Novel sst2-selective somatostatin agonists. Three-dimensional consensus structure by NMR

The 3D NMR structures of six octapeptide agonist analogues of somatostatin (SRIF) in the free form are described. These analogues, with the basic sequence H-DPhe/Phe2-c[Cys3-Xxx7-DTrp8-Lys9-Thr10-Cys14]-Thr-NH2 (the numbering refers to the position in native SRIF), with Xxx7 being Ala/Aph, exhibit potent and highly selective binding to human SRIF type 2 (sst2) receptors. The backbone of these sst2-selective analogues have the usual type-II' beta-turn reported in the literature for sst2/3/5-subtype-selective analogues. Correlating the biological results and NMR studies led to the identification of the side chains of DPhe2, DTrp8, and Lys9 as the necessary components of the sst2 pharmacophore. This is the first study to show that the aromatic ring at position 7 (Phe7) is not critical for sst2 binding and that it plays an important role in sst3 and sst5 binding. This pharmacophore is, therefore, different from that proposed by others for sst2/3/5 analogues.

177Lu-AMBA: Synthesis and characterization of a selective 177Lu-labeled GRP-R agonist for systemic radiotherapy of prostate cancer

Gastrin-releasing peptide receptors (GRP-R) are upregulated in many cancers, including prostate, breast, and lung. We describe a new radiolabeled bombesin (BBN) analog for imaging and systemic radiotherapy that has improved pharmacokinetics (PK) and better retention of radioactivity in the tumor. METHODS: DO3A-CH2CO-G-4-aminobenzoyl-Q-W-A-V-G-H-L-M-NH2 (AMBA) was synthesized and radiolabeled. The human prostate cancer cell line PC-3 was used to determine the binding (Kd), retention, and efflux of 177Lu-AMBA. Receptor specificity was determined by in vitro autoradiography in human tissues. PK and radiotherapy studies were performed in PC-3 tumor-bearing male nude mice. RESULTS: 177Lu-AMBA has a high affinity for the GRP-R (Kd, 1.02 nmol/L), with a maximum binding capacity (Bmax) of 414 fmol/10(6) cells (2.5 x 10(5) GRP-R/cell). Internalization was similar for 177Lu-AMBA (76.8%), 177Lu-BBN8 (72.9%), and 125I-[Tyr4]-BBN (74.9%). Efflux was markedly lower for 177Lu-AMBA (2.9%) compared with 177Lu-BBN8 (15.9%) and 125I-[Tyr4]-BBN (46.1%). By receptor autoradiography, Lu-AMBA binds specifically to GRP-R (0.8 nmol/L) and to the neuromedin B receptor (NMB-R) (0.9 nmol/L), with no affinity for the bb3 receptor (>1,000 nmol/L). 177Lu-AMBA was renally excreted (55 %ID 1 h [percentage injected dose at 1 h]); tumor uptake at 1 and 24 h was 6.35 %ID/g and 3.39 %ID/g, respectively. One or 2 doses of 177Lu-AMBA (27.75 MBq/dose) significantly prolonged the life span of PC-3 tumor-bearing mice (P < 0.001 and P < 0.0001, respectively) and decreased PC-3 tumor growth rate over controls. When compared using World Health Organization criteria, mice receiving 2 doses versus 1 dose of 177Lu-AMBA demonstrated a shift away from stable/progressive disease toward complete/partial response; by RECIST (Response Evaluation Criteria in Solid Tumors), median survival increased by 36% and time to progression/progression-free survival increased by 65%. CONCLUSION: 177Lu-AMBA binds with nanomolar affinity to GRP-R and NMB-R, has low retention of radioactivity in kidney, demonstrates a very favorable risk-benefit profile, and is in phase I clinical trials.

New sst4/5-selective somatostatin peptidomimetics based on a constrained tryptophan scaffold

The synthesis and biological evaluation of four peptidomimetic analogs of somatostatin based on a constrained Trp residue, 3-amino-indolo[2,3-c]azepin-2-one (Aia), are reported. It is shown that dipeptidomimetics with a D-Aia-Lys sequence, functionalized with N- and C-terminal aromatic substituents, display a good selectivity for both sst4 and sst5. This study allowed us to identify a new highly potent sst5 agonist with good selectivity over the other receptors, except versus sst4.

Novel sst5-selective somatostatin dicarba-analogues: synthesis and conformation-affinity relationships

We describe synthesis, conformational studies, and binding to the five somatostatin receptors (sst 1-5) of a few analogues of the cyclic octapeptide octreotide (1), where the disulfide bridge was replaced by a dicarba group. These analogues were prepared by on-resin RCM of linear hepta-peptides containing two allylglycine residues; first- and second-generation Grubbs catalyst efficiencies were compared. The C=C bridge was hydrogenated via two different methods. Binding experiments showed that two analogues had good affinity and high selectivity for the sst5 receptor. Three-dimensional structures of the active analogues were determined by (1)H NMR spectroscopy. Conformation-affinity relationships confirmed the importance of D-Phe(2) orientation for sst2 affinity. Moreover, helical propensities well correlates with the peptide sst5 affinity. The presence of the bulky aromatic side chain of Tyr(Bzl)(10) favored the formation of a 3(10)-helix and enhanced the sst5 selectivity suppressing the sst2 affinity. Finally, a new pharmacophore model for the sst5 was developed.

Design and in vitro characterization of highly sst2-selective somatostatin antagonists suitable for radiotargeting

Radiolabeled sst 2 and sst 3 antagonists are better candidates for tumor targeting than agonists with comparable binding characteristics (Ginj, M.; Zhang, H.; Waser, B.; Cescato, R.; Wild, D.; Erchegyi, J.; Rivier, J.; Mäcke, H. R.; Reubi, J. C. Proc. Natl. Acad. Sci. U.S.A. 2006, 103, 16436-16441.). Because most of the neuroendocrine tumors express sst 2, we used the known antagonists acetyl- pNO 2Phe (2)- c[ dCys (3)-Tyr (7)- dTrp (8)-Lys (9)-Thr (10)-Cys (14)]- dTyr (15)-NH 2 ( 1) (Bass, R. T.; Buckwalter, B. L.; Patel, B. P.; Pausch, M. H.; Price, L. A.; Strnad, J.; Hadcock, J. R. Mol. Pharmacol. 1996, 50, 709-715. Bass, R. T.; Buckwalter, B. L.; Patel, B. P.; Pausch, M. H.; Price, L. A.; Strnad, J.; Hadcock, J. R. Mol. Pharmacol. 1997, 51, 170; Erratum.) and H-Cpa (2)- c[ dCys (3)-Tyr (7)- dTrp (8)-Lys (9)-Thr (10)-Cys (14)]-2Nal (15)-NH 2 ( 7) (Hocart, S. J.; Jain, R.; Murphy, W. A.; Taylor, J. E.; Coy, D. H. J. Med. Chem. 1999, 42, 1863-1871.) as leads for analogues with increased sst 2 binding affinity and selectivity. Among the 32 analogues reported here, DOTA- pNO 2Phe (2)- c[ dCys (3)-Tyr (7)- dAph (8)(Cbm)-Lys (9)-Thr (10)-Cys (14)- dTyr (15)-NH 2 ( 3) and DOTA-Cpa (2)- c[ dCys (3)-Aph (7)(Hor)- dAph (8)(Cbm)-Lys (9)-Thr (10)-Cys (14)]- dTyr (15)-NH 2 ( 31) had the highest sst 2 binding affinity and selectivity. All of the analogues tested kept their sst 2 antagonistic properties (i.e., did not affect calcium release in vitro and competitively antagonized the agonistic effect of [Tyr (3)]octreotide). Moreover, in an immunofluorescence-based internalization assay, the new analogues prevented sst 2 internalization induced by the sst 2 agonist [Tyr (3)]octreotide without being active by themselves. In conclusion, several analogues (in particular 3, 31, and 32) have outstanding sst 2 binding and functional antagonistic properties and, because of their DOTA moiety, are excellent candidates for in vivo targeting of sst 2-expressing cancers.

THE DISPERSION AND SELECTIVE FLOCCULATION OF HEMATITE ORE

Iron ore is one of the most important ores in the world. Over the past century, most mining of iron ore has been focused on magnetite (Fe3O4). As the name suggests, magnetite is magnetic in nature and is easily separated from gangue (unwanted) minerals through magnetic separation processes. Unfortunately, the magnetite ore bodies are diminishing. Because of this, there has been a recent drive to pursue technology that can economically separate hematite (Fe2O3) from its gangue minerals as hematite is a much more abundant source of iron. Most hematite ore has a very small liberation size that is frequently less than 25μm. Beneficiation of any ore with this fine of a liberation size requires advanced processing methods and is seldom pursued. A single process, known as selective flocculation and dispersion, has been successfully implemented at a plant scale for the beneficiation of fine liberation size hematite ore. Very little is known about this process as it was discovered by the U.S. Bureau of Mines by accident. The process is driven by water chemistry and surface chemistry modifications that enhance the separation of the hematite from its gangue minerals. This dissertation focuses on the role of water chemistry and process reagents in this hematite beneficiation process. It has been shown that certain ions, including calcium and magnesium, play a significant role in the process. These ions have a significant effect on the surface chemistry as reported by zeta potential studies. It was shown that magnesium ions within the process water have a more significant impact on surface chemistry than calcium ions due to steric hindrance effects at the hematite surface. It has also been shown that polyacrylic acid dispersants, if used in the process, can increase product quality (increase iron content, decrease phosphorus content, decrease silica content) substantially. Water, surface and reagent chemistry experiments were performed at a laboratory, pilot, and full plant scale during the course of this work. Many of the conclusions developed in the laboratory and pilot scale were found to be true at the full plant scale as well. These studies are the first published in history to develop theories of water chemistry and surface chemistry interactions at a full plant scale.

Novel, potent, and radio-iodinatable somatostatin receptor 1 (sst1) selective analogues

The proposed sst(1) pharmacophore (J. Med. Chem. 2005, 48, 523-533) derived from the NMR structures of a family of mono- and dicyclic undecamers was used to design octa-, hepta-, and hexamers with high affinity and selectivity for the somatostatin sst(1) receptor. These compounds were tested for their in vitro binding properties to all five somatostatin (SRIF) receptors using receptor autoradiography; those with high SRIF receptor subtype 1 (sst(1)) affinity and selectivity were shown to be agonists when tested functionally in a luciferase reporter gene assay. Des-AA(1,4-6,10,12,13)-[DTyr(2),DAgl(NMe,2naphthoyl)(8),IAmp(9)]-SRIF-Thr-NH(2) (25) was radio-iodinated ((125)I-25) and specifically labeled sst(1)-expressing cells and tissues. 3D NMR structures were calculated for des-AA(1,4-6,10,12,13)-[DPhe(2),DTrp(8),IAmp(9)]-SRIF-Thr-NH(2) (16), des-AA(1,2,4-6,10,12,13)-[DAgl(NMe,2naphthoyl)(8),IAmp(9)]-SRIF-Thr-NH(2) (23), and des-AA(1,2,4-6,10,12,13)-[DAgl(NMe,2naphthoyl)(8),IAmp(9),Tyr(11)]-SRIF-NH(2) (27) in DMSO. Though the analogues have the sst(1) pharmacophore residues at the previously determined distances from each other, the positioning of the aromatic residues in 16, 23, and 27 is different from that described earlier, suggesting an induced fit mechanism for sst(1) binding of these novel, less constrained sst(1)-selective family members.

Synthesis and in vitro characterization of radioiodinatable benzodiazepines selective for type 1 and type 2 cholecystokinin receptors

Radiolabeled antagonists of specific peptide receptors identify a higher number of receptor binding sites than agonists and may thus be preferable for in vivo tumor targeting. In this study, two novel radioiodinated 1,4-benzodiazepines, (S)-1-(3-iodophenyl)-3-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)urea (9) and (R)-1-(3-iodophenyl)-3-(1-methyl-2-oxo-5-phenyl-2,3-dihydro-1H-benzo[e][1,4]diazepin-3-yl)urea (7), were developed. They were characterized in vitro as high affinity selective antagonists at cholecystokinin types 1 and 2 (CCK(1) and CCK(2)) receptors using receptor binding, calcium mobilization, and internalization studies. Their binding to human tumor tissues was assessed with in vitro receptor autoradiography and compared with an established peptidic CCK agonist radioligand. The (125)I-labeled CCK(1) receptor-selective compound 9 often revealed a substantially higher amount of CCK(1) receptor binding sites in tumors than the agonist (125)I-CCK. Conversely, the radioiodinated CCK(2) receptor-selective compound 7 showed generally weaker tumor binding than (125)I-CCK. In conclusion, compound 9 is an excellent radioiodinated nonpeptidic antagonist ligand for direct and selective labeling of CCK(1) receptors in vitro. Moreover, it represents a suitable candidate to test antagonist binding to CCK(1) receptor-expressing tumors in vivo.

Looking left with left neglect: the role of spatial attention when active vision selects local image features for fixation

When we actively explore the visual environment, our gaze preferentially selects regions characterized by high contrast and high density of edges, suggesting that the guidance of eye movements during visual exploration is driven to a significant degree by perceptual characteristics of a scene. Converging findings suggest that the selection of the visual target for the upcoming saccade critically depends on a covert shift of spatial attention. However, it is unclear whether attention selects the location of the next fixation uniquely on the basis of global scene structure or additionally on local perceptual information. To investigate the role of spatial attention in scene processing, we examined eye fixation patterns of patients with spatial neglect during unconstrained exploration of natural images and compared these to healthy and brain-injured control participants. We computed luminance, colour, contrast, and edge information contained in image patches surrounding each fixation and evaluated whether they differed from randomly selected image patches. At the global level, neglect patients showed the characteristic ipsilesional shift of the distribution of their fixations. At the local level, patients with neglect and control participants fixated image regions in ipsilesional space that were closely similar with respect to their local feature content. In contrast, when directing their gaze to contralesional (impaired) space neglect patients fixated regions of significantly higher local luminance and lower edge content than controls. These results suggest that intact spatial attention is necessary for the active sampling of local feature content during scene perception.

Development of an array of ion-selective microelectrodes aimed for the monitoring of extracellular ionic activities

In this study, we present the development and the characterization of a generic platform for cell culture able to monitor extracellular ionic activities (K+, NH4+) for real-time monitoring of cell-based responses, such as necrosis, apoptosis, or differentiation. The platform for cell culture is equipped with an array of 16 silicon nitride micropipet-based ion-selective microelectrodes with a diameter of either 2 or 6 microm. This array is located at the bottom of a 200-microm-wide and 350-microm-deep microwell where the cells are cultured. The characterization of the ion-selective microelectrode arrays in different standard and physiological solutions is presented. Near-Nernstian slopes were obtained for potassium- (58.6 +/- 0.8 mV/pK, n = 15) and ammonium-selective microelectrodes (59.4 +/- 3.9 mV/pNH4, n = 13). The calibration curves were highly reproducible and showed an average drift of 4.4 +/- 2.3 mV/h (n = 10). Long-term behavior and response after immersion in physiological solutions are also presented. The lifetime of the sensors was found to be extremely long with a high recovery rate.

Mapping material distribution in a heterogeneous sand tank by image analysis

Water flow and solute transport through soils are strongly influenced by the spatial arrangement of soil materials with different hydraulic and chemical properties. Knowing the specific or statistical arrangement of these materials is considered as a key toward improved predictions of solute transport. Our aim was to obtain two-dimensional material maps from photographs of exposed profiles. We developed a segmentation and classification procedure and applied it to the images of a very heterogeneous sand tank, which was used for a series of flow and transport experiments. The segmentation was based on thresholds of soil color, estimated from local median gray values, and of soil texture, estimated from local coefficients of variation of gray values. Important steps were the correction of inhomogeneous illumination and reflection, and the incorporation of prior knowledge in filters used to extract the image features and to smooth the results morphologically. We could check and confirm the success of our mapping by comparing the estimated with the designed sand distribution in the tank. The resulting material map was used later as input to model flow and transport through the sand tank. Similar segmentation procedures may be applied to any high-density raster data, including photographs or spectral scans of field profiles.

Intracellular chemical messengers and neuronal function: Two examples

Two distinct classes of neurons have been examined in the nervous system of Aplysia. The membrane properties of these neurons are regulated by intracellular signalling molecules in both a short-term and a long-term fashion.^ The role of the phosphatidylinositol cycle in the control of neuronal properties was studied in a class of bursting pacemaker cells, the left upper-quadrant bursting neurons (cells L2, L3, L4, and L6) of the abdominal ganglion of Aplysia. These cells display a regular burst-firing pattern that is controlled by cyclic changes of intracellular Ca$\sp{2+}$ that occur during the bursting rhythm. The characteristic bursting pattern of these neurons occurs within a range of membrane potentials ($-35$ to $-50$ mV) called the pacemaker range. Intracellular pressure injection of inositol 1,4,5-trisphosphate (IP$\sb3$) altered the bursting rhythm of the bursting cells. Injection of IP$\sb3$ induced a brief depolarization that was followed by a long-lasting (2-15 min) hyperpolarization. When cells were voltage-clamped at potentials within the pacemaker range, injection of IP$\sb3$ generally induced a biphasic response that had a total duration of 2-15 min. An initial inward shift in holding current (I$\sb{\rm in}$), which lasted 5-120 sec, was followed by a slow outward shift in holding current (I$\sb{\rm out}$). At membrane potentials more negative than $-40$ mV, I$\sb{\rm in}$ was associated with a small and relatively voltage-independent increase in membrane conductance. I$\sb{\rm in}$ was not blocked by bath application of TTX or Co$\sp{2+}$. Although I$\sb{\rm in}$ was activated by injection of IP$\sb3$, it was not blocked by iontophoretic injection of ethyleneglycol-bis-(beta-aminoethyl ether), N, N$\sp\prime$-tetraacetic acid (EGTA) sufficient to block the Ca$\sp{2+}$-activated inward tail current (I$\sb{\rm B}$).^ Long-term (lasting at least 24 hours) effects of adenylate cyclase activation were examined in a well characterized class of mechanosensory neurons in Aplysia. The injected cells were analyzed 24 hours later by two-electrode voltage-clamp techniques. We found that K$\sp+$ currents of these cells were reduced 24 hours after injection of cAMP. The currents that were reduced by cAMP were very similar to those found to be reduced 24 hours after behavioral sensitization. These results suggest that cAMP is part of the intracellular signal that induces long-term sensitization in Aplysia. (Abstract shortened with permission of author.) ^

Selective elevation of c-{\it myc} RNA transcripts during clonal evolution of N-methyl-N-nitrosourea induced thymic lymphomas in AKR/J mice

Untreated AKR mice develop spontaneous thymic lymphomas by 6-12 months of age. Lymphoma development is accelerated when young mice are injected with the carcinogen N-methyl-N-nitrosourea (MNU). Selected molecular and cellular events were compared during the latent period preceding "spontaneous" (retrovirally-induced) and MNU-induced thymic lymphoma development in AKR mice. These studies were undertaken to test the hypothesis that thymic lymphomas induced in the same inbred mouse strain by endogenous retroviruses and by a chemical carcinogen develop by different mechanisms.^ Immunofluorescence analysis of differentiation antigens showed that most MNU-induced lymphomas express an immature CD4-8+ profile. In contrast, spontaneous lymphomas represent each of the major lymphocyte subsets. These data suggest involvement of different target populations in MNU-induced and spontaneous lymphomas. Analyses at intervals after MNU treatment revealed selective expansion of the CD4-8+ J11d+ thymocyte subset at 8-10 weeks post-MNU in 68% of the animals examined, suggesting that these cells are targets for MNU-induced lymphomagenesis. Untreated age-matched animals showed no selective expansion of thymocyte subsets.^ Previous data have shown that both spontaneous and MNU-induced lymphomas are monoclonal or oligoclonal. Distinct rearrangement patterns of the J$\sb2$ region of the T-cell receptor $\beta$-chain showed emergence of clonal thymocyte populations beginning at 6-7 weeks after MNU treatment. However, lymphocytes from untreated animals showed no evidence of clonal expansion at the time intervals investigated.^ Activation of c-myc frequently occurs during development of B- and T- cell lymphomas. Both spontaneous and MNU-induced lymphomas showed increased c-myc transcript levels. Increased c-myc transcription was first detected at 6 weeks post-MNU, and persisted throughout the latent period. However, untreated animals showed no increases in c-myc transcripts at the time intervals examined. Another nuclear oncogene, c-fos, did not display a similar change in RNA transcription during the latent period.^ These results supports the hypothesis that MNU-induced and spontaneous tumors develop by multi-step pathways which are distinct with respect to the target cell population affected. Clonal emergence and c-myc deregulation are important steps in the development of both MNU-induced and spontaneous tumors, but the onset of these events is later in spontaneous tumor development. ^

Neuromuscular disuse and atrophy: {\it In vivo\/} magnetic resonance studies of intramuscular lipid metabolism and fluid shift changes

We postulated that neuromuscular disuse results in deleteriously affected tissue-vascular fluid exchange processes and subsequently damages the important oxidative bioenergetic process of intramuscular lipid metabolism. The in-depth research reported in the literature is somewhat limited by the ex vivo nature and sporadic time-course characterization of disuse atrophy and recovery. Thus, an in vivo controlled, localized animal model of disuse atrophy was developed in one of the hindlimbs of laboratory rabbits (employing surgically implanted tetrodotoxin (TTX)-filled mini-osmotic pump-sciatic nerve superfusion system) and tested repeatedly with magnetic resonance (MR) throughout the 2-week period of temporarily induced disuse and during the recovery period (following explantation of the TTX-filled pump) for a period of 3 weeks. Controls consisted of saline/"sham"-implanted rabbit hindlimbs. The validity of this model was established with repeated electrophysiologic nerve conduction testing using a clinically appropriate protocol and percutaneously inserted small needle stimulating and recording electrodes. Evoked responses recorded from proximal (P) and distal (D) sites to the sciatic nerve cuff in the TTX-implanted group revealed significantly decreased (p $<$ 0.001) proximal-to-distal (P/D) amplitude ratios (as much as 50-70% below Baseline/pre-implanted and sham-implanted group values) and significantly increased (p $<$ 0.01) differential latency (PL-DL) values (as much as 1.5 times the pre- and sham-implanted groups). By Day 21 of recovery, observed P/D and PL-DL levels matched Baseline/sham-implemented levels. MRI-determined cross-sectional area (CSA) values of Baseline/pre-implanted, sham- or TTX-implanted, and recovering/explanted and the corresponding contralateral hindlimb tibialis anterior (TA) muscles normalized to tibial bone (TB) CSA (in TA/TB ratios) revealed that there was a significant decline (indicative of atrophic response) from pre- and sham-implanted controls by as much as 20% (p $<$ 0.01) at Day 7 and 50-55% (p $<$ 0.001) at Day 13 of TTX-implantation. In the non-implanted contralaterals, a significant increase (indicative of hypertrophic response) by as much as 10% (p $<$ 0.025) at Day 7 and 27% (p $<$ 0.001) at Day 13 + TTX was found. The induced atrophic/hypertrophic TA muscles were observed to be fully recovered by Day 21 post-explantation as evidenced by image TA/TB ratios. End-point biopsy results from a small group of rabbits revealed comprehensive atrophy of both Type I and Type II fibers, although the heterogeneity of the response supports the use of image-guided, volume-localized proton magnetic resonance spectroscopy (MRS) to noninvasively assess tissue-level metabolic changes. MRS-determined results of a 0.25cc volume of tissue within implanted limb TA muscles under resting/pre-ischemic, ischemic-stressed, and post-ischemic conditions at timepoints during and following disuse atrophy/recovery revealed significantly increased intramuscular spectral lipid levels, as much as 2-3 times (p $<$ 0.01) the Baseline/pre-implanted values at Day 7 and 6-7 times (p $<$ 0.001) at Day 13 + TTX, which approached normal levels (compared to pre- and sham-implanted groups) by Day 21 of post-explanation recovery. (Abstract shortened by UMI.) ^