Epidermal Penetration of a Therapeutic Peptide by Lipid Conjugation; Stereo-Selective Peptide Availability of a Topical Diastereomeric Lipopeptide

In inflammatory disorders (e.g. psoriasis), local concentrations of human neutrophil elastase (HNE), also known as polymorphonuclear leukocyte elastase (HLE), possibly overwhelm its natural inhibitors leading to extracellular matrix degradation. Elevated levels of HNE have been reported in a variety of inflammatory disorders, including psoriasis. Peptidic HNE inhibitors have a common hydrophobic sequence (Ala–Ala–Pro–Val). This peptide sequence inhibits HNE competitively; however the stratum corneum presents an effective barrier to the delivery of this tetrapeptide across the skin. The current work investigates the delivery of the modified peptide whereby the tetrapeptide was lipidated to enhance its ability to penetrate the stratum corneum. The tetrapeptide was coupled to a racaemic mixture of a short chain lipoamino acid (LAA) resulting in two diastereomers of the lipoamino acid-modified tetrapeptide. The penetration of the lipopeptide mixture was assessed across human epidermis in vitro. The percentage of applied dose penetrating to the receptor over 8 h following administration was 2.53% for the d-LAA conjugate and 1.47% for the l-diastereomer, compared to 0% for the peptide. The d-diastereomer appears to be relatively stable but the l-diastereomer appears to degrade releasing possibly the tetrapeptide and peptide fragment(s). Therefore the results clearly indicate that coupling the tetrapeptide to a short chain LAA enhances its delivery across human epidermis.Australian Peptide Conference Issue.     

In vitro metabolism of opioid tetrapeptide agonists in various tissues and subcellular fractions from rats

The metabolism of three mu-selective opioid tetrapeptide agonists, Tyr-D-Arg-Phe-Nva-NH(2) (TArPN), Tyr-D-Arg-Phe-Phe-NH(2) (TArPP), and Tyr-D-Ala-Phe-Phe-NH(2) (TAPP), was investigated in different rat tissues. High metabolic activity (<20% peptide remaining after 30 min) was found against the three peptides in the kidney homogenate and against TArPN in spleen homogenate. Low metabolic activity (>80% peptide remaining after 30 min) was found for all peptides in brain homogenate and plasma, and for TArPN and TArPP in blood. The other tissue homogenates, prepared from the small and large intestine, liver and lung, all exhibited intermediate metabolic activity (20-80% peptide remaining after 30 min) against the peptides. In all tissues investigated, the tetrapeptides were metabolized at the C-terminal amide by deamidation.A further in depth metabolic investigation was performed in subcellular fractions isolated from three tissues (small intestine, liver and kidney). In the liver, the deamidation was predominantly localized to the mitochondrial/lysosomal fraction, while hydrolysis at the N-terminal Tyr residue was the major metabolic pathway in the microsomal/brush-border membrane fraction from the kidney and small intestine.     

Membrane dipeptidase is the receptor for a lung-targeting peptide identified by in vivo phage display

In vivo phage display is a powerful method to study organ- and tissue-specific vascular addresses. Using this approach, peptides capable of tissue-specific homing can be identified by performing a selection for that trait in vivo. We recently showed that the CGFECVRQCPERC (termed GFE-1) peptide can selectively bind to mouse lung vasculature after an intravenous injection. Our aim in the present study was to identify the receptor for this lung-homing peptide. By using affinity chromatography, we isolated a 55-kDa lung cell-surface protein that selectively binds to the GFE-1 peptide. Protein sequencing established the identity of the receptor as membrane dipeptidase (MDP), a cell-surface zinc metalloprotease involved in the metabolism of glutathione, leukotriene D4, and certain beta-lactam antibiotics. Phage particles displaying the GFE-1 peptide selectively bind to COS-1 cells transfected with the murine MDP cDNA. Moreover, the synthetic GFE-1 peptide could inhibit MDP activity. By establishing MDP as the receptor for the GFE-1 peptide, our results suggest potential applications for both MDP and the GFE-1 peptide in delivery of compounds to the lungs. This work also demonstrates that cell-surface proteases can be involved in tissue-specific homing.     

Conformational analysis of sea cucumber (Caudina arenicola) C globin 94-97 fragment,Pro-Glu-Leu-Leu

The tetrapeptide Pro-Glu-Leu-Leu forms the 94-97 fragment of C globin in sea cucumber. 2% Butanediol dimethacrylate-cross linked polystyrene (2% BDDMA-PS), which had been optimized, was used for the synthesis of the tetrapeptide Pro-Glu-Leu-Leu. The peptide was synthesized by using Boc-amino acid strategy. The peptide purity was checked by RP-HPLC and the peptide was characterized by (1)H NMR spectroscopy and amino acid analysis. Conformation of the peptide was studied by 1D- and 2D- homonuclear (1)H NMR, in DMSO-d6 at 300K. The conformation of the synthetic tetrapeptide (extended backbone conformation) is not in agreement with that in C globin.     

Vascular changes after intra-amniotic endotoxin in preterm lamb lungs

Chorioamnionitis is associated with preterm delivery and bronchopulmonary dysplasia (BPD), characterized by impaired alveolar and pulmonary vascular development and vascular dysfunction. To study the vascular effects in a model of chorioamnionitis, preterm lambs were exposed to 20 mg of intra-amniotic endotoxin or saline for 1, 2, 4, or 7 days and delivered at 122 days gestational age (term = 150 days). This intra-amniotic endotoxin dose was previously shown to induce lung maturation. The effect of intra-amniotic endotoxin on expression of endothelial proteins was evaluated. Muscularization of the media and collagen deposition in adventitia of small pulmonary arteries was used to assess vascular remodeling. Compared with controls, bronchoalveolar lavage fluid protein content was increased 2 days after intra-amniotic endotoxin exposure. Vascular endothelial growth factor (VEGF) 165 isoform mRNA decreased 2-4 days after intra-amniotic endotoxin. VEGF, VEGF receptor-2, endothelial nitric oxide synthase (eNOS), platelet endothelial cell adhesion molecule-1, and Tie-2 protein expression in the lung coordinately decreased 1-7 days after intra-amniotic endotoxin. Intra-amniotic endotoxin appeared to selectively decrease eNOS expression in small pulmonary vessels compared with large vessels. Medial smooth muscle hypertrophy and increased adventitial fibrosis were observed 4 and 7 days after intra-amniotic endotoxin. These results demonstrate that, in the preterm lamb lung, antenatal inflammation inhibits endothelial cell protein expression followed by vascular remodeling changes in small pulmonary arteries. Exposure to antenatal inflammation may cause vascular remodeling and contribute to the development of BPD.     

A conservative mutant of a proteolytic fragment produced during fibril formation enhances fibrillogenesis

The fibrillogenesis of a peptide corresponding to residues 35–51 of human α-lactalbumin (¹GYDTQAIVENNESTEYG¹⁷) can be dramatically enhanced by the addition of a tetrapeptide TDYG homologous to its C-terminus (TEYG). Generation of spontaneous hydrolytic products similar to this peptide was demonstrated by mass-spectrometry analysis of GYDTQAIVENNESTEYG peptide solution components during fibrillogenesis. Possible mechanisms and roles of short peptides in protein metabolism are discussed.     

The interaction of (1–4)-fragment of thymosin β4 with calmodulin-sensitive cAMP phosphodiesterase from hypothalamus

Evidence was accumulated indicating that cyclic nucleotides are involved in regulation of growth, differentiation and function of lymphoid cells. It was previously shown that the N-fragment (1–4) of thymosin 4 (Ac-Ser-Asp-Lys-Pro-OH) inhibits in vivo the entry of cell populations into S-phase. In the course of the study of the interrelationship between the immune and neuroendocrine systems we have found that the tetrapeptide caused incomplete competitive inhibition of hypothalamic calmodulin (CaM)-dependent phosphodiesterase (PDE) stimulated by CaM. In the presence of the peptide, the 20-fold increase of the constant for PDE activation by CaM was accompanied by an insignificant rise in the maximum rate of cAMP hydrolysis. The value of the inhibition constant (K_i) amounted to 600nM. In the absence of CaM, the peptide at saturating concentrations reduced the basal activity of PDE nearly 2- to 3-fold. The effect of the peptide on PDE was noncompetitive with respect to cAMP. The results support our suggestion that the tetrapeptide realizes its effects in the immuno-neuroendocrine system by the mechanism of cyclic nucleotide metabolism.     

Sustained lung activity of a novel chimeric protein, SOD2/3, after intratracheal administration

Delivery of recombinant superoxide dismutase to the lung is limited by its short half-life and poor tissue penetration. We hypothesized that a chimeric protein, SOD2/3, containing the enzymatic domain of manganese superoxide dismutase (SOD2) and the heparan-binding domain of extracellular superoxide dismutase (SOD3), would allow for the delivery of more sustained lung and pulmonary vascular antioxidant activity compared to SOD2. We administered SOD2/3 to rats by intratracheal (i.t.), intraperitoneal (i.p.), or intravenous (i.v.) routes and evaluated the presence, localization, and activity of lung SOD2/3 1 day later using Western blot, immunohistochemistry, and SOD activity gels. The effect of i.t. SOD2/3 on the pulmonary and systemic circulation was studied in vivo in chronically catheterized rats exposed to acute hypoxia. Active SOD2/3 was detected in lung 1 day after i.t. administration but not detected after i.p. or i.v. SOD2/3 administration or i.t. SOD2. The physiologic response to acute hypoxia, vasoconstriction in the pulmonary circulation and vasodilation in the systemic circulation, was enhanced in rats treated 1 day earlier with i.t. SOD2/3. These findings indicate that i.t. administration of SOD2/3 effectively delivers sustained enzyme activity to the lung as well as pulmonary circulation and has a longer tissue half-life compared to native SOD2. Further testing in models of chronic lung or pulmonary vascular diseases mediated by excess superoxide should consider the longer tissue half-life of SOD2/3 as well as its potential systemic vascular effects.     

Construction of immunogens for synthetic malaria vaccines

The immunogenicity of a peptide consisting of eight repeats of the tetrapeptide sequence NANP (Asn-Ala-Asn-Pro) contained in the circumsporozoite protein of Plasmodium falciparum was investigated in mice under different modes of presentation. This peptide was able to produce biologically active antibodies when administered with adjuvant and linked to a protein carrier. However, a (NANP) peptide polymerized by carbodiimide was found to be immunogenic in the absence of protein carrier in H-2b mice. In contrast, the (NANP)8 peptide polymerized by glutaraldehyde was not immunogenic in the same strain. Furthermore, the efficacy of murabutide in saline, as an immunological adjuvant, was compared to the efficacy of Freund's complete adjuvant.     

Pharmacokinetic investigation of a 14C-labelled beta 3/alpha tetrapeptide in rats

The solid-phase synthesis and an ADME investigation with albino and pigmented male rats of the doubly 14C-labelled beta/alpha-tetrapeptide derivative Ac-beta3 hTyr-(D)Trp-beta3 hLys-beta3 hThr-lactone (3; Fig. 3) are described. After intravenous (i.v.) and peroral (p.o.) administration of the peptide, its concentration in blood and plasma, its tissue distribution, and the metabolism and the excretion of the peptide were analyzed over a period of up to 7 days post dose. The tetrapeptide in its ring opened form, 5, has a bioavailability of ca. 25%; radioactivity is distributed in the animals in an organ-specific way, and the compound appears to pass the blood-brain barrier to a very small extent, if at all (Tables 1-3 and Figs. 2-6). Excretion (37% renal, 44% fecal, including biliary) of the tetrapeptide 4 days after i.v. administration is almost complete, with only 4.3% remaining in the carcass; 4 days after p.o. administration 97% of the dose has been excreted in the feces. Radiochromatograms taken of plasma (0.5 and 24 h after i.v. dosing) and of urine and feces extracts (0-48 h collected) reveal the presence of lactone 3 and/or the corresponding hydroxy acid 5 with essentially no or very minor other peaks, respectively, representing possible metabolites (Tables 4-6, and Fig. 7 and 8). A comparison with a previous ADME investigation of a beta-nonapeptide show that--except for the lack of metabolism--all aspects of exposure, distribution, and elimination are different (structure-specific properties). The investigated tetrapeptide 3 is a potent and highly specific agonist of the somatostatin receptor hsst4, rendering the results described herein promising for diagnostic and therapeutic applications of beta-peptides.     

Small Azurin Derived Peptide Targets Ephrin Receptors for Radiotherapy

Many lung cancer treatment regimens include radiotherapy. We sought to improve the efficacy of such treatment by invoking the targeted delivery of a model radiosensitizer (nicotinamide) to malignant tissues. Ephrin receptors (Eph), which are often overexpressed in lung cancers, were selected as the target of our delivery system. Molecular targeting was achieved utilizing a small peptide derived from the C-terminal portion of azurin, a copper-containing redox protein (“cupredoxin”) that is capable of binding to ephrin receptors. We prepared and screened a sub-library of peptides derived from the C-terminal region of azurin and found several small analogues that bound ephrin receptors EphA2, EphB2, and EphB4. One such peptide, termed AzV36, was selected for conjugation with nicotinic acid via an amide bond to form AzV36-NicL. The resulting linear peptide contains 15 residues (including unusual and d-amino acids), is very stable in human serum, and can be easily manufactured. AzV36-NicL conjugate was tested in vivo for its ability to radiosensitize Lewis lung carcinoma (LCC) in artificial metastasis and solid tumor engraftment models. The compound increased the efficacy of radiotherapy with tumor colonies being ~2–13 fold lower than with radiation alone depending on experimental schedule. In contrast, equimolar amounts of unconjugated peptide (AzV36-L) or nicotinamide alone only marginally improved radiation efficacy. The targeted delivery of radiosensitizer(s) to ephrin receptors may enhance the efficacy of radiation treatment of lung cancer and of other cancers that overexpress ephrin receptor(s).     

A novel cell adhesive protein engineered by insertion of the Arg-Gly-Asp-Ser tetrapeptide

A tetrapeptide Arg-Gly-Asp-Ser (RGDS) has been shown to be a versatile cell recognition signal of extracellular matrix components for the interaction with cells. We introduced the RGDS tetrapeptide into a truncated form of protein A, a staphylococcal immunoglobulin-binding protein, by inserting an oligonucleotide cassette encoding the tetrapeptide into the coding region of the protein A expression vector pRIT2T. The mutagenized protein was capable of not only binding to immunoglobulin G but also mediating cell attachment and spreading onto an inert substrate. Cell adhesion mediated by the mutagenized protein was inhibitable by a synthetic peptide Gly-Arg-Gly-Asp-Ser but not by a related peptide Gly-Arg-Gly-Glu-Ser, confirming that the inserted RGDS tetrapeptide served as a recognition signal for cell adhesion. Furthermore, the RGDS-containing protein was capable of adhering cells onto an immunoglobulin-coated surface which could not by itself support cell adhesion. Thus, the cell adhesive and immunoglobulin binding activities of the mutagenized protein appear to function coordinately. The protocol described here is essentially applicable to any protein and, therefore, provides a general principle in tailoring novel multifunctional proteins having cell adhesive activity.     

Investigation of S-farnesyl transferase substrate specificity with combinatorial tetrapeptide libraries

Using biased tetrapeptide libraries made up of proteinogenic amino acids of the general formula Cys-O2-X3-X4, we searched for new substrates of partly purified rat brain S-farnesyl transferase (FTase). To achieve this task, an assay was developed in which the consumption of the co-substrate (farnesyl pyrophosphate) was measured. After three steps of deconvolution including each synthesis and enzymatic assay, the most efficient substrates found under these particular conditions were Cys-Lys-Gln-Gln (peptide I) and Cys-Lys-Gln-Met (peptide II). As a control, we used another tetrapeptide library (Cys-Val-O3-X4) in which the valine position was arbitrarily fixed, corresponding to Cys-Val-Ile-Met in the CAAX box of K-RasB, although this sublibrary was only marginally active compared with Cys-Lys-X3-X4 in the first round of deconvolution. The best substrate sublibrary was Cys-Val-Thr-X4, threonine being more favourable than the aliphatic amino acids (Val, Ile, Leu, Ala) in this position. Deconvolution finally led to Cys-Val-Thr-Gln, -Met, -Thr and -Ser as the most efficient substrates of FTase. Those tetrapeptides were not substrates of a partly purified geranylgeranyl transferase 1 (GGTase1). We also investigated the influence of the -1 position (at the N-terminus of cysteine) on the specificity of the enzyme, by using a series of pentapeptides constructed on the basis of the best tetrapeptide core (peptide 1). Among this family of analogues, only His-Cys-Lys-Gln-Gln did not behave as a substrate, whereas all the other pentapeptides were measurable substrates, with Gly-, Asn- and Thr-Cys-Lys-Gln-Gln displaying kinetic constants similar to that of Cys-Lys-Gln-Gln. The present work provides strong evidence that the best tetrapeptide substrates of FTase do not necessarily belong to the classical CAAX box, in which A's are lipophilic residues, but rather contain hydrophilic amino acids in the middle of their sequences. Among them, peptides I and II are potent FTase in vitro substrates that are not recognised by GGTase1 and might be new starting points for the design of FTase inhibitors.     

Anticancer potency of small linear and cyclic tetrapeptides and pharmacokinetic investigations of peptide binding to human serum albumin

We have in the present study explored the anticancer activity against human Burkitt's lymphoma cells (Ramos) of a series of small linear and cyclic tetrapeptides containing a β^2,2‐amino acid with either two 2‐naphthyl‐methylene or two para‐CF₃‐benzyl side chains, along with their interaction with the main plasma protein human serum albumin (HSA). The cyclic and more amphipathic tetrapeptides revealed a notably higher anticancer potency against Ramos cells [50% inhibitory concentration (IC₅₀) 11–70 μM] compared to the linear tetrapeptide counterparts (IC₅₀ 18.7 to >413 μM). The most potent cyclic tetrapeptide c3 had a 16.5‐fold preference for Ramos cells compared to human red blood cells, whereas the cyclic tetrapeptide c1 both showed low hemolytic activity and displayed the overall highest (2.9‐fold) preference for Ramos cells (IC₅₀ 23 μM) compared to healthy human lung fibroblast cells (MRC‐5). Investigating the interaction of selected tetrapeptides and recently reported hexapeptides with HSA revealed that the peptides bind to drug site II of HSA in the 22–28 μM range, disregarding size and overall structure. NMR and in silico molecular docking experiments identified the lipophilic residues as responsible for the interaction, but in vitro studies showed that the anticancer potency of the peptides varied in the presence of HSA and that c3 remained the most potent peptide. Based on our findings, we call for implementing serum albumin binding in development of anticancer peptides, as it may have implications for future administration and systemic distribution of peptide‐based cancer drugs. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Adiponectin attenuates lipopolysaccharide-induced acute lung injury through suppression of endothelial cell activation

Adiponectin (APN) is an adipose tissue-derived factor with anti-inflammatory and vascular protective properties whose levels paradoxically decrease with increasing body fat. In this study, APN's role in the early development of ALI to LPS was investigated. Intratracheal LPS elicited an exaggerated systemic inflammatory response in APN-deficient (APN(-/-)) mice compared with wild-type (wt) littermates. Increased lung injury and inflammation were observed in APN(-/-) mice as early as 4 h after delivery of LPS. Targeted gene expression profiling performed on immune and endothelial cells isolated from lung digests 4 h after LPS administration showed increased proinflammatory gene expression (e.g., IL-6) only in endothelial cells of APN(-/-) mice when compared with wt mice. Direct effects on lung endothelium were demonstrated by APN's ability to inhibit LPS-induced IL-6 production in primary human endothelial cells in culture. Furthermore, T-cadherin-deficient mice that have significantly reduced lung airspace APN but high serum APN levels had pulmonary inflammatory responses after intratracheal LPS that were similar to those of wt mice. These findings indicate the importance of serum APN in modulating LPS-induced ALI and suggest that conditions leading to hypoadiponectinemia (e.g., obesity) predispose to development of ALI through exaggerated inflammatory response in pulmonary vascular endothelium.     

Targeting alphavbeta3 and alpha5beta1 for gene delivery to proliferating VSMCs: synergistic effect of TGF-beta1

TGF-beta1 levels increase after vascular injury and promote vascular smooth muscle cell (VSMC) proliferation. We define a nonviral gene delivery system that targets alphavbeta3 and alpha5beta1 integrins that are expressed on proliferating VSMCs and strongly induced by TGF-beta1. A 15-amino acid RGDNP-containing peptide from American Pit Viper venom was linked to a Lys(16) peptide as vector (molossin vector) and complexed with Lipofectamine or fusogenic peptide for delivery of luciferase or beta-galactosidase reporter genes to primary cultures of human, rabbit, and rat VSMCs. Preincubation of VSMCs with TGF-beta1 for 24 h, but not with PDGF-BB, interferon-gamma, TNF-alpha, nor PMA, increased alphavbeta3 and alpha5beta1 expressions on VSMCs and enhanced gene delivery of molossin vector. Thus beta-galactosidase activity increased from 35 +/- 5% (controls) to 75 +/- 5% after TGF-beta1 treatment, and luciferase activity increased fourfold over control values. Potential use of this system in vessel bypass surgery was examined in an ex vivo rat aortic organ culture model after endothelial damage. Molossin vector system delivered beta-galactosidase to VSMCs in the vessel wall that remained for up to 12 days posttransfection. The molossin vector system, when combined with TGF-beta1, enhances gene delivery to proliferating VSMCs and might have clinical applications for certain vasculoproliferative diseases.     

Peptide fragments derived from the beta-chain of hemoglobin (hemorphins) are centrally active in vivo

A novel tetrapeptide (hemorphin-4) and pentapeptide (hemorphin-5), derived from the beta-chain of hemoglobin, were synthesized by solid-phase methodology, purified and the amino acid sequences confirmed. The central (ICV) effects of hemorphin-4 and -5 were studied in two models of phasic and tonic nociception, the mouse warm water tail-flick assay and hindpaw formalin assay, respectively. Additionally, two physiological endpoints, central modulation of bladder motility and central effects on intestinal propulsion, were studied in rats and mice, respectively. In the tail-flick assay, both peptides (40-100 nmoles) produced a dose-related naloxone-reversible antinociceptive effect when tested 10 min after peptide administration, with the tetrapeptide being slightly more potent than the pentapeptide. No effect was noted for either peptide using the tonic nociception assay, except at a dose of 150 nmoles for hemorphin-5. Inhibition of gastrointestinal propulsion was also not affected by either peptide. However, both peptides (10-40 nmoles) inhibited micturition contractions in a dose-related and naloxone-reversible fashion, with the tetrapeptide being twice as potent as the pentapeptide. These findings provide evidence that hemorphin-4 and -5 exert naloxone-reversible opioid actions in vivo and, therefore, may be physiologically important blood-borne peptides.     

融合细胞株Eahy926与亲本人肺腺癌细胞株A549差异表达蛋白的蛋白质组学分析

融合细胞株Eahy926是人肺腺癌细胞株A549和人脐静脉内皮细胞杂交而成的永生化细胞株,具有血管内皮细胞的特性,已广泛用于内皮细胞相关研究. 本研究应用蛋白质组学技术分析融合细胞株Eahy926与亲本人肺腺癌细胞株A549的蛋白质差异表达,探讨融合细胞生物学特性变化及其机制. 对Eahy926和亲本A549的细胞总蛋白质进行双向凝胶电泳,在PDQuest软件辅助下找出差异表达蛋白质点,经肽质量指纹图谱(peptide mass fingerprinting, PMF) 和串级质谱(tandem mass spectrometry,TMS) 分析,SWISS-PROT数据库检索,成功鉴定出28个差异蛋白,如CATB、CK8、CK18、annexin A2、GRP78、HSP90、HSP60、vimentin等一些与分子伴侣、氧化应激、能量代谢、信号转导等有关,并与肿瘤细胞分裂增殖、分化凋亡、侵袭转移、免疫逃逸以及肿瘤血管生长密切关联的蛋白质. 研究发现,融合细胞株Eahy926和人肺腺癌细胞株A549的蛋白质组表达谱存在明显差异,这将有助于今后进一步探讨肿瘤细胞与内皮细胞的相互作用机制及其融合细胞的特性,筛选肿瘤增殖和转移相关蛋白质及分子标志物,亦可为肿瘤的抗血管生成治疗提供新思路.     

Activity and mechanism of action of insect oostatic peptides in flesh fly

The relationship between structure and activity of insect oostatic decapeptide (Aed-TMOF) analogues in flesh fly was analyzed. The highest oostatic activity was exhibited by the pentapetide and tetrapeptide analogues, H-Tyr-Asp-Pro-Ala-Pro-OH and H-Tyr-Asp-Pro-Ala-OH, respectively. The tetrapeptide, either native or tritiated, was used to study its metabolism in the ovaries and hemolymph and to detect putative binding sites in the flesh fly ovaries and head. A high metabolism of the tetrapeptide with a half-life in the hemolymph and ovaries less than 1h was determined. The initial limiting step in the degradation is tyrosine(1) cleavage. Other degradation products were detected only transiently in low quantities. Using tritiated tetrapeptide, we found that only very low specific binding was detected in the homogenates of ovaries and in the rough membrane preparation in the presence and absence of protease inhibitors.     

A novel vascular-targeting peptide for gastric cancer delivers low-dose TNFα to normalize the blood vessels and improve the anti-cancer efficiency of 5-fluorouracil

Various vascular-targeted agents fused with tumor necrosis factor α (TNFα) have been shown to improve drug absorption into tumor tissues and enhance tumor vascular function. TCP-1 is a peptide selected through in vivo phage library biopanning against a mouse orthotopic colorectal cancer model and is a promising agent for drug delivery. This study further investigated the targeting ability of TCP-1 phage and peptide to blood vessels in an orthotopic gastric cancer model in mice and assessed the synergistic anti-cancer effect of 5-fluorouracil (5-FU) with subnanogram TNFα targeted delivered by TCP-1 peptide. In vivo phage targeting assay and in vivo colocalization analysis were carried out to test the targeting ability of TCP-1 phage/peptide. A targeted therapy for improvement of the therapeutic efficacy of 5-FU and vascular function was performed through administration of TCP-1/TNFα fusion protein in this model. TCP-1 phage exhibited strong homing ability to the orthotopic gastric cancer after phage injection. Immunohistochemical staining suggested that and TCP-1 phage/TCP-1 peptide could colocalize with tumor vascular endothelial cells. TCP-1/TNFα combined with 5-FU was found to synergistically inhibit tumor growth, induce apoptosis and reduce cell proliferation without evident toxicity. Simultaneously, subnanogram TCP-1/TNFα treatment normalized tumor blood vessels. Targeted delivery of low-dose TNFα by TCP-1 peptide can potentially modulate the vascular function of gastric cancer and increase the drug delivery of chemotherapeutic drugs.