Cell-penetrating γ-peptide/antimicrobial undecapeptide conjugates with anticancer activity

In this study, we combined a cell-penetrating γ-peptide, PEG-1, with antimicrobial undecapeptides in order to provide compounds with anticancer properties against MDA-MB-231 human breast cancer cells. We demonstrated that the conjugates were more cytotoxic than Ac-PEG-1 and the parent undecapeptides. We also evaluated the toxicity of the conjugates against non-malignant cells. The peptide conjugate with the best biological profile was BP77-PEG-1, which, at 10 μM, showed a 71% growth inhibition in MDA-MB-231 cells and only a 17% inhibition in non-malignant cells. Therefore, this study suggests that PEG-1 mediated the undecapeptide delivery into cancer cells and that these conjugates are the proof-of-concept of this strategy to generate improved anticancer drugs based on peptides.     

Synthesis and in vitro characterization of semitelechelic poly[N-(2-hydroxypropyl)methacrylamide]-trastuzumab conjugates targeted to breast cancer

Trastuzumab (TRZ) is a humanized monoclonal antibody that targets the extracellular domain of the human epidermal growth factor receptor type 2 (Her2). Semitelechelic (ST) poly[N-(2-hydroxypropyl)methacrylamide]-TRZ conjugates are successfully synthesized and evaluated as a potential drug delivery system that actively targets Her2-overexpressing cancer cells. The ST backbone shows favorable characteristics when conjugated to TRZ. The conjugate exhibits comparable and prolonged anticancer activity when compared to free TRZ in Her2 overexpressing breast cancer cell lines.     

Conjugation of curcumin onto hyaluronic acid enhances its aqueous solubility and stability

Polymer-drug conjugates have gained much attention largely to circumvent lower drug solubility and to enhance drug stability. Curcumin is widely known for its medicinal properties including its anticancer efficacy. One of the serious drawbacks of curcumin is its poor water solubility which leads to reduced bioavailability. With a view to address these issues, we synthesized hyaluronic acid-curcumin (HA-Cur) conjugate. The drug conjugate was characterized using FT-IR, NMR, Dynamic light scattering and TEM techniques. The conjugates, interestingly found to assembles as micelles in aqueous phase. The formation of micelles seems to improve the stability of the drug in physiological pH. We also assessed cytotoxicity of the conjugate using L929 fibroblast cells and quantified by MTT assay.     

Evaluation of the DNA Alkylation Properties of a Chlorambucil-Conjugated Cyclic Pyrrole-Imidazole Polyamide

Hairpin pyrrole-imidazole polyamides (hPIPs) and their chlorambucil (Chb) conjugates (hPIP-Chbs) can alkylate DNA in a sequence-specific manner, and have been studied as anticancer drugs. Here, we conjugated Chb to a cyclic PIP (cPIP), which is known to have a higher binding affinity than the corresponding hPIP, and investigated the DNA alkylation properties of the resulting cPIP-Chb using the optimized capillary electrophoresis method and conventional HPLC product analysis. cPIP-Chb conjugate 3 showed higher alkylation activity at its binding sites than did hPIP-Chb conjugates 1 and 2 . Subsequent HPLC analysis revealed that the alkylation site of conjugate 3 , which was identified by capillary electrophoresis, was reliable and that conjugate 3 alkylates the N3 position of adenine as do hPIP-Chbs. Moreover, conjugate 3 showed higher cytotoxicity against LNCaP prostate cancer cells than did conjugate 1 and cytotoxicity comparable to that of conjugate 2 . These results suggest that cPIP-Chbs could be novel DNA alkylating anticancer drugs.     

Well‐Defined Polymer–Paclitaxel Prodrugs by a Grafting‐from‐Drug Approach

We report on the design of a polymeric prodrug of the anticancer agent paclitaxel (PTX) by a grafting‐from‐drug approach. A chain transfer agent for reversible addition fragmentation chain transfer (RAFT) polymerization was efficiently and regioselectively linked to the C2′ position of paclitaxel, which is crucial for its bioactivity. Subsequent RAFT polymerization of a hydrophilic monomer yielded well‐defined paclitaxel–polymer conjugates with high drug loading, water solubility, and stability. The versatility of this approach was further demonstrated by ω‐end post‐functionalization with a fluorescent tracer. In vitro experiments showed that these conjugates are readily taken up into endosomes where native PTX is efficiently cleaved off and then reaches its subcellular target. This was confirmed by the cytotoxicity profile of the conjugate, which matches those of commercial PTX formulations based on mere physical encapsulation.     

Targeting Cancer with PCPA‐Drug Conjugates: LSD1 Inhibition‐Triggered Release of 4‐Hydroxytamoxifen

Targeting cancer with small molecule prodrugs should help overcome problems associated with conventional cancer‐targeting methods. Herein, we focused on lysine‐specific demethylase 1 (LSD1) to trigger the controlled release of anticancer drugs in cancer cells, where LSD1 is highly expressed. Conjugates of the LSD1 inhibitor trans‐2‐phenylcyclopropylamine (PCPA) were used as novel prodrugs to selectively release anticancer drugs by LSD1 inhibition. As PCPA‐drug conjugate (PDC) prototypes, we designed PCPA‐tamoxifen conjugates 1 a and 1 b , which released 4‐hydroxytamoxifen in the presence of LSD1 in vitro. Furthermore, 1 a and 1 b inhibited the growth of breast cancer cells by the simultaneous inhibition of LSD1 and the estrogen receptor without exhibiting cytotoxicity toward normal cells. These results demonstrate that PDCs provide a useful prodrug method that may facilitate the selective release of drugs in cancer cells.     

A Photoactivatable Platinum(IV) Complex Targeting Genomic DNA and Histone Deacetylases

We report toxic effects of a photoactivatable platinum(IV) complex conjugated with suberoyl‐bis‐hydroxamic acid in tumor cells. The conjugate exerts, after photoactivation, two functions: activity as both a platinum(II) anticancer drug and histone deacetylase (HDAC) inhibitor in cancer cells. This approach relies on the use of a Pt^IV pro‐drug, acting by two independent mechanisms of biological action in a cooperative manner, which can be selectively photoactivated to a cytotoxic species in and around a tumor, thereby increasing selectivity towards cancer cells. These results suggest that this strategy is a valuable route to design new platinum agents with higher efficacy for photodynamic anticancer chemotherapy.     

Feasibility of polymer-drug conjugates for non-cancer applications

Polymer-drug conjugates have been intensely studied in the context of improving cancer chemotherapy and yet the only polymer-drug conjugate on the market (Movantik^®) has a different therapeutic application (relieving opioid-induced constipation). In parallel, a number of studies have recently been published proposing the use of this approach for treating diseases other than cancer. In this commentary, we analyse the many and very diverse applications that have been proposed for polymer-drug conjugates (ranging from inflammation to cardiovascular diseases) and the rationales underpinning them. We also highlight key design features to be considered when applying polymer-drug conjugates to these new therapeutic areas.     

Targeting EGFR Overexpression at the Surface of Colorectal Cancer Cells by Exploiting Amidated BODIPY-Peptide Conjugates

Three BODIPY-peptide conjugates designed to target the epidermal growth factor receptor (EGFR) at the extracellular domain were synthesized, and their specificity for binding to EGFR was investigated. Peptide sequences containing seven amino acids, GLARLLT ( 2) and KLARLLT ( 4 ), and 13 amino acids, GYHWYGYTPQNVI ( 3 ), were conjugated to carboxyl BODIPY dye ( 1 ) by amide bond formation in up to 73% yields. The BODIPY-peptide conjugates and their “parent” peptides were determined to bind to EGFR experimentally using SPR analysis and were further investigated using computational methods (AutoDock). Results of SPR, competitive binding and docking studies propose that conjugate 6 including the GYHWYGYTPQNVI sequence binds to EGFR more effectively than conjugates 5 and 7 , bearing the smaller peptide sequences. Findings in human carcinoma HEp2 cells overexpressing EGFR showed nontoxic behavior in the presence of activated light (1.5 J cm⁻²) and in the absence of light for all BODIPYs. Furthermore, conjugate 6 showed about five-fold higher accumulation within HEp2 cells compared with conjugates 5 and 7 , localizing preferentially in the cell ER and lysosomes. Our findings suggest that BODIPY-peptide conjugate 6 is a promising contrast agent for detection of colorectal cancer and potentially other EGFR-overexpressing cancers.     

Preparation and properties of insoluble forms of bacteriophage T4 lysozyme and chicken egg white lysozyme

Bacteriophage T4 lysozyme and chicken egg white lysozyme were covalently bound to cyanogen bromide activated Sepharose and to glutaraldehyde activated polyacrylhydrazido-Sepharose. The latter method seemed less favorable for T4 lysozyme, since the poly-acrylhydrazido-agarose conjugate exhibited low activity compared to the agarose conjugate. Whole bacteria (M.luteus and chloroform-treatedE. coli B cells) and the soluble uncross-linked peptidoglycan polymer fromM. luteus were used as substrates. Both types of conjugates exhibited low specific activity (lytic activity) toward insoluble substrates (cells), but surprisingly high specific activity toward the soluble substrate (hydrolytic activity). Product analysis showed that the enzyme conjugates retained their specificity of action, i.e., the same products were formed, and their rates of production were the same as those observed with the soluble (native) enzyme. The cell wall disaccharide-tetrapeptide GlcNAc-MurNAc-L-ala-D-gIu-(A₂pm-D-Ala) (C₆) inhibits the hydrolytic activity of both the native and the agarose bound T4 lysozyme. Only a slightly increased thermal stability was observed upon immobilization of T4 lysozyme, whereas the stability of the enzyme during storage and handling was greatly improved. The pH optimum of the lytic activity of Sepharose-T4 lysozyme was shifted about 1 pH unit to the alkaline side, compared to that found for the soluble enzyme, whereas no pH shift was observed for the polyacrylhydrazido-Sepharose conjugate. The optimum of the hydrolytic activity of Sepharose-T4 lysozyme was shifted to the acidic side. The pH optima of the lytic activity of the various lysozymes toward the bacterial cells were all very similar (>7), and differed greatly from the pH optima (<6) observed for their hydrolytic activities toward the negatively charged soluble peptidoglycan polymer. It is proposed that the observed differences in pH optima primarily reflect the basically different properties measured, i.e., the β(1–4) cleaving activity (hydrolytic activity), and dissolution process of the damaged cells (lytic activity).     

Reduction-sensitive liposomes from a multifunctional lipid conjugate and natural phospholipids: reduction and release kinetics and cellular uptake

The development of targeted and triggerable delivery systems is of high relevance for anticancer therapies. We report here on reduction-sensitive liposomes composed of a novel multifunctional lipidlike conjugate, containing a disulfide bond and a biotin moiety, and natural phospholipids. The incorporation of the disulfide conjugate into vesicles and the kinetics of their reduction were studied using dansyl-labeled conjugate 1 in using the dansyl fluorescence environmental sensitivity and the Fo?rster resonance energy transfer from dansyl to rhodamine-labeled phospholipids. Cleavage of the disulfide bridge (e.g., by tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), l-cysteine, or glutathione (GSH)) removed the hydrophilic headgroup of the conjugate and thus changed the membrane organization leading to the release of entrapped molecules. Upon nonspecific uptake of vesicles by macrophages, calcein release from reduction-sensitive liposomes consisting of the disulfide conjugate and phospholipids was more efficient than from reduction-insensitive liposomes composed only of phospholipids. The binding of streptavidin to the conjugates did not interfere with either the subsequent reduction of the disulfide bond of the conjugate or the release of entrapped molecules. Breast cancer cell line BT-474, overexpressing the HER2 receptor, showed a high uptake of the reduction-sensitive doxorubicin-loaded liposomes functionalized with the biotin-tagged anti-HER2 antibody. The release of the entrapped cargo inside the cells was observed, implying the potential of using our system for active targeting and delivery.     

Influence of verapamil on pharmacokinetics of pristimerin in rats

The aim of this study was to investigate the effects of verapamil on the pharmacokinetics of pristimerin in rats. A sensitive and reliable LC‐MS/MS method was developed and applied for the determination of pristimerin in rat plasma. The pharmacokinetics of orally administered pristimerin with and without verapamil pretreatment was investigated. The results indicated that, when the rats were pretreated with verapamil, the peak concentration of pristimerin increased from 189.13 to 277.53 ng/mL (46.7%), and the AUC_0–t increased by approximately 82.0%. Additionally, the effects of verapamil on the absorption and metabolic stability of pristimerin were investigated using the Caco‐2 cell transwell model and rat liver microsomes. A markedly higher transport of pristimerin across the Caco‐2 cells was observed in the basolateral‐to‐apical direction and was abrogated in the presence of the P‐gp inhibitor, verapamil. These results indicated that P‐gp might be involved in the absorption of pristimerin. Of note, the metabolic half‐life of pristimerin was prolonged by the pretreatment with verapamil. In conclusion, verapamil could affect the pharmacokinetics of pristimerin, and it might work through increasing the absorption of pristimerin by inhibiting P‐gp, or through slowing down the metabolism of pristimerin in the rat liver. Copyright © 2015 John Wiley & Sons, Ltd.     

Novel Polyhedral Silsesquioxanes [POSS(OH)32] as Anthracycline Nanocarriers—Potential Anticancer Prodrugs

Anthracyclines belong to the anticancer drugs that are widely used in chemotherapy. However, due to their systemic toxicity they also exert dangerous side effects associated mainly with cardiovascular risks. The pathway that is currently often developed is their chemical and physical modification via formation of conjugated or complexed prodrug systems with a variety of nanocarriers that can selectively release the active species in cancer cells. In this study, six new nanoconjugates were synthesized with the use of polyhedral oligosilsesquioxanes [POSS(OH)₃₂] as nanocarriers of the anticancer drugs anthracyclines—doxorubicin (DOX) and daunorubicin (DAU). These prodrug conjugates are also equipped with poly(ethylene glycol) (PEG) moieties of different structure and molecular weight. Water-soluble POSS, succinic anhydride modified (SAMDOX and SAMDAU) with carboxylic function, and PEGs (PEG1, PEG2 and PEGB3) were used for the synthesis. New nanoconjugates were formed via ester bonds and their structure was confirmed by NMR spectroscopy (¹H-NMR, ¹³C-NMR, ¹H-¹³C HSQC, DOSY and ¹H-¹H COSY), FTIR and DLS. Drug release rate was evaluated using UV-Vis spectroscopy at pH of 5.5. Release profiles of anthracyclines from conjugates 4–9 point to a range of 10 to 75% (after 42 h). Additionally, model NMR tests as well as diffusion ordered spectroscopy (DOSY) confirmed formation of the relevant prodrugs. The POSS-anthracycline conjugates exhibited prolonged active drug release time that can lead to the possibility of lowering administered doses and thus giving them high potential in chemotherapy. Drug release from conjugate 7 after 42 h was approx. 10%, 33% for conjugate 4, 47% for conjugate 5, 6, 8 and 75% for conjugate 9.     

Dual-targeting conjugates designed to improve the efficacy of radiolabeled peptides

Radiolabeled regulatory peptides are useful tools in nuclear medicine for the diagnosis (imaging) and therapy of cancer. The specificity of the peptides towards GPC receptors, which are overexpressed by cancer cells, and their favorable pharmacokinetic profile make them ideal vectors to transport conjugated radionuclides to tumors and metastases. However, after internalization of the radiopeptide into cancer cells and tumors, a rapid washout of a substantial fraction of the delivered radioactivity is often observed. This phenomenon may represent a limitation of radiopeptides for clinical applications. Here, we report the synthesis, radiolabeling, stability, and in vitro evaluation of a novel, dual-targeting peptide radioconjugate designed to enhance the cellular retention of radioactivity. The described trifunctional conjugate is comprised of a Tc-99m SPECT reporter probe, a cell membrane receptor-specific peptide, and a second targeting entity directed towards mitochondria. While the specificity of the first generation of dual-targeting conjugates towards its extracellular target was demonstrated, intracellular targeting could not be confirmed probably due to non-specific binding or hindered passage through the membrane of the organelle. The work presented describes a novel approach with potential to improve the efficacy of radiopharmaceuticals by enhancing the intracellular retention of radioactivity.     

One-pot thiol–amine bioconjugation to maleimides: simultaneous stabilisation and dual functionalisation

Maleimide chemistry is widely used in the site-selective modification of proteins. However, hydrolysis of the resultant thiosuccinimides is required to provide robust stability to the bioconjugates. Herein, we present an alternative approach that affords simultaneous stabilisation and dual functionalisation in a one pot fashion. By consecutive conjugation of a thiol and an amine to dibromomaleimides, we show that aminothiomaleimides can be generated extremely efficiently. Furthermore, the amine serves to deactivate the electrophilicity of the maleimide, precluding further reactivity and hence generating stable conjugates. We have applied this conjugation strategy to peptides and proteins to generate stabilised trifunctional conjugates. We propose that this stabilisation-dual modification strategy could have widespread use in the generation of diverse conjugates.

An alternative approach to maleimide conjugate stabilisation is presented, by the consecutive addition of a thiol and an amine to dibromomaleimides. The amine serves to simultaneously deactivate the maleimide and enable dual functionalisation.     

Cyclic peptide conjugate of curcumin and doxorubicin as an anticancer agent

The hydrophobicity of curcumin creates hurdle towards its use in the anticancer therapy. Herein, we synthesized a curcumin-doxorubicin conjugated cyclic peptide scaffold to improve the solubility of curcumin and create a conjugate containing two anticancer agents. A solid-phase Fmoc/tBu solid phase methodology was used to synthesize a cell-penetrating nuclear targeting peptide with free thiol and amine groups, which was coupled with the activated doxorubicin (Dox) and curcumin, affording Dox-peptide-curcumin conjugate (DPCC) (10). The antiproliferative activity of the conjugate was evaluated in human leukemia carcinoma cell (CCRF-CEM), human ovarian carcinoma cell (SKOV-3), and normal kidney cell line (LLCPK). Cyclic peptide-doxorubicin conjugate (7) and DPCC (10) did not inhibit the proliferation of normal kidney LLCPK cells after 72?h incubation, but were cytotoxic in CCRF-CEM (73% and 41%, respectively) and SKOV-3 (55% and 30%, respectively) cells while Dox was cytotoxic (60–79%) in all three cell lines under similar conditions, suggesting selectivity of these compounds towards cancer cells.     

Revealing the metabolic mechanism of dandelion extract against A549 cells using UPLC-QTOF MS

Dandelion extract exhibits potential anticancer activity and is expected to be a new type of natural anticancer drug. However, the effect mechanism of dandelion extract to lung cancer cells is still unclear. Here, untargeted metabolomics approach based on LC–MS was used to characterize the metabolic responses of A549 cells to dandelion extract exposure and to provide new clues for the antitumor mechanism of dandelion extract from the metabolomics perspective. A total of 16 differentially expressed and time-related metabolites were identified between dandelion extract exposure and control groups. The perturbed metabolic pathways of A549 cells after dandelion extract exposure mainly include the glycerophospholipid metabolism and purine metabolism. These results concluded that dandelion extract may exert anticancer activity by affecting malignant proliferation, disturbing the stability of cell membrane structure, reducing the adhesion of tumor cells to extracellular matrix and fibronectin, and finally inducing tumor cell death.     

促性腺激素释放激素类似物-紫杉醇靶向抗肿瘤缀合物的合成及评价

以促性腺激素释放激素类似物(GnRHa)为靶向配体, 以紫杉醇为抗癌因子, 分别以硫醚键和二硫键为连接臂, 设计合成了2个靶向抗肿瘤缀合物. 研究了缀合物的肿瘤细胞增殖抑制活性和GnRH受体结合活性, 结果表明, 2个缀合物均具有较强的抗肿瘤活性和GnRH受体亲和力; 另外, 血浆稳定性实验结果显示, 以硫醚键偶联的缀合物1在血浆中孵育24 h, 原型保留仍在50%以上, 具有较高的稳定性.     

Hyper-branched multifunctional carbon nanotubes carrier for targeted liver cancer therapy

The systemic toxicity of anticancer drugs regularly restricts the use of conventional chemotherapy to treat cancer. In this study, the limitations overcome by profitably fabricating a multifunctional nanocarrier system to carry the anticancer drug into the specific location of the cancer cells. The polyethylene glycol (PEG) was functionalized in the carboxylated multiwalled carbon nanotubes (MWCNT-COOH) through an esterification reaction (MWCNT-PEG). The targeting ligand of folic acid (FA) was covalently bonded with hyperbranched poly-L-lysine (HBPLL) using adipic acid (AA) as a cross-linking agent. Doxorubicin (DOX), an anticancer drug, was effectively loaded on MWCNT-PEG-AA-HBPLL-FA carrier loading, and in-vitro drug release was investigated by UV–Vis spectrophotometer. The chemical functionalization, morphological properties, crystalline nature, surface charge, and thermal stability of the synthesized materials were studied by FT-IR, FE-SEM, HR-TEM, DLS, and TGA techniques. In-vitro cytotoxicity and anticancer properties of DOX-loaded nanocarrier were studied in human liver cancer (HepG2) cells and human embryonic kidney (HEK293) cells. The activities of caspases (caspase ?3, ?8 & ?9) were analyzed using luminometry. The intrinsic apoptosis pathway proteins (Bcl-2 & BAX) were determined by western blot and RT-PCR analysis. The synthesized DOX-loaded nanocarriers exhibited increased cytotoxicity and apoptosis in liver HepG2 cells. The results suggest that the DOX-loaded nanocarrier possesses strong anticancer properties and could be an applicable and potential drug carrier for liver cancer chemotherapy.