RGD Peptide Conjugates Research Articles

Impact of bifunctional chelators on biological properties of 111In-labeled cyclic peptide RGD dimers

The present study describes the synthesis and biological evaluation of (111)In(DOTA-3P-RGD(2)) (DOTA = 1,4,7,10-tetraazacyclo-dodecane-1,4,7,10-tetraacetic acid; 3P-RGD(2) = PEG(4)-E[PEG(4)-c(RGDfK)](2); PEG(4) = 15-amino-4,7,10,13-tetraoxapentadecanoic acid), (111)In(DTPA-3P-RGD(2)) (DTPA = diethylenetriaminepentaacetic acid) and (111)In(DTPA-Bn-3P-RGD(2)) (DTPA-Bn = 2-(p-thioureidobenzyl)-diethylenetriaminepentaacetic acid) as potential radiotracers for imaging tumor integrin α(v)β(3) expression in athymic nude mice bearing U87MG glioma xenografts. The aim of the study is to assess the impact of the bifunctional chelator (BFC) (DOTA vs. DTPA or DTPA-Bn) on the biodistribution characteristics of the (111)In-labeled 3P-RGD(2). IC(50) values of DOTA-3P-RGD(2), DTPA-3P-RGD(2) and DTPA-Bn-3P-RGD(2) were determined to be 1.3 ± 0.2, 1.4 ± 0.3, 1.3 ± 0.3 nM, respectively, against (125)I-c(RGDyK) bound to U87MG human glioma cells. Radiotracers were prepared by reacting (111)InCl(3) with the RGD peptide conjugates in NH(4)OAc buffer (100 mM, pH 5.5). For DOTA-3P-RGD(2), successful radiolabeling could be completed by heating the reaction mixture at 100°C for 15-20 min. For DTPA-3P-RGD(2) and DTPA-Bn-3P-RGD(2), the radiolabeling was almost instantaneous at room temperature. The specific activity was ~50 mCi/mg (or ~100 mCi/μmol) for (111)In(DOTA-3P-RGD(2)) and ~200 mCi/mg (or ~400 mCi/μmol) for (111)In(DTPA-3P-RGD(2)). The results from biodistribution studies showed that all the three radiotracers have high tumor uptake and excellent tumor-to-background (T/B) ratios up to 4-h postinjection. After that time point, both (111)In(DTPA-3P-RGD(2)) and (111)In(DTPA-Bn-3P-RGD(2)) showed a much faster tumor washout and poorer T/B ratios than (111)In(DOTA-3P-RGD(2)). The tumor uptake of (111)In(DOTA-3P-RGD(2)) is integrin α(v)β(3)- and RGD-specific. (111)In(DOTA-3P-RGD(2)) is metabolically stable while only ~25% of (111)In(DTPA-Bn-3P-RGD(2)) remains intact in the feces during 2-h period. On the basis of results from this study, it was concluded that (111)In(DTPA-3P-RGD(2)) can be an effective integrin α(v)β(3)-targeted radiotracer if the high-specific activity is required. However, DOTA remains to be the BFC of choice for the development of therapeutic lanthanide radiotracers.

3D QSAR of novel estrogen–RGD peptide conjugates: Getting insight into structural dependence of anti-osteoporosis activity and side effect of estrogen in ERT

To explore the structural dependence of the oral potency and side effect of estrogen–RGD peptide conjugates, here six novel conjugates were prepared via introducing RGD-tetrapeptides into both 3- and 17-positions of estradiol, and introducing RGD-octapeptides into 3-position of estrone. In an ovariectomized mouse model they exhibited higher anti-osteoporosis activity and lower side effect than estrogen. For 3D QSAR analysis the anti-osteoporosis activities of nine known conjugates estrogen–RGD tetrapeptide conjugates were also provided. Using Cerius 2 module their 3D QSAR analysis was performed, four equations with high r 2 values were established, and the structural dependence of the oral potency and side effect of them was elucidated.

Synthesis, evaluation and 3D QSAR analysis of novel estradiol–RGD octapeptide conjugates with oral anti-osteoporosis activity

To enhance the potency, reduce the side effects and improve oral property of estradiol in estrogen replacement therapy (ERT), 6 novel estradiol–RGD octapeptide conjugates have been prepared. In an ovariectomized mouse osteoporotic model, at an oral dosage of 110.3 nmol/kg per day, their anti-osteoporosis activity was significantly higher than that of estradiol and estradiol–RGD tetrapeptide conjugates, and their risks of thrombogenesis and endometrial hyperplasia were significantly lower than that of estradiol and estradiol–RGD tetrapeptide conjugates. Using QSAR module of Cerius2, the 3D QSAR was performed for both femur weights and femur ash weights of estradiol–RGD peptide conjugates receiving mice. The r 2 of the 3D QSAR equations up to 0.995 and 0.988 indicates that they are capable of predicting a comparatively exact anti-osteoporosis activity for a conjugate.

Synthesis and Biological Characterisation of Targeted Pro‐Apoptotic Peptide

We report herein the synthesis and in vitro assay of new, multimeric RGD-peptide conjugates for cell-targeted drug delivery. We generated a peptide scaffold comprising two functional domains, one a tumour blood vessel "homing" motif and the other a programmed cell-death-inducing peptide sequence. RGD peptides were selected to direct the molecular conjugate to alpha(V)beta(3) integrin-containing tumour cells. The pro-apoptotic (Lys-Leu-Ala-Lys-Leu-Ala-Lys)(2) peptide was found to be nontoxic outside cells, but toxic when internalized into targeted cells as it disrupted the mitochondrial membrane. The synthesis of these targeted pro-apoptotic conjugates was carried out by assembling three different units (that is, scaffold, RGD units and pro-apoptotic peptide) through chemoselective ligations. We show that one compound displays significant biological effect in alpha(V)beta(3) integrin-containing tumour cells.

Improved Anti-Osteoporosis Potency and Reduced Endometrial Membrane Hyperplasia During Hormone Replacement Therapy with Estrogen−RGD Peptide Conjugates

To improve the specificity and potency of estrogen replacement therapy therapeutics while also minimizing the side effects such as bone resorption and thickening of the uterine wall, a series of novel estrogen-derived conjugates estradiol-3-RGD, estradiol-17-RGD, and estrone-3-RGD peptides have been prepared. In a mouse model, intraperitoneal (i.p.) administration of these estrogen-RGD peptide conjugates resulted in decreased serum concentrations of calcium and alkaline phosphatase, as well as increased levels of calcium, phosphorus, and minerals in the mouse femur. Furthermore, the anti-osteoporosis action of these conjugates followed a dose-dependent manner and was accompanied with no observable effects on endometrial cell hyperplasia. In addition to all of these compounds exhibiting biological activity when administered by the i.p. route, we were particularly pleased to note that the estradiol-3-RGD and estradiol-17-RGD conjugates were both orally active.

Delivery of the p38 MAPkinase Inhibitor SB202190 to Angiogenic Endothelial Cells: Development of Novel RGD-Equipped and PEGylated Drug−Albumin Conjugates Using Platinum(II)-Based Drug Linker Technology

Endothelial cells play an important role in inflammatory disorders, as they control the recruitment of leukocytes into inflamed tissue and the formation of new blood vessels. Activation of p38MAP kinase results in the production of proinflammatory cytokines and the expression of adhesion molecules. P38MAP kinase inhibitors are therefore considered important candidates for the treatment of inflammatory disorders. In the present study, we propose a novel strategy to counteract these processes by delivery of the p38MAP kinase inhibitor SB202190 into angiogenic endothelial cells. A drug-targeting conjugate was developed by conjugation of SB202190 to human serum albumin (HSA) using a novel platinum-based linker. Specificity for angiogenic endothelial cells was introduced by conjugation of cyclic RGD-peptides via bifunctional polyethylene glycol linkers. The final products contained an average of nine SB202190 and six RGDPEG groups per albumin. The platinum-based linker displayed high stability in buffers and culture medium, but released SB202190 slowly upon competition with sulfur-containing ligands like glutathione. RGDPEG-SB-HSA bound to alpha(v3)-integrin expressing endothelial cells (human umbilical cord vein endothelial cells) with low nanomolar affinity and was subsequently internalized. When HUVEC were treated with TNF to induce inflammatory events, pretreatment with RGDPEG-SB-HSA partially inhibited proinflammatory gene expression (IL-8, E-selectin; 30% inhibition) and secretion of cytokines (IL-8, 34% inhibition). We conclude that the developed RGDPEG-SB-HSA conjugates provide a novel means to counteract inflammation disorders such as rheumatoid arthritis.

Dual-acting agents that possess free radical scavenging and antithrombotic activities: Design, synthesis, and evaluation of phenolic tetrahydro-β-carboline RGD peptide conjugates

A new approach to construct a single dual-acting agent is described. Compounds 6a– c are potent free radical scavengers as demonstrated by the EC 50 values in PC12 cell survival assay in term of NO, H 2O 2, and OH scavenging activity. The Ach-induced vaso-relaxation assay further confirms the potent NO scavenging activity of compounds 6a– c. In addition, 6a– c are efficacious in a rat arterial thrombosis, and are active in ADP- or PAF-induced in vitro platelet aggregation assay, suggesting that compounds 6a– c also possess anti-thrombotic activities. Since both free radical and thrombogenesis are important risk factors in myocardial ischemic/reperfusion injuries, these dual-acting agents having both free radical scavenging and antithrombolic activities may potentially be beneficial toward their treatment.

Preparation and functional evaluation of RGD-modified proteins as alpha(v)beta(3) integrin directed therapeutics.

Tumor blood vessels can be selectively targeted by RGD-peptides that bind to alpha(v)beta(3) integrin on angiogenic endothelial cells. By inhibiting the binding of these integrins to its natural ligands, RGD-peptides can serve as antiangiogenic therapeutics. We have prepared multivalent derivatives of the cyclic RGD-peptide c(RGDfK) by covalent attachment of the peptide to side chain amino groups of a protein. These RGDpep-protein conjugates inhibited alpha(v)beta(3)-mediated endothelial cell adhesion in vitro, while conjugates prepared with a control RAD-peptide showed no activity. Radiobinding and displacement studies with endothelial cells demonstrated an increased affinity of the RGDpep-protein conjugates compared to the free peptide, with IC(50) values ranging from 23 to 0.6 nM, depending on the amount of coupled RGDpep per protein. Compared to the parental RGD-peptide and the related RGD-peptide ligand c(RGDfV), the RGDpep-protein conjugates showed a considerable increase in affinity (IC(50) parent RGDpep: 818 nM; IC(50) c(RGDfV): 158 nM). We conclude that the conjugation of RGD-peptides to a protein, resulting in products that can bind multivalently, is a powerful approach to increase the affinity of peptide ligands for alpha(v)beta(3)/alpha(v)beta(5) integrins.