Interactions between corticotropin releasing factor signaling and prophylactic antibiotics on measures of intestinal function in weaned and transported pigs.

The study objective was to evaluate the interaction between corticotrophin releasing factor (CRF) receptor signaling and prophylactic antibiotic administration on intestinal physiology in newly weaned and transported pigs. Pigs (n = 56; 5.70 ± 1.05 kg) were weaned (20.49 ± 0.64 d), a blood sample was taken, and then pigs were given an intraperitoneal injection of saline (SAL; n = 28 pigs) or a CRF receptor antagonist (CRFA; n = 28 pigs; 30 µg/kg body weight; Astressin B), and then were transported in a livestock trailer for 12 h and 49 min. A second and third intraperitoneal injection was given at 4 h 42 min and 11 h 36 min into the transport process, respectively. Following transport, 4 SAL and 4 CRFA pigs were blood sampled and euthanized. The remaining 48 pigs were individually housed and given dietary antibiotics [AB; n = 12 SAL and 12 CRFA pigs; chlortetracycline (441 ppm) + tiamulin (38.6 ppm)] or no dietary antibiotics (NAB; n = 12 SAL and 12 CRFA pigs) for 14 d post-transport. Blood was collected at 12 h and on d 3, 7, and 14, and then pigs were euthanized on d 7 (n = 24) and d 14 (n = 24) post-weaning and transport. Circulating cortisol was reduced (p = 0.05) in CRFA pigs when compared to SAL pigs post-weaning and transport. On d 7, jejunal villus height and crypt depth was greater overall (p < 0.05) in AB-fed pigs versus NAB-fed pigs. On d 14, ileal crypt depth was reduced (p = 0.02) in CRFA pigs when compared to SAL pigs. Jejunal CRF mRNA abundance tended to be reduced (p = 0.09) on d 7 in CRFA pigs versus SAL pigs. On d 14, jejunal tumor necrosis factor-alpha was reduced (p = 0.01) in AB-fed pigs versus NAB-fed pigs. On d 7, change in glucose short-circuit current tended to be increased (p = 0.07) in CRFA pigs fed the AB diet when compared to CRFA pigs fed the NAB diet. In conclusion, CRFA pigs and pigs fed AB had some similar biological intestinal function measures post-weaning and transport.

Peripheral CRF-R1/CRF-R2 antagonist, astressin C, induces a long-lasting blockade of acute stress-related visceral pain in male and female rats.

Peptide CRF antagonists injected peripherally alleviate stress-induced visceral hypersensitivity (SIVH) to colorectal distension (CRD) in rodents. Here we further evaluated the dose and time-dependent inhibitory activity of several long-acting peptide CRF receptor antagonists related to astressin on SIVH, focusing on astressin C (AstC), which previously showed high efficacy on stress-related alterations of HPA axis and gut secretomotor functions. Male and female Sprague-Dawley rats pretreated subcutaneously (SC) with AstC were injected intraperitoneally (IP) with CRF 15 min later. The visceromotor responses (VMR) to graded phasic CRD (10, 20, 40 and 60 mmHg) were monitored at basal, 15 min and up to 1-8 days after pretreatment. Two other astressin analogs, hexanoyl-astressin D (Hex-AstD) and [CαMeVal19,32]-AstC, were also tested. The response to IP CRF was sex-dependent with female rats requiring a higher dose to exhibit visceral hyperalgesia. Pretreatment with AstC (30-1000 µg/kg) resulted in a dose-related inhibition of IP CRF-induced SIVH and diarrhea in both sexes. The highest dose prevented SIVH and diarrhea up to 5-7 days after a single SC injection and was lost on day 7 (females) and day 8 (males) but reinstated after a second injection of AstC on day 8 or 9 respectively. [CαMeVal19,32]-AstC and Hex-AstD (1000 µg/kg in males) also prevented SIVH. These data show the potent long-lasting anti-hyperalgesic effect of AstC in an acute model of SIVH in both male and female rats. This highlights the potential of long-acting peripheral CRF antagonists to treat stress-sensitive irritable bowel syndrome.

Protective effects of urocortin 2 against caerulein-induced acute pancreatitis.

Because little is known about the role of corticotropin-releasing factor (CRF) agonists in regulating responses in pancreatitis, we evaluated the effects of urocortin 2 (UCN2) and stressin1 in caerulein-induced acute pancreatitis (AP) model in rats. Male rats were pretreated with UCN2 or stressin1 for 30 min followed by induction of AP with supraphysiologic doses of caerulein. Serum amylase and lipase activity, pancreatic tissue necrosis, immune cell infiltrate, nuclear factor (NF)-κB activity, trypsin levels, and intracellular Ca2+ ([Ca2+]i) were ascertained. UCN2, but not stressin1 attenuated the severity of AP in rats. UCN2, but not stressin1, reduced serum amylase and lipase activity, cell necrosis and inflammatory cell infiltration in AP. NF-κB activity in pancreatic nuclear extracts increased in AP and UCN2 treatment reduced caerulein-induced increases in NF-κB activity by 42%. UCN2 treatment prevented caerulein-induced degradation of IκB-α in the cytosolic fraction as well as increased levels of p65 subunit of NF-κB in the cytosolic fraction. Pancreatic UCN2 levels decreased in AP compared with saline. UCN2 evoked [Ca2+]i responses in primary acinar cells and abolished caerulein-evoked [Ca2+]i responses at 0.1nM, and decreased by ~50% at 1.0nM caerulein. UCN2 stimulation resulted in redistribution of a portion of F-actin from the apical to the basolateral pole. UCN2 prevented the massive redistribution of F-actin observed with supraphysiologic doses of caerulein. UCN2, but not stressin1 attenuated severity of an experimental pancreatitis model. The protective effects of UCN2, including anti-inflammatory and anti-necrotic effects involve activation of the CRF2 receptor, [Ca2+]i signaling, and inhibition of NF-κB activity.

Safety, Biodistribution, and Radiation Dosimetry of 68Ga-OPS202 in Patients with Gastroenteropancreatic Neuroendocrine Tumors: A Prospective Phase I Imaging Study.

Preclinical and preliminary clinical evidence indicates that radiolabeled somatostatin (sst) receptor antagonists perform better than agonists in detecting neuroendocrine tumors (NETs). We performed a prospective phase I/II study to evaluate the sst receptor antagonist 68Ga-OPS202 (68Ga-NODAGA-JR11; NODAGA = 1,4,7-triazacyclononane,1-glutaric acid-4,7-acetic acid and JR11 = Cpa-c(dCys-Aph(Hor)-dAph(Cbm)-Lys-Thr-Cys)-dTyr-NH2)) for PET imaging. Here, we report the results of phase I of the study. Methods: Patients received 2 single 150-MBq intravenous injections of 68Ga-OPS202 3-4 wk apart (15 µg of peptide at visit 1 and 50 µg at visit 2). At visit 1, a dynamic PET/CT scan over the kidney was obtained during the first 30 min after injection, and static whole-body scans were obtained at 0.5, 1, 2, and 4 h after injection; at visit 2, a static whole-body scan was obtained at 1 h. Blood samples and urine were collected at regular intervals to determine 68Ga-OPS202 pharmacokinetics. Safety, biodistribution, radiation dosimetry, and the most appropriate imaging time point for 68Ga-OPS202 were assessed. Results: Twelve patients with well-differentiated gastroenteropancreatic (GEP) NETs took part in the study. 68Ga-OPS202 cleared rapidly from the blood, with a mean residence time of 2.4 ± 1.1 min/L. The organs with the highest mean dose coefficients were the urinary bladder wall, kidneys, and spleen. The calculated effective dose was 2.4E-02 ± 0.2E-02 mSv/MBq, corresponding to 3.6 mSv, for a reference activity of 150 MBq. Based on total numbers of detected malignant lesions, the optimal time window for the scan was between 1 and 2 h. For malignant liver lesions, the time point at which most patients had the highest mean tumor contrast was 1 h. 68Ga-OPS202 was well tolerated; adverse events were grade 1 or 2, and there were no signals of concern from laboratory blood or urinalysis tests. Conclusion:68Ga-OPS202 showed favorable biodistribution and imaging properties, with optimal tumor contrast between 1 and 2 h after injection. Dosimetry analysis revealed that the dose delivered by 68Ga-OPS202 to organs is similar to that delivered by other 68Ga-labeled sst analogs. Further evaluation of 68Ga-OPS202 for PET/CT imaging of NETs is therefore warranted.

Distribution of corticotropin-releasing factor (CRF) receptor binding in the mouse brain using a new, high-affinity radioligand, [125 I]-PD-Sauvagine.

The corticotropin-releasing factor (CRF) family of peptides includes CRF and three urocortins, which signal through two distinct G-protein coupled receptors, CRF1 and CRF2 . Although the cellular distribution of CRF receptor expression has been well characterized at the mRNA level, the localization of receptor protein, and, by inference, of functional receptors, has been limited by a lack of reliable immunohistochemical evidence. Recently, a CRF-related peptide, termed PD-sauvagine, was isolated from the skin of the frog, Pachymedusa dacnicolor, and validated as a high-affinity ligand for CRF receptor studies. A radiolabeled analog, [125 I]-PD-sauvagine, with high signal-to-noise ratio, was used in autoradiographic studies to map the distribution of CRF receptor binding sites in the mouse brain. Through the use of receptor-deficient mice and subtype-specific antagonists, CRF1 and CRF2 binding sites were isolated, and found to be readily reconcilable with regional patterns of mRNA expression. Binding site distributions within a given structure sometimes differed from mRNA patterns, however, particularly in laminated structures of the isocortex, hippocampus, and cerebellum, presumably reflecting the trafficking of receptors to their operational homes on neuronal (mostly dendritic) processes. Binding patterns of [125 I]-PD-sauvagine provided independent assessments of controversial receptor localizations, failing to provide support for CRF1 expression in central autonomic components of the limbic forebrain, the locus coeruleus and cerebellar Purkinje cells, or for CRF2 in any aspect of the cerebellar cortex. Though lacking in ideal resolution, in vitro binding of the PD-sauvagine radioligand currently provides the most sensitive and accurate available tool for localizing CRF receptors in rodent brain.

Prospective Clinical Applications of CRF Peptide Antagonists.

It is noteworthy that thirty three years of efforts in corticotropin releasing factor (CRF) research by academia and the pharmaceutical industry resulted in several thousand papers and patents, yet little progress has been made to identify and market diagnostic or therapeutic CRF peptides and small molecule ligands. We document the potential relevance of CRF peptide antagonists to reinvigorate stress/anxiety affected "anatomy systems" in order to boost their efficacy.

Highly Increased 125I-JR11 Antagonist Binding In Vitro Reveals Novel Indications for sst2 Targeting in Human Cancers.

There is recent in vitro and in vivo evidence that somatostatin receptor subtype 2 (sst2) antagonists are better tools to target neuroendocrine tumors (NETs) than sst2 agonists. Indeed, antagonists bind to a greater number of sst2 sites than agonists. Whether sst2 antagonists could be used successfully to target non-NETs, expressing low sst2 density, is unknown. Here, we compare quantitatively 125I-JR11 sst2 antagonist binding in vitro with that of the sst2 agonist 125I-Tyr3-octreotide in large varieties of non-NET and NET. METHODS: In vitro receptor autoradiography was performed with 125I-JR11 and 125I-Tyr3-octreotide in cancers from prostate, breast, colon, kidney, thyroid, and lymphoid tissues as well as NETs as reference. RESULTS: In general, 125I-JR11 binds to many more sst2 sites than 125I-Tyr3-octreotide. In 13 breast cancers, 8 had a low binding (mean density, 844 ± 168 dpm/mg of tissue) with the agonist whereas 12 had a high binding (mean density, 4,447 ± 1,128 dpm/mg of tissue) with the antagonist. All 12 renal cell cancers showed a low binding of sst2 with the agonist (mean density, 348 ± 49 dpm/mg of tissue) whereas all cases had a high sst2 binding with the antagonist (mean density, 3,777 ± 582 dpm/mg of tissue). One of 5 medullary thyroid cancers was positive with the agonist, whereas 5 of 5 were positive with the antagonist. In 15 non-Hodgkin lymphomas, many more sst2 sites were labeled with the antagonist than with the agonist. In 14 prostate cancers, none had sst2 binding with the agonist and only 4 had a weak binding with the antagonist. None of 17 colon cancers showed sst2 sites with the agonist, and only 3 cases were weakly positive with the antagonist. In the various tumor types, adjacent sst2-expressing tissues such as vessels, lymphocytes, nerves, mucosa, or stroma were more strongly labeled with the antagonist than with the agonist. The reference NET cases, incubated with a smaller amount of tracer, were also found to have many more sst2 sites measured with the antagonist. CONCLUSION: All renal cell cancers and most breast cancers, non-Hodgkin lymphomas, and medullary thyroid cancers represent novel indications for the in vivo radiopeptide targeting of sst2 by sst2 antagonists, comparable to NET radiotargeting with sst2 agonists.

A minimized human insulin-receptor-binding motif revealed in a Conus geographus venom insulin.

Insulins in the venom of certain fish-hunting cone snails facilitate prey capture by rapidly inducing hypoglycemic shock. One such insulin, Conus geographus G1 (Con-Ins G1), is the smallest known insulin found in nature and lacks the C-terminal segment of the B chain that, in human insulin, mediates engagement of the insulin receptor and assembly of the hormone's hexameric storage form. Removal of this segment (residues B23-B30) in human insulin results in substantial loss of receptor affinity. Here, we found that Con-Ins G1 is monomeric, strongly binds the human insulin receptor and activates receptor signaling. Con-Ins G1 thus is a naturally occurring B-chain-minimized mimetic of human insulin. Our crystal structure of Con-Ins G1 reveals a tertiary structure highly similar to that of human insulin and indicates how Con-Ins G1's lack of an equivalent to the key receptor-engaging residue PheB24 is mitigated. These findings may facilitate efforts to design ultrarapid-acting therapeutic insulins.

Solid-state NMR sequential assignment of the ß-endorphin peptide in its amyloid form.

Insights into the three-dimensional structure of hormone fibrils are crucial for a detailed understanding of how an amyloid structure allows the storage of hormones in secretory vesicles prior to hormone secretion into the blood stream. As an example for various hormone amyloids, we have studied the endogenous opioid neuropeptide ß-endorphin in one of its fibril forms. We have achieved the sequential assignment of the chemical shifts of the backbone and side-chain heavy atoms of the fibril. The secondary chemical shift analysis revealed that the ß-endorphin peptide adopts three ß-strands in its fibril state. This finding fosters the amyloid nature of a hormone at the atomic level.

Structural Basis for the Inhibition of Voltage-gated Sodium Channels by Conotoxin µO§-GVIIJ.

Cone snail toxins are well known blockers of voltage-gated sodium channels, a property that is of broad interest in biology and therapeutically in treating neuropathic pain and neurological disorders. Although most conotoxin channel blockers function by direct binding to a channel and disrupting its normal ion movement, conotoxin µO§-GVIIJ channel blocking is unique, using both favorable binding interactions with the channel and a direct tether via an intermolecular disulfide bond. Disulfide exchange is possible because conotoxin µO§-GVIIJ contains anS-cysteinylated Cys-24 residue that is capable of exchanging with a free cysteine thiol on the channel surface. Here, we present the solution structure of an analog of µO§-GVIIJ (GVIIJ[C24S]) and the results of structure-activity studies with synthetic µO§-GVIIJ variants. GVIIJ[C24S] adopts an inhibitor cystine knot structure, with two antiparallel ß-strands stabilized by three disulfide bridges. The loop region linking the ß-strands (loop 4) presents residue 24 in a configuration where it could bind to the proposed free cysteine of the channel (Cys-910, rat NaV1.2 numbering; at site 8). The structure-activity study shows that three residues (Lys-12, Arg-14, and Tyr-16) located in loop 2 and spatially close to residue 24 were also important for functional activity. We propose that the interaction of µO§-GVIIJ with the channel depends on not only disulfide tethering via Cys-24 to a free cysteine at site 8 on the channel but also the participation of key residues of µO§-GVIIJ on a distinct surface of the peptide.

Characterization of Multisubstituted Corticotropin Releasing Factor (CRF) Peptide Antagonists (Astressins).

CRF mediates numerous stress-related endocrine, autonomic, metabolic, and behavioral responses. We present the synthesis and chemical and biological properties of astressin B analogues {cyclo(30-33)[D-Phe(12),Nle(21,38),C(α)MeLeu(27,40),Glu(30),Lys(33)]-acetyl-h/r-CRF(9-41)}. Out of 37 novel peptides, 17 (2, 4, 6-8, 10, 11, 16, 17, 27, 29, 30, 32-36) and 16 (3, 5, 9, 12-15, 18, 19, 22-26, 28, 31) had k(i) to CRF receptors in the high picomolar and low nanomole ranges, respectively. Peptides 1, 2, and 11 inhibited h/rCRF and urocortin 1-induced cAMP release from AtT20 and A7r5 cells. When Astressin C 2 was administered to adrenalectomized rats at 1.0 mg subcutaneously, it inhibited ACTH release for >7 d. Additional rat data based on the inhibitory effect of (2) on h/rCRF-induced stimulation of colonic secretory motor activity and urocortin 2-induced delayed gastric emptying also indicate a safe and long-lasting antagonistic effect. The overall properties of selected analogues may fulfill the criteria expected from clinical candidates.

Characterization of a Pachymedusa dacnicolor-Sauvagine analog as a new high-affinity radioligand for corticotropin-releasing factor receptor studies.

The corticotropin-releasing factor (CRF) peptide family comprises the mammalian peptides CRF and the urocortins as well as frog skin sauvagine and fish urophyseal urotensin. Advances in understanding the roles of the CRF ligand family and associated receptors have often relied on radioreceptor assays using labeled CRF ligands. These assays depend on stable, high-affinity CRF analogs that can be labeled, purified, and chemically characterized. Analogs of several of the native peptides have been used in this context, most prominently including sauvagine from the frog Phyllomedusa sauvageii (PS-Svg). Because each of these affords both advantages and disadvantages, new analogs with superior properties would be welcome. We find that a sauvagine-like peptide recently isolated from a different frog species, Pachymedusa dacnicolor (PD-Svg), is a high-affinity agonist whose radioiodinated analog, [(125)ITyr(0)-Glu(1), Nle(17)]-PD-Svg, exhibits improved biochemical properties over those of earlier iodinated agonists. Specifically, the PD-Svg radioligand binds both CRF receptors with comparably high affinity as its PS-Svg counterpart, but detects a greater number of sites on both type 1 and type 2 receptors. PD-Svg is also ∼10 times more potent at stimulating cAMP accumulation in cells expressing the native receptors. Autoradiographic localization using the PD-Svg radioligand shows robust specific binding to rodent brain and peripheral tissues that identifies consensus CRF receptor-expressing sites in a greater number and/or with greater sensitivity than its PS-Svg counterpart. We suggest that labeled analogs of PD-Svg may be useful tools for biochemical, structural, pharmacological, and anatomic studies of CRF receptors.

Α- and ß-subunit composition of voltage-gated sodium channels investigated with µ-conotoxins and the recently discovered µO§-conotoxin GVIIJ.

We investigated the identities of the isoforms of the α (NaV1)- and ß (NaVß)-subunits of voltage-gated sodium channels, including those responsible for action potentials in rodent sciatic nerves. To examine α-subunits, we used seven µ-conotoxins, which target site 1 of the channel. With the use of exogenously expressed channels, we show that two of the µ-conotoxins, µ-BuIIIB and µ-SxIIIA, are 50-fold more potent in blocking NaV1.6 from mouse than that from rat. Furthermore, we observed that µ-BuIIIB and µ-SxIIIA are potent blockers of large, myelinated A-fiber compound action potentials (A-CAPs) [but not small, unmyelinated C-fiber CAPs (C-CAPs)] in the sciatic nerve of the mouse (unlike A-CAPs of the rat, previously shown to be insensitive to these toxins). To investigate ß-subunits, we used two synthetic derivatives of the recently discovered µO§-conotoxin GVIIJ that define site 8 of the channel, as previously characterized with cloned rat NaV1- and NaVß-subunits expressed in Xenopus laevis oocytes, where it was shown that µO§-GVIIJ is a potent inhibitor of several NaV1-isoforms and that coexpression of NaVß2 or -ß4 (but not NaVß1 or -ß3) totally protects against block by µO§-GVIIJ. We report here the effects of µO§-GVIIJ on 1) sodium currents of mouse NaV1.6 coexpressed with various combinations of NaVß-subunits in oocytes; 2) A- and C-CAPs of mouse and rat sciatic nerves; and 3) sodium currents of small and large neurons dissociated from rat dorsal root ganglia. Our overall results lead us to conclude that action potentials in A-fibers of the rodent sciatic nerve are mediated primarily by NaV1.6 associated with NaVß2 or NaVß4.

Selective agonists of somatostatin receptor subtype 1 or 2 injected peripherally induce antihyperalgesic effect in two models of visceral hypersensitivity in mice.

Somatostatin interacts with five G-protein-coupled receptor (sst1-5). Octreotide, a stable sst2≫3≥5 agonist, exerts a visceral anti-hyperalgesic effect in experimental and clinical studies. Little is known on the receptor subtypes involved. We investigated the influence of the stable sst1-5 agonist, ODT8-SST and selective receptor subtype peptide agonists (3 or 10µg/mouse) injected intraperitoneally (ip) on visceral hypersensitivity in mice induced by repeated noxious colorectal distensions (four sets of three CRD, each at 55mmHg) or corticotropin-releasing factor receptor 1 agonist, cortagine given between two sets of graded CRD (15, 30, 45, and 60mmHg, three times each pressure). The mean visceromotor response (VMR) was assessed using a non-invasive manometry method and values were expressed as percentage of the VMR to the 1st set of CRD baseline or to the 60mmHg CRD, respectively. ODT8-SST (10µg) and the sst2 agonist, S-346-011 (3 and 10µg) prevented mechanically induced visceral hypersensitivity in the three sets of CRD, the sst1 agonist (10µg) blocked only the 2nd set and showed a trend at 3µg while the sst4 agonist had no effect. The selective sst2 antagonist, S-406-028 blocked the sst2 agonist but not the sst1 agonist effect. The sst1 agonist (3 and 10µg) prevented cortagine-induced hypersensitivity to CRD at each pressure while the sst2 agonist at 10µg reduced it. These data indicate that in addition to sst2, the sst1 agonist may provide a novel promising target to alleviate visceral hypersensitivity induced by mechanoreceptor sensitization and more prominently, stress-related visceral nociceptive sensitization.

Brain somatostatin receptor 2 mediates the dipsogenic effect of central somatostatin and cortistatin in rats: role in drinking behavior.

Intracerebroventricular injection of stable somatostatin (SST) agonists stimulates food and water intake in rats. We investigated the receptor subtype(s) involved in the dipsogenic effect of intracerebroventricular injection of SST agonists, mechanisms of action, and role. In nonfasted and non-water-deprived male rats with chronic intracerebroventricular cannula, intake of water without food or food without water was monitored separately to avoid any interactions compared with intracerebroventricular vehicle. SST-14 and cortistatin (CST-14) (1 µg/rat icv) increased water intake by 3.1- and 2.7-fold, respectively, while both peptides did not alter food intake at 1 h postinjection in the light phase. By contrast, the stable pan-somatostatin agonist ODT8-SST (1 µg/rat icv) increased both water and food intake by 4.9- and 3.7-fold, respectively. S-346-011, a selective receptor 2 (sst2) agonist (1 µg/rat icv) induced water ingestion, while sst1 or sst4 agonist, injected under the same conditions, did not. The sst2 antagonist S-406-028 (1 µg/rat icv) prevented the 1-h water intake induced by intracerebroventricular ODT8-SST and CST-14. Losartan (100 µg/rat icv), an angiotensin receptor 1 (AT1) antagonist, completely blocked the water consumption induced by intracerebroventricular ODT8-SST, whereas intracerebroventricular injection of S-406-028 did not modify the intracerebroventricular ANG II-induced dipsogenic response. The sst2 antagonist reduced by 40% the increase of the 3-h water intake in the early dark phase. These data indicate that SST-14 and CST-14 interact with sst2 to exert a potent dipsogenic effect, which is mediated downstream by angiotensin-AT1 signaling. These data also indicate that sst2 activation by brain SST-14 and/or CST-14 may play an important role in the regulation of drinking behavior.

Comparison of somatostatin receptor agonist and antagonist for peptide receptor radionuclide therapy: a pilot study.

UNLABELLED: Preclinical and clinical studies have indicated that somatostatin receptor (sst)-expressing tumors demonstrate higher uptake of radiolabeled sst antagonists than of sst agonists. In 4 consecutive patients with advanced neuroendocrine tumors, we evaluated whether treatment with (177)Lu-labeled sst antagonists is feasible. METHODS: After injection of approximately 1 GBq of (177)Lu-DOTA-[Cpa-c(DCys-Aph(Hor)-DAph(Cbm)-Lys-Thr-Cys)-DTyr-NH2] ((177)Lu-DOTA-JR11) and (177)Lu-DOTATATE, 3-dimensional voxel dosimetry analysis based on SPECT/CT was performed. A higher tumor-to-organ dose ratio for (177)Lu-DOTA-JR11 than for (177)Lu-DOTATATE was the prerequisite for treatment with (177)Lu-DOTA-JR11. RESULTS: Reversible minor adverse effects of (177)Lu-DOTA-JR11 were observed. (177)Lu-DOTA-JR11 showed a 1.7-10.6 times higher tumor dose than (177)Lu-DOTATATE. At the same time, the tumor-to-kidney and tumor-to-bone marrow dose ratio was 1.1-7.2 times higher. All 4 patients were treated with (177)Lu-DOTA-JR11, resulting in partial remission in 2 patients, stable disease in 1 patient, and mixed response in the other patient. CONCLUSION: Treatment of neuroendocrine tumors with radiolabeled sst antagonists is clinically feasible and may have a significant impact on peptide receptor radionuclide therapy.

Expression and functional characterization of membrane-integrated mammalian corticotropin releasing factor receptors 1 and 2 in Escherichia coli.

Corticotropin-Releasing Factor Receptors (CRFRs) are class B1 G-protein-coupled receptors, which bind peptides of the corticotropin releasing factor family and are key mediators in the stress response. In order to dissect the receptors' binding specificity and enable structural studies, full-length human CRFR1α and mouse CRFR2ß as well as fragments lacking the N-terminal extracellular domain, were overproduced in E. coli. The characteristics of different CRFR2ß-PhoA gene fusion products expressed in bacteria were found to be in agreement with the predicted ones in the hepta-helical membrane topology model. Recombinant histidine-tagged CRFR1α and CRFR2ß expression levels and bacterial subcellular localization were evaluated by cell fractionation and Western blot analysis. Protein expression parameters were assessed, including the influence of E. coli bacterial hosts, culture media and the impact of either PelB or DsbA signal peptide. In general, the large majority of receptor proteins became inserted in the bacterial membrane. Across all experimental conditions significantly more CRFR2ß product was obtained in comparison to CRFR1α. Following a detergent screen analysis, bacterial membranes containing CRFR1α and CRFR2ß were best solubilized with the zwitterionic detergent FC-14. Binding of different peptide ligands to CRFR1α and CRFR2ß membrane fractions were similar, in part, to the complex pharmacology observed in eukaryotic cells. We suggest that our E. coli expression system producing functional CRFRs will be useful for large-scale expression of these receptors for structural studies.

Corticotropin-releasing factor peptide antagonists: design, characterization and potential clinical relevance.

Elusive for more than half a century, corticotropin-releasing factor (CRF) was finally isolated and characterized in 1981 from ovine hypothalami and shortly thereafter, from rat brains. Thirty years later, much has been learned about the function and localization of CRF and related family members (Urocortins 1, 2 and 3) and their 2 receptors, CRF receptor type 1 (CRFR1) and CRF receptor type 2 (CRFR2). Here, we report the stepwise development of peptide CRF agonists and antagonists, which led to the CRFR1 agonist Stressin1; the long-acting antagonists Astressin2-B which is specific for CRFR2; and Astressin B, which binds to both CRFR1 and CRFR2.This analog has potential for the treatment of CRF-dependent diseases in the periphery, such as irritable bowel syndrome.

Anxiolytic-like effects of antisauvagine-30 in mice are not mediated by CRF2 receptors.

The role of brain corticotropin-releasing factor type 2 (CRF2) receptors in behavioral stress responses remains controversial. Conflicting findings suggest pro-stress, anti-stress or no effects of impeding CRF2 signaling. Previous studies have used antisauvagine-30 as a selective CRF2 antagonist. The present study tested the hypotheses that 1) potential anxiolytic-like actions of intracerebroventricular (i.c.v.) administration of antisauvagine-30 also are present in mice lacking CRF2 receptors and 2) potential anxiolytic-like effects of antisauvagine-30 are not shared by the more selective CRF2 antagonist astressin2-B. Cannulated, male CRF2 receptor knockout (n = 22) and wildtype littermate mice (n = 21) backcrossed onto a C57BL/6J genetic background were tested in the marble burying, elevated plus-maze, and shock-induced freezing tests following pretreatment (i.c.v.) with vehicle, antisauvagine-30 or astressin2-B. Antisauvagine-30 reduced shock-induced freezing equally in wildtype and CRF2 knockout mice. In contrast, neither astressin2-B nor CRF2 genotype influenced shock-induced freezing. Neither CRF antagonist nor CRF2 genotype influenced anxiety-like behavior in the plus-maze or marble burying tests. A literature review showed that the typical antisauvagine-30 concentration infused in previous intracranial studies (∼1 mM) was 3 orders greater than its IC50 to block CRF1-mediated cAMP responses and 4 orders greater than its binding constants (Kd , Ki ) for CRF1 receptors. Thus, increasing, previously used doses of antisauvagine-30 also exert non-CRF2-mediated effects, perhaps via CRF1. The results do not support the hypothesis that brain CRF2 receptors tonically promote anxiogenic-like behavior. Utilization of CRF2 antagonists, such as astressin2-B, at doses that are more subtype-selective, can better clarify the significance of brain CRF2 systems in stress-related behavior.

Key role of CRF in the skin stress response system.

The discovery of corticotropin-releasing factor (CRF) or CRH defining the upper regulatory arm of the hypothalamic-pituitary-adrenal (HPA) axis, along with the identification of the corresponding receptors (CRFRs 1 and 2), represents a milestone in our understanding of central mechanisms regulating body and local homeostasis. We focused on the CRF-led signaling systems in the skin and offer a model for regulation of peripheral homeostasis based on the interaction of CRF and the structurally related urocortins with corresponding receptors and the resulting direct or indirect phenotypic effects that include regulation of epidermal barrier function, skin immune, pigmentary, adnexal, and dermal functions necessary to maintain local and systemic homeostasis. The regulatory modes of action include the classical CRF-led cutaneous equivalent of the central HPA axis, the expression and function of CRF and related peptides, and the stimulation of pro-opiomelanocortin peptides or cytokines. The key regulatory role is assigned to the CRFR-1α receptor, with other isoforms having modulatory effects. CRF can be released from sensory nerves and immune cells in response to emotional and environmental stressors. The expression sequence of peptides includes urocortin/CRF→pro-opiomelanocortin→ACTH, MSH, and ß-endorphin. Expression of these peptides and of CRFR-1α is environmentally regulated, and their dysfunction can lead to skin and systemic diseases. Environmentally stressed skin can activate both the central and local HPA axis through either sensory nerves or humoral factors to turn on homeostatic responses counteracting cutaneous and systemic environmental damage. CRF and CRFR-1 may constitute novel targets through the use of specific agonists or antagonists, especially for therapy of skin diseases that worsen with stress, such as atopic dermatitis and psoriasis.