The Holy Grail of Polymer Therapeutics for Cancer Therapy: An Overview on the Pharmacokinetics and Bio Distribution

In recent years, multifaceted clinical benefits of polymeric therapeutics have been reported. Over the past decades, cancer has been one of the leading causes of mortality in the world. Many clinically approved chemotherapeutics encounter potential challenges against deadly cancer. Moreover, safety and efficacy of anticancer agents have been limited by undesirable pharmacokinetics and biodistribution. To address these limitations, various polymer drug conjugates are being studied and developed to improve the antitumor efficacy. Among other therapeutics, polymer therapeutics are well established platforms that circumvent anticancer therapeutics from enzymatic metabolism via direct conjugation to therapeutic molecules. Interestingly, polymer therapeutics meets an unmet need of small molecules. Further clinical study showed that polymer-drug conjugation can achieve desired pharmacokinetics and biodistribution properties of several anticancer drugs. The present retrospective review mainly enlightens the most recent preclinical and clinical studies include safety, stability, pharmacokinetic behavior and distribution of polymer therapeutics.

Trans-dichlorooxovandium (IV) complex as a novel photoinducible DNA interstrand crosslinker for cancer therapy

Although DNA interstrand crosslinking (ICL) agents such as mitomycin C, cisplatin and psoralen serve as potent anticancer drugs, these agents are known to have dose-limiting toxic effects on normal cells. Moreover, tumor resistance to these agents has been reported. Here, we show that trans-dichlorooxovanadium (IV) complex of pyrenyl terpyridine (VDC) is a novel photoinducible DNA crosslinking agent. By a combination of in vitro and ex vivo experiments including plasmid-based assays, we find that VDC forms monoadducts on the DNA and can be activated by UV-A and visible light to generate DNA interstrand crosslinks. VDC efficiently activates Fanconi anemia (FA) pathway of DNA interstrand crosslink repair. Strikingly, photoinduction of VDC induces prolonged activation of cell cycle checkpoint and a high degree of cell death in homologous recombination (HR)/ICL repair defective cells. Moreover, VDC specifically targets cells that express pathological RAD51C mutants. These data imply that VDC can be potentially used for cancer therapy and suggest that tumors arising in patients with gene mutations in FA and HR repair pathway can be specifically targeted by a photoactivatable VDC.

Sequence specific recognition and cleavage of DNA by synthetic molecules

DNA recognition is an essential biological process responsible for the regulation of cellular functions including protein synthesis and cell division and is implicated in the mechanism of action of some anticancer drugs. Studies directed towards defining the elements responsible for sequence specific DNA recognition through the study of the interactions of synthetic organic ligands with DNA are described.

DNA recognition by poly-N-methylpyrrolecarboxamides was studied by the synthesis and characterization of a series of molecules where the number of contiguous N-methylpyrrolecarboxamide units was increased from 2 to 9. The effect of this incremental change in structure on DNA recognition has been investigated at base pair resolution using affinity cleaving and MPE•Fe(II) footprinting techniques. These studies led to a quantitative relationship between the number of amides in the molecule and the DNA binding site size. This relationship is called the n + 1 rule and it states that a poly-N methylpyrrolecarboxamide molecule with n amides will bind n + 1 base pairs of DNA. This rule is consistent with a model where the carboxamides of these compounds form three center bridging hydrogen bonds between adjacent base pairs on opposite strands of the helix. The poly-N methylpyrrolecarboxamide recognition element was found to preferentially bind poly dA•poly dT stretches; however, both binding site selection and orientation were found to be affected by flanking sequences. Cleavage of large DNA is also described.

One approach towards the design of molecules that bind large sequences of double helical DNA sequence specifically is to couple DNA binding subunits of similar or diverse base pair specificity. Bis-EDTA-distamycin-fumaramide (BEDF) is an octaamide dimer of two tri-N methylpyrrolecarboxamide subunits linked by fumaramide. DNA recognition by BEDF was compared to P7E, an octaamide molecule containing seven consecutive pyrroles. These two compounds were found to recognize the same sites on pBR322 with approximately the same affinities demonstrating that fumaramide is an effective linking element for Nmethylpyrrolecarboxamide recognition subunits. Further studies involved the synthesis and characterization of a trimer of tetra-N-methylpyrrolecarboxamide subunits linked by β-alanine ((P4)_(3)E). This trimerization produced a molecule which is capable of recognizing 16 base pairs of A•T DNA, more than a turn and a half of the DNA helix.

DNA footprinting is a powerful direct method for determining the binding sites of proteins and small molecules on heterogeneous DNA. It was found that attachment of EDTA•Fe(II) to spermine creates a molecule, SE•Fe(II), which binds and cleaves DNA sequence neutrally. This lack of specificity provides evidence that at the nucleotide level polyamines recognize heterogeneous DNA independent of sequence and allows SE•Fe(II) to be used as a footprinting reagent. SE•Fe(II) was compared with two other small molecule footprinting reagents, EDTA•Fe(II) and MPE•Fe(II).

Studies toward the total synthesis of ritterazine B : the investigation of alkynylation reactions for use in the synthesis of the western fragment

The ritterazine and cephalostatin natural products have biological activities and structures that are interesting to synthetic organic chemists. These products have been found to exhibit significant cytotoxicity against P388 murine leukemia cells, and therefore have the potential to be used as anticancer drugs. The ritterazines and cephalostatins are steroidal dimers joined by a central pyrazine ring. Given that the steroid halves are unsymmetrical and highly oxygenated, there are several challenges in synthesizing these compounds in an organic laboratory.

Ritterazine B is the most potent derivative in the ritterazine family. Its biological activity is comparable to drugs that are being used to treat cancer today. For this reason, and the fact that there are no reported syntheses of ritterazine B to date, our lab set out to synthesize this natural product.

Herein, efforts toward the synthesis of the western fragment of ritterazine B are described. Two different routes are explored to access a common intermediate. An alkyne conjugate addition reaction was initially investigated due to the success of this key reaction in the synthesis of the eastern fragment. However, it has been found that a propargylation reaction has greater reactivity and yields, and has the potential to reduce the step count of the synthesis of the western fragment of ritterazine B.

Design, synthesis, and biological activity of rhodium metalloinsertors

Deficiencies in the mismatch repair (MMR) pathway are associated with several types of cancers, as well as resistance to commonly used chemotherapeutics. Rhodium metalloinsertors have been found to bind DNA mismatches with high affinity and specificity in vitro, and also exhibit cell-selective cytotoxicity, targeting MMR-deficient cells over MMR-proficient cells.

Here we examine the biological fate of rhodium metalloinsertors bearing dipyridylamine ancillary ligands. These complexes are shown to exhibit accelerated cellular uptake which permits the observation of various cellular responses, including disruption of the cell cycle and induction of necrosis, which occur preferentially in the MMR-deficient cell line. These cellular responses provide insight into the mechanisms underlying the selective activity of this novel class of targeted anti-cancer agents.

In addition, ten distinct metalloinsertors with varying lipophilicities are synthesized and their mismatch binding affinities and biological activities studied. While they are found to have similar binding affinities, their cell-selective antiproliferative and cytotoxic activities vary significantly. Inductively coupled plasma mass spectrometry (ICP-MS) experiments show that all of these metalloinsertors localize in the nucleus at sufficient concentrations for binding to DNA mismatches. Furthermore, metalloinsertors with high rhodium localization in the mitochondria show toxicity that is not selective for MMR-deficient cells. This work supports the notion that specific targeting of the metalloinsertors to nuclear DNA gives rise to their cytotoxic and antiproliferative activities that are selective for cells deficient in MMR.

To explore further the basis of the unique selectivity of the metlloinsertors in targeting MMR-deficient cells, experiments were conducted using engineered NCI-H23 lung adenocarcinoma cells that contain a doxycycline-inducible shRNA which suppresses the expression of the MMR gene MLH1. Here we use this new cell line to further validate rhodium metalloinsertors as compounds capable of differentially inhibiting the proliferation of MMR-deficient cancer cells over isogenic MMR-proficient cells. General DNA damaging agents, such as cisplatin and etoposide, in contrast, are less effective in the induced cell line defective in MMR.

Finally, we describe a new subclass of metalloinsertors with enhanced potency and selectivity, in which the complexes show Rh-O coordination. In particular, it has been found that both Δ and Λ enantiomers of [Rh(chrysi)(phen)(DPE)]²⁺ bind to DNA with similar affinities, suggesting a possible different binding conformation than previous metalloinsertors. Remarkably, all members of this new family of compounds have significantly increased potency in a range of cellular assays; indeed, all are more potent than the FDA-approved anticancer drugs cisplatin and MNNG. Moreover, these activities are coupled with high levels of selectivity for MMR-deficient cells.

虎眼万年青化学成分及抗癌药与DNA相互作用的电喷雾质谱研究

首先，对虎眼万年青中化学成分进行了分离提取研究，采用柱层析和薄层层析等方法，并利用电喷雾质谱技术跟踪洗脱流分，首次从虎眼万年青氯仿提取物中分离得到两个新化合物。其一为单菇内酷loliolide，通过FT－ICRMS高分辨质谱、IR和NMR等手段对其结构进行了确定，并通过IHNMR和旋光法确定了其绝对构型。此外，还利用电喷雾多级串联质谱（ESI一MSn）技术对其质谱裂解规律进行了系统研究，其分析结果与NMR解析结果完全一致，建立了该类化合物结构解析的简便、快速的质谱新方法；另一化合物为生物碱类化合物，采用电喷雾多级串联质谱技术对其质谱裂解规律进行了详细研究，并对其结构进行了初步推断，进一步的NMR结构确定正在进行中。其次，对虎眼万年青多糖成分进行了分离提取研究，并结合体外抑瘤活性评价实验，确定了两种具有抑瘤生物活性的多糖成分OC一2一1一c和OC一2一卜d。并通过SDS凝胶电泳和糖基组成分析，初步证明了虎眼万年青中的活性多糖成分为阿拉伯半乳糖蛋白（AGPs），为进一步研究虎眼万年青抗肿瘤活性提供了基础数据和理论指导。此外，还对虎眼万年青中的微量元素及其溶出率进行了初步的研究，研究结果表明，其水煎液中Cu/Zn比值明显低于癌症患者血清中的Cu/Zn比值，因此，虎眼万年青可能有助于调节癌症患者体内的Cu、Zn平衡，从而达到抗癌目的；而虎眼万年青中Se、Ge含量很低，不能作为两种微量元素的药用来源。为了进一步研究抗癌药物的作用机理，我们采用电喷雾质谱技术，对三种临床疗效较好的抗癌药物与DNA相互作用进行了系统的研究，确定了复合物的化学计量比及其结合的特异性，并利用竞争实验方法，研究了抗癌药物的相对结合强度。质谱实验结果与液相行为完全一致，其中抗癌药物米托葱醒为首次利用质谱进行研究。最后，采用电喷雾质谱技术，对硝酸盐离子簇合物进行了详细研究，发现了双电荷簇合物离子，并且在离子簇合物中还发现了魔数规律的存在。利用电喷雾多级串联质谱技术，研究了双电荷簇合物离子的质谱裂解规律，并对其稳定性进行了分析。此外，还讨论了溶液浓度和毛细管温度对离子簇合物形成的影响规律。

Interactions of Rare Earth-Amino Acid Complexes with Nucleic Acids

A number of metal-based anticancer drugs are designed to target nucleic acids. Therefore, the elucidation of their interactions with nucleic acids is important for rational design of new anticancer agents with high selectivity and low toxicity, which has been received much attention in this field. Lanthanide complexes have the potential to be therapeutic agents due to their unique magnetic, optical, electronic, and coordinate characteristics. However, lanthanide ions are easy to hydrolysis under physiological pH, which makes it difficult to study rare earth complexes nucleic acids selectivity. Recent studies have shown that natural amino acids can form stable complexes with rare earth ions under near physiological condition and the complexes have high solubility. This review summarizes the current progress in rare earth-amino acid complexes binding to nuclelic acids and their selectivity.

Biodegradable amphiphilic polymer-drug conjugate micelles

The coupling of drugs to macromolecular carriers received an important impetus from Ringsdorf's notion of polymer-drug conjugates. Several water-soluble polymers, poly(ethylene glycol), poly[N-(2-hydroxypropyl) methacrylamidel, poly(L-glutamic acid) and dextran, are studied intensively and have been utilized successfully in clinical research. The promising results arising from clinical trials with polymer-drug conjugates (e.g., paclitaxel, doxorubicin, camptothecins) have provided a firm foundation for other synthetic polymers, especially biodegradable polymers, used as drug delivery vehicles. This review discusses biodegradable polymeric micelles as an alternative drug-conjugate system. Particular focus is on A-B or B-A-B type biodegradable amphiphilic block copolymer such as polylactide, morpholine-2,5-dione derivatives and cyclic carbonates, which can form a core-shell micellar structure, with the hydrophobic drug-binding segment forming the hydrophobic core and the hydrophilic segment as a hydrated outer shell. Polymeric micelles can be designed to avoid uptake by cells of reticuloendothelial system and thus enhance their blood lifetime via the enhanced permeability and retention effect.

Interaction of echinomycin with guanine: electrochemistry and spectroscopy studies

The interaction of antitumor antibiotic, echinomycin (Echi) with guanine (Gua) was thoroughly investigated by adsorptive transfer stripping cyclic voltammetry, ultraviolet and visible adsorption spectra (UV/Vis) and Fourier-transform infrared spectroscopy (FTIR). Electrochemistry provided a simple tool for verifying the occurrence of interaction between Echi and Gua. Echi could be accumulated from the solution and give well-defined electrochemical signals in 0.1 M phosphate buffer solution (pH 7.0) only when Gua was present on the surface of the electrochemically pretreated glass carbon electrode (GCE), suggesting a strong binding of Echi to Gua. All the acquired spectral data showed that a new adduct between Echi and Gua was formed, and two pairs of adjacent intermolecular hydrogen bonds between the Ala backbone atoms in Echi and Gua (Ala-NH to Gua-N3 and Gua-NH2 to Ala-CO) played a dominating role in the interaction. Electrochemistry coupled with spectroscopy techniques could provide a relatively easy way to obtain useful insights into the molecular mechanism of drug-DNA interactions, which should be important in the development of new anticancer drugs with specific base recognition.

A novel anti-tumor protein extracted from Meretrix meretrix Linnaeus induces cell death by increasing cell permeability and inhibiting tubulin polymerization

Discovery and development of new pharmaceuticals from marine organisms are attracting increasing interest. Several agents derived from marine organisms are under preclinical and clinical evaluation as potential anticancer drugs. We extracted and purified a novel anti-tumor protein from the coelomic fluid of Meretrix meretrix Linnaeus by ammonium sulphate fractionation, ion exchange and hydrophobic interaction chromatography. The molecular weight of the highly purified protein, designated MML, was 40 kDa as determined by SDS-PAGE analysis. MML exhibited significant cytotoxicity to several cancer cell types, including human hepatoma BEL-7402, human breast cancer MCF-7 and human colon cancer HCT116 cells. However, no inhibitory effect was found when treating murine normal fibroblasts NIH3T3 and benign human breast MCF-10A cells with MML. The cell death induced by MML was characterized by cell morphological changes. The induction of apoptosis of BEL-7402 cells by MML was weak by DNA ladder assay. The possible mechanisms of its anti-tumor effect might be the changes in cell membrane permeability and inhibition of tubulin polymerization. MML may be developed as a novel, highly selective and effective anti-cancer drug.

Sequence analysis and functional study of thymidylate synthase from zebrafish, Danio rerio

The thymidylate synthase (TS), an important target for many anticancer drugs, has been cloned from different species. But the cDNA property and function of TS in zebrafish are not well documented. In order to use zebrafish as an animal model for screening novel anticancer agents, we isolated TS cDNA from zebrafish and compared its sequence with those from other species. The open reading frame (ORF) of zebrafish TS cDNA sequence was 954 nucleotides, encoding a 318-amino acid protein with a calculated molecular mass of 36.15 kDa. The deduced amino acid sequence of zebrafish TS was similar to those from other organisms, including rat, mouse and humans. The zebrafish TS protein was expressed in Escherichia coli and purified to homogeneity. The purified zebrafish TS showed maximal activity at 28 degrees C with similar K-m value to human TS. Western immunoblot assay confirmed that TS was expressed in all the developmental stages of zebrafish with a high level of expression at the 1-4 cell stages. To study the function of TS in zebrafish embryo development, a short hairpin RNA (shRNA) expression vector, pSilencer 4.1-CMV/TS, was constructed which targeted the protein-coding region of zebrafish TS mRNA. Significant change in the development of tail and epiboly was found in zebrafish embryos microinjected pSilencer4.1-CMV/TS siRNA expression vector.

Synteza oraz właściwości fizykochemiczne bis-interkalatorów naftalimidowych

Wydział Chemii

Badania spektroskopowe, strukturalna i aktywność przeciwdrobnoustrojowa nowych soli kwasu lasalowego z aminami

Wydział Chemii

Chemotherapy-Induced Activation of ADAM-17: A Novel Mechanism of Drug Resistance in Colorectal Cancer

Purpose: We have shown previously that exposure to anticancer drugs can trigger the activation of human epidermal receptor survival pathways in colorectal cancer (CRC). In this study, we examined the role of ADAMs (a disintegrin and metalloproteinases) and soluble growth factors in this acute drug resistance mechanism.

Experimental Design: In vitro and in vivo models of CRC were assessed. ADAM-17 activity was measured using a fluorometric assay. Ligand shedding was assessed by ELISA or Western blotting. Apoptosis was assessed by flow cytometry and Western blotting.

Results: Chemotherapy (5-fluorouracil) treatment resulted in acute increases in transforming growth factor-a, amphiregulin, and heregulin ligand shedding in vitro and in vivo that correlated with significantly increased ADAM-17 activity. Small interfering RNA–mediated silencing and pharmacologic inhibition confirmed that ADAM-17 was the principal ADAM involved in this prosurvival response. Furthermore, overexpression of ADAM-17 significantly decreased the effect of chemotherapy on tumor growth and apoptosis. Mechanistically, we found that ADAM-17 not only regulated phosphorylation of human epidermal receptors but also increased the activity of a number of other growth factor receptors, such as insulin-like growth factor-I receptor and vascular endothelial growth factor receptor.

Conclusions: Chemotherapy acutely activates ADAM-17, which results in growth factor shedding, growth factor receptor activation, and drug resistance in CRC tumors. Thus, pharmacologic inhibition of ADAM-17 in conjunction with chemotherapy may have therapeutic potential for the treatment of CRC.