Investigation of bombesin peptide as a targeting ligand for the gastrin releasing peptide (GRP) receptor

Gastrin releasing peptide (GRP) receptor (GRPR), a bombesin family receptor, is overexpressed in many cancers including breast, prostate, pancreatic and lung. The targeting of therapeutics to GRPR can be achieved using the full-length (14 amino acid) GRP analogue Bombesin (BBN) or the truncated BBN(6–14) sequence, both of which bind GRPR with high affinity and specificity. In this study, we have investigated the level of GRPR expression in various cancerous (Caco-2, HeLa, LNCap, MDA-MB-231, and PC-3) and non-cancerous (WPMY-1) cell lines using a western blotting approach. Such information is currently lacking in the literature, and is therefore of importance for the in vitro assessment of GRPR targeted therapeutics. Of the cell lines assessed, the PC-3 (prostate cancer) and Caco-2 (colon cancer) cell lines demonstrated the highest and lowest levels of GRPR expression respectively. Using this information, we further investigated the cellular uptake of carboxyfluorescein-labelled BBN and BBN(6–14) peptides by flow cytometry and confocal microscopy using cell lines that express GRPR (Caco-2, HeLa, PC-3). The uptake of each of these peptides was similar, suggesting that the shorter BBN(6–14) peptide is sufficient for GRPR targeting. Further, the uptake of these peptides could be inhibited by competition with unlabelled BBN peptides, suggesting their cellular uptake is GRPR-mediated, while the level of BBN uptake (as measured by flow cytometry) was found to be directly proportional to the level of GRPR expression. Overall, the information obtained from these studies provides useful information for the in vitro assessment of GRPR targeted therapeutics.     

Conformational analysis in solution of gastrin releasing peptide

Gastrin releasing peptide (GRP) is the first peptide isolated from porcine gastric and intestinal tissues and is homologous to the carboxyl terminus of bombesin (Bn) isolated from the skin of the frog Bombina bombina. It is a member of the Bn-like peptides, which are important in numerous biological and pathological processes. The Bn-like peptides show high sequence homology in their C-terminal regions, but they have different selectivity for their receptors. In particular, GRP selectively binds to the GRP receptor (GRPR). However, the molecular basis for this selectivity remains largely unknown. Here, we report the three-dimensional structure of GRP. Hopefully, it could be helpful in a better understanding of the binding selectivity between GRP and GRPR.     

Gastrin releasing peptide reduces meal size in rats

The satiety-eliciting effect of gastrin-releasing peptide (GRP), a putative mammalian counterpart of bombesin (BBS), was examined in mildly food-deprived rats. Intraperitoneal injections of GRP resulted in a significant decrease of 30-minute food intake at 2, 4, 8 and 16 μg/kg, while 1 μg/kg had no reliable effect. Intraperitoneal GRP at 4 and 8 μg/kg did not suppress 30-minute water consumption by thirsty rats. When the dose-effect curves of GRP and BBS are compared on a molar scale, GRP is approximately 30% less potent than BBS in suppressing food intake. The two dose-effect curves are similar in shape and their regression lines have parallel slopes. These data lend further support to the hypothesis that GRP is a mammalian counterpart of BBS and strengthen the argument that they may function as endogenous satiety factors.     

Catabolism of gastrin releasing peptide and substance P by gastric membrane-bound peptidases

The catabolism of two gastric neuropeptides, the C-terminal decapeptide of gastrin releasing peptide-27 (GRP10) and substance P (SP), by membrane-bound peptidases of the porcine gastric corpus and by porcine endopeptidase-24.11 ("enkephalinase") has been investigated. GRP10 was catabolized by gastric muscle peptidases (specific activity 1.8 nmol min-1 mg-1 protein) by hydrolysis of the His8-Leu9 bond and catabolism was inhibited by phosphoramidon (I50 approx. 10(-8) M), a specific inhibitor of endopeptidase-24.11. The same bond in GRP10 was cleaved by purified endopeptidase-24.11, and hydrolysis was equally sensitive to inhibition by phosphoramidon. SP was catabolized by gastric muscle peptidases (specific activity 1.7 nmol min-1 mg-1 protein) by hydrolysis of the Gln6-Phe7, Phe7-Phe8 and Gly9-Leu10 bonds, which is identical to the cleavage of SP by purified endopeptidase-24.11. The C-terminal cleavage of GRP10 and SP would inactivate the peptides. It is concluded that a membrane-bound peptidase in the stomach wall catabolizes and inactivates GRP10 and SP and that, in its specificity and sensitivity to phosphoramidon, this peptidase resembles endopeptidase-24.11.     

Distribution and projections of neurons with immunoreactivity for both gastrin-releasing peptide and bombesin in the guinea-pig small intestine

Summary Bombesin-like and gastrin-releasing peptide (GRP)-like immunoreactivities were localized in nerves of the guinea-pig small intestine and celiac ganglion with the use of antibodies raised against the synthetic peptides. The anti-bombesin serum (preincubated to avoid cross reactivity with substance P) and the anti-GRP serum revealed the same population of neurons. Preincubation of the antibombesin serum with bombesin abolished the immunoreactivity in nerves while absorption of the anti-GRP serum with either bombesin or the 14–27 C-terminal of GRP only reduced the immunoreactivity. The immunoreactivity was abolished by incubation with GRP 1–27.Immunoreactive nerves were found in the myenteric plexus, circular muscle, submucous plexus and in the celiac ganglion. Faintly reactive nerve cell bodies were found in the myenteric ganglia (3.2% of all neurons) but not in submucous ganglia. After all ascending and descending pathways in the myenteric plexus had been cut, reactive terminals disappeared in the myenteric plexus, circular muscle (including the deep muscular plexus) and the submucous plexus on the anal side. After the mesenteric nerves were cut no changes were observed in the intestinal wall but the reactive fibres in celiac ganglia disappeared. It is deduced that GRP/bombesin-immunoreactive nerve cell bodies in myenteric ganglia project from the myenteric plexus to other myenteric ganglia situated further anally (average length 12 mm), anally to the circular muscle (average length 9 mm), anally to submucous ganglia (average length 13 mm) and external to the intestine to the celiac ganglia.It is concluded that the GRP/bombesin-reactive neurons in the intestinal wall represent a distinct population of enteric neurons likely to be involved in controlling motility and in the coordination of other intestinal functions.     

A synthetic peptide containing loop 4 of nerve growth factor for targeted gene delivery

BACKGROUND: Gene delivery vectors that restrict the expression of a therapeutic gene to a particular type of cells are critical to gene therapy in a complex structure, such as the central nervous system. We constructed a nonviral vector for targeted gene transfer to cells expressing nerve growth factor (NGF) receptor TrkA. METHODS AND RESULTS: The vector was a synthetic chimeric peptide composed of a targeting moiety derived from NGF loop 4 and a DNA-binding moiety of 10 lysine residues. The peptide activated signal transduction pathways of the NGF receptor TrkA in PC12 cells and supported the survival of the cells after serum deprivation. After forming complexes with plasmid DNA, the peptide dose-dependently increased reporter gene expression in PC12 cells, which could be inhibited by excess NGF. The peptide-mediated gene expression was not affected in PC12 cells by co-incubation with a blocking antibody against the low-affinity NGF receptor p75 and was significantly enhanced in NIH3T3 cells stably transfected with TrkA cDNA, suggesting the involvement of the high-affinity NGF receptor TrkA without the participation of p75. Moreover, the peptide did not assist gene transfer in TrkA-poor, but TrkB- and/or TrkC-positive primary cerebellar granule neurons and primary cortical glial cells. CONCLUSIONS: The chimeric peptide reported will be useful in gene delivery to and gene therapy of the nervous system and other tissues/organs with cells expressing TrkA.     

Targeted delivery of atorvastatin via asialoglycoprotein receptor (ASGPR)

Targeted drug delivery platforms can increase the concentration of drugs in specific cell populations, reduce adverse effects, and hence improve the therapeutic effect of drugs. Herein, we designed two conjugates by installing the targeting ligand GalNAc (N-acetylgalactosamine) onto atorvastatin (AT). Compared to the parent drug, these two conjugates, termed G2-AT and G2-K-AT, showed increased hepatic cellular uptake. Moreover, both conjugates were able to release atorvastatin, and consequently showed dramatic inhibition of β-hydroxy-β-methylglutaryl-CoA (HMG-CoA) reductase and increased LDL receptors on cell surface.     

Sequence analysis and feeding responses evoked by the large molecular form of gastrin releasing peptide (GRP) in the rat GRP-29

Microisolation techniques utilizing several reverse phase high performance liquid chromatography (HPLC) steps have resulted in the purification of two rat gastrin releasing peptide (GRP) forms suitable for microsequence and mass spectral analysis. The sequence of the larger form is APVSTGAGGGTVLAKMYPRGSHWAVGHLM-amide and the smaller form is GSHWAVGHLM-amide which is the carboxyl terminal decapeptide of the larger peptide. The peptides were synthesized and their feeding patterns e.g. first meal size (MS), intermeal interval (IMI) and satiety ratio (SR, IMI/MS) were determined in overnight food-, but not water deprived, male Sprague Dawley rats. The peptides were administered in the femoral vein (0, 0.21, 0.41 and 1.03 nmol/kg) immediately before presenting the rats with a 10% sucrose solution. We found that (1) GRP-10 (all doses) and GRP-29 (0.41 nmol/kg) reduced first MS, (2) both peptides prolonged IMI length and (3) both peptides increased the SR to similar extents. In conclusion, GRP-10 and GRP-29 are the two endogenous forms of GRP in the rat intestine and they reduce short term feeding to similar extents when administered intravenously.     

Design of cyclic RGD-conjugated Aib-containing amphipathic helical peptides for targeted delivery of small interfering RNA

To achieve the targeted delivery of siRNA, five conjugates of Aib-containing amphipathic helical peptides with mono-, di-, and trivalent cRGDfC [cyclo(-Arg-Gly-Asp-d-Phe-Cys-)], which is known to bind to α_Vβ₃ integrin, at several positions of the amphipathic helical peptide were designed and synthesized. Among the five conjugates, the monovalent cRGDfC conjugating at position 20 of the amino acid sequence of the helical peptide through the formation of a disulfide bond (PI) and the divalent cRGDfC conjugating at positions 2 and 14 of the amino acid sequence of the helical peptide through the formation of disulfide bonds (PIII) significantly enhanced the delivery of fluorescence-labeled siRNA into A549 cells as the peptide/siRNA complex formed by electrostatic interaction. The cellular uptake of the PI/siRNA complex was mediated by both endocytic and non-endocytic pathways, whereas that of the PIII/siRNA complex was enabled by endocytosis. Furthermore, the cellular uptake of the PI/siRNA complex might involve specific interactions of the RGD group with the α_Vβ₃ integrin receptor. Next, the RNAi effect of the peptide/siRNA complex on luciferase expression in A549-Luc cells was examined. Luciferase expression was significantly decreased in the presence of the complex at the concentration of 1.0 μM PI/10 nM siRNA. In contrast, the PIII/siRNA complex did not show the RNAi effect under the same conditions. However, extending the incubation time led to the suppression of the luciferase expression in the presence of the PIII/siRNA complex. Considering that the cellular uptake of the PIII/siRNA complex is mediated by the endocytic pathway, the release of siRNA from the endosome into the cytosol might require a long time. We present herein a useful and unique tool for the delivery of siRNA.     

A poly(ethylene) glycolylated peptide for ocular delivery compacts DNA into nanoparticles for gene delivery to post‐mitotic tissues in vivo

Background

We have previously shown that a novel synthetic peptide for ocular delivery (POD) can efficiently compact DNA and deliver it to cells in vitro. This observation prompted us to develop use of POD as a nonviral vector in vivo.

Methods

POD peptide was modified using poly(ethylene) glycol (PEG‐POD) and used to compact DNA into nanoparticles that were then analysed using electron microscopy, dynamic light scattering, and fluorescent labeling. Transfection efficiency and localization were determined 48 h post‐injection into the subretinal space of the mouse eye using luciferase and LacZ, respectively. Efficiency of ocular transfection was compared to two other PEGylated peptides: PEG‐TAT and PEG‐CK30.

Results

PEG‐POD can compact DNA and form discrete nanoparticles of approximately 136 nm that can penetrate and transduce the retinal pigment epithelium (RPE) in vivo. PEG‐POD significantly increased expression of plasmid DNA by 215‐fold, PEG‐TAT by 56.52‐fold, and PEG‐CK30 by 24.73‐fold relative to DNA injected alone. In all cases β‐galactosidase was observed primarily in the RPE layer after subretinal injection. Electrophysiological analyses of PEG‐POD transduced retina indicates an absence of PEG‐POD‐mediated toxicity. PEG‐POD can protect plasmid DNA from DNaseI digestion, resulting in significant transfection of the lung after intravenous injection in mice.

Conclusions

PEG‐POD was found to significantly increase gene delivery relative to both DNA alone and other pegylated peptides. These findings highlight the use of pegylated peptides, and specifically PEG‐POD, as novel gene delivery vectors. Copyright © 2009 John Wiley & Sons, Ltd.     

Reduction/pH dual-sensitive PEGylated hyaluronan nanoparticles for targeted doxorubicin delivery

To minimize the side effect of chemotherapy, a novel reduction/pH dual-sensitive drug nanocarrier, based on PEGylated dithiodipropionate dihydrazide (TPH)-modified hyaluronic acid (PEG-SS-HA copolymer), was developed for targeted delivery of doxorubicin (DOX) to hepatocellular carcinoma. The copolymer was synthesized by reductive amination via Schiff's base formation between TPH-modified HA and galactosamine-conjugated poly(ethylene glycol) aldehyde/methoxy poly(ethylene glycol) aldehyde. Conjugation of DOX to PEG-SS-HA copolymer was accomplished through the hydrazone linkage formed between DOX and PEG-SS-HA, and confirmed by FTIR and ¹H NMR spectra. The polymer–DOX conjugate could self-assemble into spherical nanoparticles (∼150 nm), as indicated by TEM and DLS. In vitro release studies showed that the DOX-loaded nanoparticles could release DOX rapidly under the intracellular levels of pH and glutathiose. Cellular uptake experiments demonstrated that the nanoparticles could be efficiently internalized by HepG2 cells. These results indicate that the PEG-SS-HA copolymer holds great potential for targeted intracellular delivery of DOX.     

Increase of the trophoblast giant cells with prolactin-releasing peptide (PrRP) receptor expression in p53-null mice

Trophoblast giant cells in the mouse placentas are polyploid cells that form as a result of endoreduplication. The giant cells form the outermost layer of the extraembryonic compartment and produce a number of pregnancy-specific hormones, including prolactin family members. Here we demonstrate that trophoblast giant cells are increased, and display upregulation of prolactin releasing peptide (PrRP) receptor in the p53-null (p53(-/-)) embryonic placentas. At day 13.5 of gestation, the weight of p53(-/-) placentas was less than that of both wild-type and p53(+/-) placentas. In p53(-/-) placentas, the spongiotrophoblast layer was significantly decreased in thickness, and the trophoblast giant cells were observed not only in the outer layer of placentas but in both the spongiotrophoblast layer and the labyrinthine layer. The giant cells spread over the spongiotrophoblast and labyrinthine layer in p53(-/-) placentas displayed more intensive expression of immunoreactive PrRP receptor than in wild-type placentas. Previous studies indicated that the association between PrRP and PrRP receptor physiologically involves in the expression and secretion of the peptide hormones, including prolactin and growth hormones. These results suggest that p53 may regulate the differentiation of trophoblast giant cells, and may control the physiological PrRP stimuli in mouse placentas.     

Discovery of potent and selective peptide agonists at the GRP-preferring bombesin receptor (BB2).

Analogues of the nonselective bombesin receptor synthetic agonist H-D-Phe-Gln-Trp-Ala-Val-betaAla-His-Phe-Nle-NH2 were prepared and their biological activity assessed at the NMB-preferring/bombesin receptor (NMB-R: BB1), the GRP-preferring/bombesin receptor (GRP-R: BB2) and the orphan receptor bombesin receptor subtype-3 (BRS-3; BB3). Progressive N-terminal deletions identified the minimum C-terminal sequences required for maintaining a significant agonist effect, whilst an alanine scan, targeted changes in stereochemistry and other pertinent substitutions identified key side-chain and stereochemical requirements for activation. Key structural elements required for functional potency at BB1 BB2 and BB3, and for selectivity between these receptor subtypes were established. Synthetic peptides were discovered. which were highly potent agonists at BB2 and extremely selective over both BB1 and BB3.     

Synthesis,biological studies and molecular dynamics of new anticancer RGD-based peptide conjugates for targeted drug delivery

New cyclic RGD peptide-anticancer agent conjugates, with different chemical functionalities attached to the parent peptide were synthesized in order to evaluate their biological activities and to provide a comparative study of their drug release profiles. The Integrin binding c(RGDfK) penta-peptide was used for the synthesis of Camptothecin (CPT) carbamate and Chlorambucil (CLB) amide conjugates. Substitution of the amino acid Lys with Ser resulted in a modified c(RGDfS) with a new attachment site, which enabled the synthesis of an ester CLB conjugate. Functional versatility of the conjugates was reflected in the variability of their drug release profiles, while the conserved RGD sequence of a selective binding to the α_v integrin family, likely preserved their recognition by the Integrin and consequently their favorable toxicity towards targeted cancer cells. This hypothesis was supported by a computational analysis suggesting that all conjugates occupy conformational spaces similar to that of the Integrin bound bio-active parent peptide.     

Epidermal growth factor (EGF) receptor targeted delivery of PEGylated adenovirus

A biotin-polyethylene glycol (PEG)-epidermal growth factor (EGF) conjugate was immobilized onto the surface of avidin-modified adenovirus (ADV-Avi) via biotin-avidin interaction to deliver ADV specifically to EGF receptor over-expressing cancer cells. ADV-Avi/biotin-PEG-EGF complexes showed greatly enhanced intracellular uptake of ADV particles for an EGF receptor positive cell line (A431 cells), compared to naked or PEG alone immobilized ADV. ADV coding an exogenous GFP gene was used to quantitatively evaluate the level of GFP expression. ADV-Avi/biotin-PEG-EGF complexes also exhibited significantly increased extent of GFP expression for A431 cells, but not for MCF-7 cells (an EGF receptor deficient cell line), suggesting that retargeting of ADV to specific cells occurred by tethering of a cell-specific targeting ligand to the distal end of a PEG chain anchored onto the surface of ADV. This study demonstrates that ADV-Avi/biotin-PEG-EGF construct systems can be applied for cell-specific delivery of ADV with simultaneously reducing innate immune responses.     

Acid‐activated nonviral peptide vector for gene delivery

Nonviral vector–based gene therapy is a promising strategy for treating a myriad of diseases. Cell‐penetrating peptides are gaining increasing attention as vectors for nucleic acid delivery. However, most studies have focused more on the transfection efficiency of these vectors than on their specificity and toxicity. To obtain ideal vectors with high efficiency and safety, we constructed the vector stearyl‐TH by attaching a stearyl moiety to the N‐terminus of the acid‐activated cell penetrating peptide TH in this study. Under acidic conditions, stearyl‐TH could bind to and condense plasmids into nanoparticle complexes, which displayed significantly enhanced cellular uptake and transfection efficiencies. In contrast, stearyl‐TH lost the capacities of DNA binding and transfection at physiological pH. More importantly, stearyl‐TH and the complexes formed by stearyl‐TH and plasmids displayed no obvious toxicity at physiological pH. Consequently, the high transfection efficiency under acidic conditions and low toxicity make stearyl‐TH a potential nucleic acid delivery vector for gene therapy.     

Immunocytochemical identification of some regulatory peptides (gastrin,gastrin-releasing peptide,neurotensin and vasoactive intestinal polypeptide) in the Harderian gland of the green frog,Rana esculenta

Summary The presence and distribution of gastrin-, gastrin-releasing peptide-, neurotensin-and vasoactive intestinal polypeptide-like immunoreactivity in the Harderian gland ofRana esculenta were studied at different times of the annual cycle. Gastrin-releasing peptide, neurotensin and vasoactive intestinal polypeptide-like substances were found either in the glandular cells, or in the nerve fibers surrounding the glandular acini. Gastrin-like immunoreactivity was confined to the glandular cells. The immunoreactivity varied during the annual cycle, with the greatest concentration being noted during the recovery phase of glandular secretory activity.