central retinal thickness was significantly thinner at baseline (p=0.02) and throughout the study period in these eyes compared with those with normal perifoveal capillaries. Both groups responded with a significant gain of best-corrected visual acuity to ranibizumab treatment (7.6±3.3 and 6.3±1.3 ETDRS letters, respectively). Eyes with previous panretinal laser photocoagulation displayed a comparable final outcome regarding function and morphology, requiring a similar intensity of intravitreal injections. CONCLUSIONS Perifoveal capillary drop-out did not limit the gain of visual acuity from intravitreal ranibizumab treatment. The reduction of central retina thickness was similar to that seen in eyes with normal perifoveal capillaries. Central retinal thickness in eyes with perifoveal capillary drop-out was generally reduced. However, this did not affect their benefit from treatment. Ranibizumab did not increase the amount of perifoveal capillary loss.

Time-Resolved Ultra–High Resolution Optical Coherence Tomography for Real-Time Monitoring of Selective Retina Therapy

Purpose: Selective retina therapy (SRT) is a novel treatment for retinal pathologies, solely targeting the retinal pigment epithelium (RPE). During SRT, the detection of an immediate tissue reaction is challenging as tissue effects remain limited to intracellular RPE photodisruption. Time-resolved ultra-high axial resolution optical coherence tomography (OCT) is thus evaluated for the monitoring of dynamic optical changes at and around the RPE during SRT. Methods: An experimental OCT system with an ultra-high axial resolution of 1.78 µm was combined with an SRT system and time-resolved OCT M-scans of the target area were recorded from four patients undergoing SRT. OCT scans were analyzed and OCT morphology was correlated with findings in fluorescein angiography, fundus photography and cross-sectional OCT. Results: In cases where the irradiation caused RPE damage proven by fluorescein angiography, the lesions were well discernible in time-resolved OCT images but remained invisible in fundus photography and cross-sectional OCT acquired after treatment. If RPE damage was introduced, all applied SRT pulses led to detectable signal changes in the time-resolved OCT images. The extent of optical signal variation seen in the OCT data appeared to scale with the applied SRT pulse energy. Conclusion: The first clinical results proved that successful SRT irradiation induces detectable changes in the OCT M-scan signal while it remains invisible in conventional ophthalmoscopic imaging. Thus, real-time high-resolution OCT is a promising modality to monitor and analyze tissue effects introduced by selective retina therapy and may be used to guide SRT in an automatic feedback mode.

Report of the WPA section of pharmacopsychiatry on the relationship of antiepileptic drugs with suicidality in epilepsy

INTRODUCTION This report from the World Psychiatric Association Section on Pharmacopsychiatry examines the possible relationship of antiepileptic drugs with suicide-related clinical features and behaviors in patients with epilepsy. MATERIALS AND METHODS A systematic review of the MEDLINE search returned 1039 papers, of which only 8 were considered relevant. A critical analysis of the Food and Drug Administration (FDA) report on the increase risk for patients under antiepileptics to manifest suicidality is also included in this report. RESULTS The analysis of these studies revealed that the data are not supportive of the presence of a "class effect" on suicide-related behavior; on the contrary, there are some data suggesting such an effect concerning treatment with topiramate, lamotrigine, and levetiracetam for which further research is needed. DISCUSSION For the majority of people with epilepsy, anticonvulsant treatment is necessary and its failure for any reason is expected to have deleterious consequences. Therefore, clinicians should inform patients and their families of this increased risk of suicidal ideation and behavior, but should not overemphasize the issue. Specific subgroups of patients with epilepsy might be at a higher risk, and deserve closer monitoring and follow-up. Future research with antiepileptics should specifically focus on depression and suicidal thoughts.

PreImplantation Factor bolsters neuroprotection via modulating Protein Kinase A and Protein Kinase C signaling

A synthetic peptide (sPIF) analogous to the mammalian embryo-derived PreImplantation Factor (PIF) enables neuroprotection in rodent models of experimental autoimmune encephalomyelitis and perinatal brain injury. The protective effects have been attributed, in part, to sPIF's ability to inhibit the biogenesis of microRNA let-7, which is released from injured cells during central nervous system (CNS) damage and induces neuronal death. Here, we uncover another novel mechanism of sPIF-mediated neuroprotection. Using a clinically relevant rat newborn brain injury model, we demonstrate that sPIF, when subcutaneously administrated, is able to reduce cell death, reverse neuronal loss and restore proper cortical architecture. We show, both in vivo and in vitro, that sPIF activates cyclic AMP dependent protein kinase (PKA) and calcium-dependent protein kinase (PKC) signaling, leading to increased phosphorylation of major neuroprotective substrates GAP-43, BAD and CREB. Phosphorylated CREB in turn facilitates expression of Gap43, Bdnf and Bcl2 known to have important roles in regulating neuronal growth, survival and remodeling. As is the case in sPIF-mediated let-7 repression, we provide evidence that sPIF-mediated PKA/PKC activation is dependent on TLR4 expression. Thus, we propose that sPIF imparts neuroprotection via multiple mechanisms at multiple levels downstream of TLR4. Given the recent FDA fast-track approval of sPIF for clinical trials, its potential clinical application for treating other CNS diseases can be envisioned.