Targeting histone deacetylases for the treatment of immune, endocrine and metabolic disorders

The 'histone code' is a well-established hypothesis describing the idea that specific patterns of post-translational modifications to histones act like a molecular "code" recognised and used by non-histone proteins to regulate specific chromatin functions. One modification which has received significant attention is that of histone acetylation. The enzymes which regulate this modification are described as histone acetyltransferases or HATs, and histone deacetylases or HDACs. Due to their conserved catalytic domain HDACs have been actively targeted as a therapeutic target. The proinflammatory environment is increasingly being recognised as a critical element for both degenerative diseases and cancer. The present review will discuss the current knowledge surrounding the clinical potential & current development of histone deacetylases for the treatment of diseases for which a proinflammatory environment plays important roles, and the molecular mechanisms by which such inhibitors may play important functions in modulating the proinflammatory environment. © 2009 Bentham Science Publishers Ltd.

Gene expression analysis of diagnostic biopsies predicts pathological response to neoadjuvant chemoradiotherapy of esophageal cancer

OBJECTIVE: This study explored gene expression differences in predicting response to chemoradiotherapy in esophageal cancer. PURPOSE:: A major pathological response to neoadjuvant chemoradiation is observed in about 40% of esophageal cancer patients and is associated with favorable outcomes. However, patients with tumors of similar histology, differentiation, and stage can have vastly different responses to the same neoadjuvant therapy. This dichotomy may be due to differences in the molecular genetic environment of the tumor cells. BACKGROUND DATA: Diagnostic biopsies were obtained from a training cohort of esophageal cancer patients (13), and extracted RNA was hybridized to genome expression microarrays. The resulting gene expression data was verified by qRT-PCR. In a larger, independent validation cohort (27), we examined differential gene expression by qRT-PCR. The ability of differentially-regulated genes to predict response to therapy was assessed in a multivariate leave-one-out cross-validation model. RESULTS: Although 411 genes were differentially expressed between normal and tumor tissue, only 103 genes were altered between responder and non-responder tumor; and 67 genes differentially expressed >2-fold. These included genes previously reported in esophageal cancer and a number of novel genes. In the validation cohort, 8 of 12 selected genes were significantly different between the response groups. In the predictive model, 5 of 8 genes could predict response to therapy with 95% accuracy in a subset (74%) of patients. CONCLUSIONS: This study has identified a gene microarray pattern and a set of genes associated with response to neoadjuvant chemoradiation in esophageal cancer. The potential of these genes as biomarkers of response to treatment warrants further investigation. Copyright © 2009 by Lippincott Williams & Wilkins.

The role of chemotherapy in the treatment of adult soft tissue sarcomas

Adult soft tissue sarcomas are relatively rare tumours which are curable with radical surgery. Approximately 50% of patients will develop inoperable disease or metastases for which chemotherapy may be inappropriate. Only two cytotoxic agents - doxorubicin and ifosfamide - have activity in > 20% of patients. For both these agents there is evidence of a dose-response relationship. There is currently no good evidence that combination chemotherapy confers a clinical benefit compared with single agents. Outside a clinical trial, standard first-line therapy should be with single agent doxorubicin at a dose intensity ≥ 70 mg2 every 3 weeks. Approximately 25% of patients may be expected to respond to this regimen. There is the suggestion that responses may occur to ifosfamide in patients who progress on doxorubicin. The role of chemotherapy in the adjuvant setting remains uncertain. Several trials have suggested a modest relapse-free and overall survival benefit for the use of post-operative chemotherapy and a recent overview of 14 randomised trials confirms a small though significant benefit. These benefits have to be weighed against the toxicity of chemotherapy. The importance of treating all patients with soft tissue sarcomas in clinical trials is stressed. There is an urgent need to define new active agents to treat this disease.

Long-term outcomes following neoadjuvant chemoradiotherapy for esophageal cancer

OBJECTIVE: We present and analyze long-term outcomes following multimodal therapy for esophageal cancer, in particular the relative impact of histomorphologic tumor regression and nodal status. PATIENTS AND METHODS: A total of 243 patients [(adenocarcinoma (n = 170) and squamous cell carcinoma (n = 73)] treated with neoadjuvant chemoradiotherapy in the period 1990 to 2004 were followed prospectively with a median follow-up of 60 months. Pathologic stage and tumor regression grade (TRG) were documented, the site of first failure was recorded, and Kaplan-Meier survival curves were plotted. RESULTS: Thirty patients (12%) did not undergo surgery due to disease progression or deteriorated performance status. Forty-one patients (19%) had a complete pathologic response (pCR), and there were 31(15%) stage I, 69 (32%) stage II, and 72 (34%) stage III cases. The overall median survival was 18 months, and the 5-year survival was 27%. The 5-year survival of patients achieving a pCR was 50% compared with 37% in non-pCR patients who were node-negative (P = 0.86). Histomorphologic tumor regression was not associated with pre-CRT cTN stage but was significantly (P < 0.05) associated with ypN stage. By multivariate analysis, ypN status (P = 0.002) was more predictive of overall survival than TRG (P = 0.06) or ypT stage (P = 0.39). CONCLUSION: Achieving a node-negative status is the major determinant of outcome following neoadjuvant chemoradiotherapy. Histomorphologic tumor regression is less predictive of outcome than pathologic nodal status (ypN), and the need to include a primary site regression score in a new staging classification is unclear. © 2007 Lippincott Williams & Wilkins, Inc.

Bioavailability study of oral and intravenous OGT 719, a novel nucleoside analogue with preferential activity in the liver

Purpose: Although oral fluoropyrimidine pro-drugs are increasingly being administered in preference to intravenous nucleoside analogues in cancer chemotherapy, their activation in malignant liver tissue may be insufficient. OGT 719 (1-galactopyranosyl-5-fluorouracil) is a novel nucleoside analogue, preferentially localized in hepatocytes and hepatoma cells via the asialoglycoprotein receptor. The aim of this study was to assess the systemic bioavailability of this rationally designed drug in 16 patients with advanced solid cancers. Method: Crossover pharmacokinetic study of oral (400 or 800 mg) and intravenous (250 mg/m 2) OGT 719. Results: Linear pharmacokinetics and oral bioavailability of approximately 25% were observed at the dose levels used in this study. Like other 5-FU prodrugs, considerable interpatient variability was observed in bioavailability following oral dosing. The mean half-life for oral doses was 4 h. OGT 719 was well tolerated. No objective tumour responses were demonstrated. Conclusion: The systemic bioavailability and half-life of oral OGT 719 are sufficient to merit dose escalation studies with frequent daily dosing. Subsequent efficacy studies should be performed in patients with primary and secondary liver malignancies.

Bioavailability study of oral and intravenous OGT 719, a novel nucleoside analogue with preferential activity in the liver

Purpose: Although oral fluoropyrimidine pro-drugs are increasingly being administered in preference to intravenous nucleoside analogues in cancer chemotherapy, their activation in malignant liver tissue may be insufficient. OGT 719 (1-galactopyranosyl-5-fluorouracil) is a novel nucleoside analogue, preferentially localized in hepatocytes and hepatoma cells via the asialoglycoprotein receptor. The aim of this study was to assess the systemic bioavailability of this rationally designed drug in 16 patients with advanced solid cancers. Method: Crossover pharmacokinetic study of oral (400 or 800 mg) and intravenous (250 mg/m 2) OGT 719. Results: Linear pharmacokinetics and oral bioavailability of approximately 25% were observed at the dose levels used in this study. Like other 5-FU prodrugs, considerable interpatient variability was observed in bioavailability following oral dosing. The mean half-life for oral doses was 4 h. OGT 719 was well tolerated. No objective tumour responses were demonstrated. Conclusion: The systemic bioavailability and half-life of oral OGT 719 are sufficient to merit dose escalation studies with frequent daily dosing. Subsequent efficacy studies should be performed in patients with primary and secondary liver malignancies.

Management of endocrine resistant breast cancer

Metastatic breast cancer (MBC) may present de novo but more commonly develops in women initially presenting with early breast cancer despite the widespread use of adjuvant hormonal and cytotoxic chemotherapy. MBC is incurable. Hormone sensitive MBC eventually becomes resistant to endocrine therapy in most women. Anthracyclines are the agents of choice in the treatment of endocrine resistant MBC. With the widespread use of anthracyclines in the adjuvant setting, taxanes have become the agents of choice for many patients. Recently capecitabine has become established as a standard of care for patients pretreated with anthracyclines and taxanes. However, a range of agents have activity as third line treatment. These include gemcitabine, vinorelbine and platinum analogues. The sequential use of non-cross resistant single agents rather than combination therapy is preferable in most women with MBC. Even though combination therapy can improve response rates and increase progression free interval, there is no robust evidence to indicate an advantage in terms of overall survival. Moreover, combination therapy is associated with a higher toxicity rate and poor quality of life. There is no role for dose-intense therapy, high dose therapy or maintenance chemotherapy outside the context of a clinical trial. The introduction of trastuzumab, monoclonal antibody targeting growth factor receptors, has improved the therapeutic options for women with tumours overexpressing HER2/neu. DNA micro-array profiles of tumours can potentially help to individualise therapy in future. Molecular targeted therapy has the potential to revolutionise the management of MBC.

Chemotherapy for upper gastrointestinal tumours

The aim of this review is to identify current chemotherapy treatment for tumours of the oesophagus, stomach, pancreas, and liver. The role of both neoadjuvant, adjuvant, and palliative chemotherapy regimens will be discussed. This review will be of interest to oncologists in clarifying current issues regarding chemotherapy, and to physicians in other medical specialties, to increase their general understanding of benefits and drawbacks of chemotherapy in this patient group.

Outcomes in gastric and junctional cancer using neoadjuvant and adjuvant chemotherapy (epirubicin, oxaliplatin, and capecitabine) and radical surgery

Background The MAGIC/UK Medical Research Council (MRC) trial set the standard of care for treatment of resectable gastric and junctional adenocarcinoma, demonstrating that perioperative chemotherapy with epirubicin, cisplatin and 5-fluorouracil (ECF) confers a survival benefit over surgery alone. The randomized ECF for advanced and locally advanced esophagogastric cancer (REAL-2) trial showed that, in the metastatic setting, the EOX regimen (epirubicin, oxaliplatin and capecitabine) is as effective as ECF, with a favourable toxicity profile. Methods Consecutive patients with resectable gastric or junctional adenocarcinoma treated with perioperative EOX, between 2007 and 2012, were retrospectively analysed. Results Fifty-nine patients (12 female, 47 male), commenced EOX therapy; 47 underwent surgery. A good pathological response was seen in 34 %, (16/47). Disease recurrence occurred in 19 patients (19/47, 40 %). Median overall survival was 22 months, with 4-year survival of 47 %. Chemotoxicities were consistent with those previously reported for this regimen. Conclusion This study in a high-volume centre demonstrates that EOX in resectable gastric and junctional adenocarcinoma is associated with a reasonable safety profile, and efficacy consistent with that reported for ECF.

A germline polymorphism of thymine DNA glycosylase induces genomic instability and cellular transformation

Thymine DNA glycosylase (TDG) functions in base excision repair, a DNA repair pathway that acts in a lesion-specific manner to correct individual damaged or altered bases. TDG preferentially catalyzes the removal of thymine and uracil paired with guanine, and is also active on 5-fluorouracil (5-FU) paired with adenine or guanine. The rs4135113 single nucleotide polymorphism (SNP) of TDG is found in 10% of the global population. This coding SNP results in the alteration of Gly199 to Ser. Gly199 is part of a loop responsible for stabilizing the flipped abasic nucleotide in the active site pocket. Biochemical analyses indicate that G199S exhibits tighter binding to both its substrate and abasic product. The persistent accumulation of abasic sites in cells expressing G199S leads to the induction of double-strand breaks (DSBs). Cells expressing the G199S variant also activate a DNA damage response. When expressed in cells, G199S induces genomic instability and cellular transformation. Together, these results suggest that individuals harboring the G199S variant may have increased risk for developing cancer.

Adenoviral Gene Therapy for Advanced Head and Neck Cancer

Advanced stage head and neck cancers (HNC) with distant metastasis, as well as prostate cancers (PC), are devastating diseases currently lacking efficient treatment options. One promising developmental approach in cancer treatment is the use of oncolytic adenoviruses, especially in combination therapy with conventional cancer therapies. The safety of the approach has been tested in many clinical trials. However, antitumor efficacy needs to be improved in order to establish oncolytic viruses as a viable treatment alternative. To be able to test in vivo the effects on anti-tumor efficiency of a multimodal combination therapy of oncolytic adenoviruses with the standard therapeutic combination of radiotherapy, chemotherapy and Cetuximab monoclonal antibody (mAb), a xenograft HNC tumor model was developed. This model mimics the typical clinical situation as it is initially sensitive to cetuximab, but resistance develops eventually. Surprisingly, but in agreement with recent findings for chemotherapy and radiotherapy, a higher proportion of cells positive for HNC cancer stem cell markers were found in the tumors refractory to cetuximab. In vitro as well as in vivo results found in this study support the multimodal combination therapy of oncolytic adenoviruses with chemotherapy, radiotherapy and monoclonal antibody therapy to achieve increased anti-tumor efficiency and even complete tumor eradication with lower treatment doses required. In this study, it was found that capsid modified oncolytic viruses have increased gene transfer to cancer cells as well as an increased antitumor effect. In order to elucidate the mechanism of how oncolytic viruses promote radiosensitization of tumor cells in vivo, replicative deficient viruses expressing several promising radiosensitizing viral proteins were tested. The results of this study indicated that oncolytic adenoviruses promote radiosensitization by delaying the repair of DNA double strand breaks in tumor cells. Based on the promising data of the first study, two tumor double-targeted oncolytic adenoviruses armed with the fusion suicide gene FCU1 or with a fully human mAb specific for human Cytotoxic T Lymphocyte-Associated Antigen 4 (CTLA-4) were produced. FCU1 encodes a bifunctional fusion protein that efficiently catalyzes the direct conversion of 5-FC, a relatively nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil and 5-fluorouridine monophosphate, bypassing the natural resistance of certain human tumor cells to 5-fluorouracil. Anti-CTLA4 mAb promotes direct killing of tumor cells via apoptosis and most importantly immune system activation against the tumors. These armed oncolytic viruses present increased anti-tumor efficacy both in vitro and in vivo. Furthermore, by taking advantage of the unique tumor targeted gene transfer of oncolytic adenoviruses, functional high tumor titers but low systemic concentrations of the armed proteins were generated. In addition, supernatants of tumor cells infected with Ad5/3-24aCTLA4, which contain anti-CTLA4 mAb, were able to effectively immunomodulate peripheral blood mononuclear cells (PBMC) of cancer patients with advanced tumors. -- In conclusion, the results presented in this thesis suggest that genetically engineered oncolytic adenoviruses have great potential in the treatment of advanced and metastatic HNC and PC.

Thymidine inhibits the incorporation of 5-fluoro-2'-deoxyuridine to DNA of mouse mammary tumour

5-Fluoro-2'-deoxyuricine is incorporated into DNA of mouse breast tumour Image . The incorporation is inhibited by thymidine. Part of the fluorodeoxyuridine is cleaved to fluorouracil and is incorporated into RNA. This incorporation is enhanced by thymidine. The result suggests that the major mechanism of action of the fluorouracil is due to its incorporation into RNA. FUra, 5-Fluorouracil; FdUR, 5-Fluoro-2'-deoxyuridine; FdUMP, 5-Fluoro-2'-deoxyuridine-5'-monophosphate.

Investigation of biodegradable and biocompatible castor oil poly(mannitol-citric-sebacate) polyester as a drug carrier

Castor oil-based poly(mannitol-citric sebacate) was synthesized by simple, catalyst-free melt condensation process using monomers having potential to be metabolized in vivo. The polymer was characterized using various techniques and the tensile and hydration properties of the polymers were also determined. The biocompatibility of the polymer was tested using human foreskin fibroblasts cells. The in vitro degradation studies show that the time for complete degradation of the polymer was more than 21 days. The usage of castor oil polyester as a drug carrier was analysed by doping the polymer with 5-fluorouracil model drug and the release rate was studied by varying the percentage loading of drugs and the pH of the PBS solution medium. The cumulative drug-release profiles exhibited a biphasic release with an initial burst release and cumulative 100% release within 42 h. To understand the role of the polymer as a drug carrier in the release behaviour, drug-release studies were conducted with another drug, isoniazid. The release behaviour of isoniazid drug from the same polymer matrix followed an nth order kinetic model and 100% cumulative release was achieved after 12 days. The variation in the release behaviour for two model drugs from the same polymer matrix suggests a strong interaction between the polymer and the drug molecule.

Dual Drug Conjugate Loaded Nanoparticles for the Treatment of Cancer

Two antineoplastic agents, Imatinib (IM) and 5-Fluorouracil (FU) were conjugated by hydrolysable linkers through an amide bond and entrapped in polymeric Human Serum Albumin (HSA) nanoparticles. The presence of dual drugs in a common carrier has the advantage of reaching the site of action simultaneously and acting at different phases of the cell cycle to arrest the growth of cancer cells before they develop chemoresistance. The study has demonstrated an enhanced anticancer activity of the conjugate, and conjugate loaded stealth HSA nanoparticles (NPs) in comparison to the free drug in A-549 human lung carcinoma cell line and Zebra fish embryos (Danio rerio). Hydrolysability of the conjugate has also been demonstrated with complete hydrolysis being observed after 12 h. In vivo pharmacodynamics study in terms of tumor volume and pharmacokinetics in mice for conjugate (IM-SC-FU) and conjugate loaded nanoparticles showed significant anti-cancer activity. The other parameters evaluated were particle size (86nm), Poly Dispersive Index (PDI) (0.209), zeta potential (-49mV), drug entrapment efficiency (96.73%) and drug loading efficiency (89%). Being in stealth mode gives the potential for the NPs to evade Reticulo-Endothelial system (RES), achieve passive targeting by Enhanced Permeation Retention (EPR) effect with controlled release of the therapeutic agent. As the conjugate cleaves into individual drugs in the tumor environment, this promises better suppression of cancer chemoresistance by delivering dual drugs with different modes of action at the same site, thereby synergistically inhibiting the growth of cancerous tissue.

5-氟尿嘧啶-β-环糊精结肠靶向前体药物的研究

5-氟尿嘧啶(5-Fluorouracil， 5-FU)是一种抗代谢药物，广泛用于临床治疗结直肠癌、胃癌、乳腺癌等多种癌症，但其首过代谢显著、亲脂性较低，选择性差、毒副作用大。为克服这些缺点人们对5-FU进行了大量的修饰工作，包括小分子修饰以及与各种载体形成微球、微囊、纳米粒、共价前药等。 环糊精(Cyclodextrin，简称CD)，可被结肠中的糖苷酶特异性地降解成小分子糖，而胃和小肠中由于缺乏相应的酶而使环糊精不被降解，这一特性在结肠药物的靶向输送及释放中有重要应用价值。环糊精中含有丰富的羟基，易进行化学修饰，将药物与环糊精通过共价键结合制成前药，使其在胃和小肠中不降解，而在盲结肠中被特异性的酶降解释出药物，达到结肠靶向释药的目的。研究表明，环糊精作为一种前药载体为结肠靶向释药和缓释、控释系统提供了一种有效的手段。 本工作选择5-氟尿嘧啶为模型药物、β-环糊精作为载体，通过中间体5-FU羧酸衍生物的制备及其与β-环糊精的偶联，合成了系列5-FU-β-CD前体药物，并利用紫外、红外、质谱、核磁、元素分析、热分析等手段对其进行结构表征。同时，还研究了前体药物的体外释药性质。具体内容包括： 1. 含有羧基的5-FU衍生物中间体的合成：(5-氟尿嘧啶-1-基)-乙酸(FUAC)、3-(5-氟尿嘧啶-1-基)-丙酸(FUPC)、5-(5-氟尿嘧啶-1-基)-戊酸(FUVC)的合成。 2. 中间体5-FU的羧酸衍生物与β-CD的偶联：分别通过以6-OTs-β-CD为中间体的取代法和活化酯法，合成了第一面取代和第二面取代的5-FU-β-CD大分子前体药物。在二面取代的前体药物制备中，通过改变原料的比例，合成了系列不同取代度(DS)的2-[(5-氟尿嘧啶-1-基)-乙酰基] -β-环糊精结合物。 3. 对上述前体药物进行体外释放研究：分别考察了前体药物在不同pH缓冲溶液中的水解行为及其在小鼠胃肠道人工体液中的酶解行为，并通过UV-Vis及HPLC对前体药物释放情况进行检测分析。 5-Fluorouracil(5-Fu), commonly known as a broad-spectrum antineoplastic drug, has been widely used in the treatment of various kinds of cancer including colon cancer for 40 years. However, this antitumor agent exhibits serious adverse effects, such as their marrow toxicity, gastrointestinal reaction and low selectivity in their clinical use. In order to improve its antitumor activity and reduce its toxicity, the compound was modified in various ways, including the formation of conjugated prodrugs with kinds of carrier, microsphere and nanoparticles etc. Cyclodextrins(CDs) are known to be barely capable of being hydrolyzed and only slightly absorbed in passing through the stomach and small intestine; however they are fermented into small saccharides by colonic microflora and thus absorbed as small saccharides in the large intestine. This biodegradation property of CDs may be useful as a colon-targeting carrier, and thus CD prodrugs may serve as a source of site-specific delivery of drugs to colon. It was demonstrated that prodrugs of CDs can provide a versatile means for construction of not only colon targeted delivery systems, but also delayed release systems. 5-Fluorouracil was taken as a model drug and β-CD as the carrier in this study. Series prodrugs of 5-FU was prepared through the preparation of reactive 5-FU derivatives containing carboxyl group and coupling to hydroxyl groups of CD. The structures of the conjugates were charactered by using IR, UV–vis, ESI-MS, 1H, 13C-NMR spectra, elemental analyses, and thermal analysis. In vitro hydrolysis behavior in aqueous solution and in rat gastrointestinal tract contents of the conjugates were also investigated. The main content of this dissertation includes following aspects: 1. The preparation of 5-FU derivatives containing carboxyl group: 5-Fluorouracil- acetic acid(FUAC)、3-(5-FU-1)-propionic acid (FUPC)、and 5-(5-FU-1)-valeric acid(FUVC). 2. The coupling of 5-FU derivatives to β-CD: 5-FU was selectively conjugated onto the primary or secondary hydroxyl groups of β-CD through an ester linkage, by the substitution of 6-OTs-β-CD and the activated ester method respectively. For the secondary face conjugation, the degree of substitution(DS) can be controlled by changing the mole ratio of the starting materials(FUAC and β-CD). 3. In vitro release behavior of the conjugates in aqueous solution and in rat gastro- intestinal tract contents of the conjugates were investigated, and the reaction was monitored and analyzed by using UV-Vis and HPLC methods.