Mutual Prodrugs - Codrugs.

This review encapsulates an extensive variety of substances identified as mutual prodrugs or codrugs, wherein two, or sometimes three, biologically active moieties are linked using an assortment of metabolically unstable bridging entities. Following the administration of the mutual prodrugs, these undergo a bridge cleavage releasing the active molecules, which then elicit their respective biological effects. In some cases, the released drugs act synergistically, other times the biological activity of only one of the drugs is elicited, and in such cases, the accompanying drug serves only as a carrier, which may have an affinity to the desired receptor. The most promising results are commonly observed when the two released drugs are efficacious at similar concentrations and particularly when the two drugs are effective against similar diseases. For instance, the best results are observed, when two analgesics, two anticancer agents, two drugs for the treatment of cardiac conditions, etc., are the substances comprising the codrug. Mutual prodrugs/ codrugs described herein have been reported, primarily since the year 2000, as potential drugs for use against a plethora of diseases including pain, inflammation, cancer, bacterial infections, sickle cell anemia, Alzheimer's disease, and others.

An evaluation of the interaction of pixantrone with formaldehyde-releasing drugs in cancer cells.

PURPOSE: Pixantrone is a synthetic aza-anthracenedione currently used in the treatment of non-Hodgkin's lymphoma. The drug is firmly established as a poison of the nuclear enzyme topoisomerase II, however, pixantrone can also generate covalent drug-DNA adducts following activation by formaldehyde. While pixantrone-DNA adducts form proficiently in vitro, little evidence is presently at hand to indicate their existence within cells. The molecular nature of these lesions within cancer cells exposed to pixantrone and formaldehyde-releasing prodrugs was characterized along with the cellular responses to their formation. METHODS: In vitro crosslinking assays, [14C] scintillation counting analyses and alkaline comet assays were applied to characterize pixantrone-DNA adducts. Flow cytometry, cell growth inhibition and clonogenic assays were used to measure cancer cell kill and survival. RESULTS: Pixantrone-DNA adducts were not detectable in MCF-7 breast cancer cells exposed to [14C] pixantrone (10-40 µM) alone, however the addition of the formaldehyde-releasing prodrug AN9 yielded readily measurable levels of the lesion at ~ 1 adduct per 10 kb of genomic DNA. Co-administration with AN9 completely reversed topoisomerase II-associated DNA damage induction by pixantrone yet potentiated cell kill by the drug, suggesting that pixantrone-DNA adducts may promote a topoisomerase II-independent mechanism of cell death. Pixantrone-DNA adduct-forming treatments generally conferred mild synergism in multiple cell lines in various cell death and clonogenic assays, while pixantrone analogues either incapable or relatively defective in forming DNA adducts demonstrated antagonism when combined with AN9. CONCLUSIONS: The features unique to pixantrone-DNA adducts may be leveraged to enhance cancer cell kill and may be used to guide the design of pixantrone analogues that generate adducts with more favorable anticancer properties.

Dimeric Drugs.

This review intends to summarize the structures of an extensive number of symmetrical-dimeric drugs, having two monomers, linked via a bridging entity emphasizing the versatility of biologically active substances reported to possess dimeric structures. The major number of these compounds consists of anticancer agents, antibiotics/ antimicrobials, and anti-AIDS drugs. Other symmetrical-dimeric drugs include antidiabetics, antidepressants, analgesics, anti-inflammatories, drugs for the treatment of Alzheimer's disease, anticholesterolemics, estrogenics, antioxidants, enzyme inhibitors, anti- Parkinsonians, laxatives, antiallergy compounds, cannabinoids, etc. Most of the articles reviewed do not compare the activity/potency of the dimers to that of their corresponding monomers. Only in limited cases, various suggestions have been made to justify the unexpectedly higher activity of the dimers vs. that of the corresponding monomers. These suggestions include statistical effects, the presence of dimeric receptors, binding of a dimer to two receptors simultaneously, and others. It is virtually impossible to predict which dimers will be preferable to their respective monomers, or which linking bridges will lead to the most active compounds. It is expected that the extensive variety of substances mentioned, and the assortment of their biological activities should be of interest to academic and industrial medicinal chemists.

Valproic Acid Prodrug Affects Selective Markers, Augments Doxorubicin Anticancer Activity and Attenuates Its Toxicity in a Murine Model of Aggressive Breast Cancer.

We studied the unique inhibitor of the histone deacetylases (HDAC) valproate-valpromide of acyclovir (AN446) that upon metabolic degradation release the HDAC inhibitor (HDACI) valproic acid (VPA). Among the HDAC inhibitors that we have tested, only AN446, and to a lesser extent VPA, synergized with doxorubicin (Dox) anti-cancer activity. Romidepsin (Rom) was additive and the other HDACIs tested were antagonistic. These findings led us to test and compare the anticancer activities of AN446, VPA, and Rom with and without Dox in the 4T1 triple-negative breast cancer murine model. A dose of 4 mg/kg once a week of Dox had no significant effect on tumor growth. Rom was toxic, and when added to Dox the toxicity intensified. AN446, AN446 + Dox, and VPA + Dox suppressed tumor growth. AN446 and AN446 + Dox were the best inhibitory treatments for tumor fibrosis, which promotes tumor growth and metastasis. Dox increased fibrosis in the heart and kidneys, disrupting their function. AN446 most effectively suppressed Dox-induced fibrosis in these organs and protected their function. AN446 and AN446 + Dox treatments were the most effective inhibitors of metastasis to the lungs, as measured by the gap area. Genes that control and regulate tumor growth, DNA damage and repair, reactive oxygen production, and generation of inflammation were examined as potential therapeutic targets. AN446 affected their expression in a tissue-dependent manner, resulting in augmenting the anticancer effect of Dox while reducing its toxicity. The specific therapeutic targets that emerged from this study are discussed.

Comparison of the tissue distribution and metabolism of AN1284, a potent anti-inflammatory agent, after subcutaneous and oral administration in mice.

This study is to compare the tissue distribution and metabolism of AN1284 after subcutaneous and oral administration at doses causing maximal reductions in IL-6 in plasma and tissues of mice. Anti-inflammatory activity of AN1284 and its metabolites was detected in lipopolysaccharide (LPS) activated RAW 264.7 macrophages. Mice were given AN1284 by injection or gavage, 15 min before LPS. IL-6 protein levels were measured after 4 h. Using a liquid chromatography/mass spectrometry method we developed, we showed that AN1284 is rapidly metabolized to the indole (AN1422), a 7-OH derivative (AN1280) and its glucuronide. AN1422 has weaker anti-inflammatory activity than AN1284 in LPS-activated macrophages and in mice. AN1284 (0.5 mg/kg) caused maximal reductions in IL-6 in the plasma, brain, and liver when injected subcutaneously and after gavage only in the liver. Similar reductions in the plasma and brain required a dose of 2.5 mg/kg, which resulted in 5.5-fold higher hepatic levels than after injection of 0.5 mg/kg, but 7, 11, and 19-fold lower ones in the plasma, brain, and kidneys, respectively. Hepatic concentrations produced by AN1284 were 2.5 mg/kg/day given by subcutaneously implanted mini-pumps that were only 12% of the peak levels seen after acute injection of 0.5 mg/kg. Similar hepatic concentrations were obtained by (1 mg/kg/day), administered in the drinking fluid. These were sufficient to decrease hepatocellular damage and liver triglycerides in previous experiments in diabetic mice. AN1284 can be given orally by a method of continuous release to treat chronic liver disease, and its preferential concentration in the liver should limit any adverse effects.

A switch in mechanism of action prevents doxorubicin-mediated cardiac damage.

Cancer patients treated with doxorubicin are at risk of congestive heart failure due to doxorubicin-mediated cardiotoxicity via topoisomerase IIß poisoning. Acute cardiac muscle damage occurs in response to the very first dose of doxorubicin, however, cardioprotection has been reported after co-treatment of doxorubicin with acyloxyalkyl ester prodrugs. The aim of this study was to examine the role played by various forms of acute cardiac damage mediated by doxorubicin and determine a mechanism for the cardioprotective effect of formaldehyde-releasing prodrug AN-9 (pivaloyloxymethyl butyrate). Doxorubicin-induced cardiac damage in BALB/c mice bearing mammary tumours was established with a single dose of doxorubicin (4 or 16 mg/kg) administered alone or in combination with AN-9 (100 mg/kg). AN-9 protected the heart from doxorubicin-induced myocardial apoptosis and also significantly reduced dsDNA breaks, independent from the level of doxorubicin biodistribution to the heart. Covalent incorporation of [14C]doxorubicin into DNA showed that the combination treatment yielded significantly higher levels of formaldehyde-mediated doxorubicin-DNA adducts compared to doxorubicin alone, yet this form of damage was associated with cardioprotection from apoptosis. The cardiac transcriptomic analysis indicates that the combination treatment initiates inflammatory response signalling pathways. Doxorubicin and AN-9 combination treatments were cardioprotective, yet preserved doxorubicin-mediated anti-tumour proliferation and apoptosis in mammary tumours. This was associated with a switch in doxorubicin action from cardiac topoisomerase IIß poisoning to covalent-DNA adduct formation. Co-administration of doxorubicin and formaldehyde-releasing prodrugs, such as AN-9, may be a promising cardioprotective therapy while maintaining doxorubicin activity in primary mammary tumours.

Cardioprotection by AN-7, a prodrug of the histone deacetylase inhibitor butyric acid: Selective activity in hypoxic cardiomyocytes and cardiofibroblasts.

The anticancer prodrug butyroyloxymethyl diethylphosphate (AN-7), upon metabolic hydrolysis, releases the histone deacetylase inhibitor butyric acid and imparts histone hyperacetylation. We have shown previously that AN-7 increases doxorubicin-induced cancer cell death and reduces doxorubicin toxicity and hypoxic damage to the heart and cardiomyocytes. The cardiofibroblasts remain unprotected against both insults. Herein we examined the selective effect of AN-7 on hypoxic cardiomyocytes and cardiofibroblasts and investigated mechanisms underlying the cell specific response. Hypoxic cardiomyocytes and cardiofibroblasts or H2O2-treated H9c2 cardiomyoblasts, were treated with AN-7 and cell damage and death were evaluated as well as cell signaling pathways and the expression levels of heme oxygenase-1 (HO-1). AN-7 diminished hypoxia-induced mitochondrial damage and cell death in hypoxic cardiomyocytes and reduced hydrogen peroxide damage in H9c2 cells while increasing cell injury and death in hypoxic cardiofibroblasts. In the cell line, AN-7 induced Akt and ERK survival pathway activation in a kinase-specific manner including phosphorylation of the respective downstream targets, GSK-3ß and BAD. Hypoxic cardiomyocytes responded to AN-7 treatment by enhanced phosphorylation of Akt, ERK, GSK-3ß and BAD and a significant 6-fold elevation in HO-1 levels. In hypoxic cardiofibroblasts, AN-7 did not activate Akt and ERK beyond the effect of hypoxia alone and induced a limited (~1.5-fold) increase in HO-1. The cell specific differences in kinase activation and in heme oxygenase-1 upregulation may explain, at least in part, the disparate outcome of AN-7 treatment in hypoxic cardiomyocytes and hypoxic cardiofibroblasts.

Synthesis and Biological Evaluation of Derivatives of Indoline as Highly Potent Antioxidant and Anti-inflammatory Agents.

We describe the preparation and evaluation of novel indoline derivatives with potent antioxidant and anti-inflammatory activities for the treatment of pathological conditions associated with chronic inflammation. The indolines are substituted at position 1 with chains carrying amino, ester, amide, or alcohol groups, and some have additional substituents, Cl, MeO, Me, F, HO, or BnO, on the benzo ring. Concentrations of 1 pM to 1 nM of several compounds protected RAW264.7 macrophages against H2O2 induced cytotoxicity and LPS induced elevation of NO, TNF-α, and IL-6. Several derivatives had anti-inflammatory activity at 1/100th of the concentration of unsubstituted indoline. Four compounds with ester, amine, amide, or alcohol side chains injected subcutaneously in mice at a dose of 1 µmol/kg or less, like dexamethasone (5.6 µmol/kg) prevented LPS-induced cytokine elevation in the brain and peripheral tissues. Subcutaneous injection of 100 µmol/kg of these compounds caused no noticeable adverse effects in mice during 3 days of observation.

Comparison of the anticancer properties of a novel valproic acid prodrug to leading histone deacetylase inhibitors.

The HDAC inhibitory activity of valproic acid (VPA) has led to on-going evaluation of it as an anticancer agent. The histone deacetylase (HDAC) inhibitor AN446, a prodrug of VPA, releases the acid upon metabolic degradation. AN446 is >60-fold more potent than VPA in killing cancer cells in vitro. Herein, we compare the activities of AN446, as an anticancer agent, to those of representative types from each of the four major classes of HDAC inhibitors (HDACIs): vorinostat, romidepsin, entinostat, and VPA. AN446 exhibited the greatest selectivity and HDAC inhibitory activity against cancer cells. In glioblastoma cells only AN446, and in MDA-MB-231 cells only AN446 and VPA interacted in synergy with doxorubicin (Dox). AN446 was superior to the studied HDACIs in inducing DNA-damage in cancer cells, while in normal astrocytes and cardiomyoblasts AN446 was the least toxic. AN446 was the only HDACI tested that exhibited selective HDAC inhibitory activity that was high in cancer cells and low in noncancerous cells. This discriminating inhibition correlated with the toxicity of the HDACIs, suggesting that their effects could be attributed to HDAC inhibition. In cardiomyoblasts, the HDACIs tested, except for AN446, hampered DNA repair by reducing the level of Rad 51. VPA and AN446 were the most effective HDACIs in inhibiting in vitro migration and invasion. The advantages of AN446 shown here, position it as a potentially improved HDACI for treatment of glioblastoma and triple negative breast cancer.

AN-7, a butyric acid prodrug, sensitizes cutaneous T-cell lymphoma cell lines to doxorubicin via inhibition of DNA double strand breaks repair.

We previously found that the novel histone deacetylase inhibitor (HDACI) butyroyloxymethyl diethylphosphate (AN-7) had greater selectivity against cutaneous T-cell lymphoma (CTCL) than SAHA. AN-7 synergizes with doxorubicin (Dox), an anthracycline antibiotic that induces DNA breaks. This study aimed to elucidate the mechanism underlying the effect of AN-7 on Dox-induced double-strand DNA breaks (DSBs) in CTCL, MyLa and Hut78 cell lines. The following markers/assays were employed: comet assay; western blot of γH2AX and p-KAP1; immunofluorescence of γH2AX nuclear foci; Western blot of repair protein; quantification of DSBs-repair through homologous recombination. DSB induction by Dox was evidenced by an increase in DSB markers, and DSBs-repair, by their subsequent decrease. The addition of AN-7 slightly increased Dox induction of DSBs in MyLa cells with no effect in Hut78 cells. AN-7 inhibited the repair of Dox-induced DSBs, with a more robust effect in Hut78. Treatment with AN-7 followed by Dox reduced the expression of DSB-repair proteins, with direct interference of AN-7 with the homologous recombination repair. AN-7 sensitizes CTCL cell lines to Dox, and when combined with Dox, sustains unrepaired DSBs by suppressing repair protein expression. Our data provide a mechanistic rationale for combining AN-7 with Dox or other DSB inducers as a therapeutic modality in CTCL.

Effects of histone deacetylase inhibitory prodrugs on epigenetic changes and DNA damage response in tumor and heart of glioblastoma xenograft.

The histone deacetylase (HDAC) inhibitory prodrugs of butyric (AN7) and valproic (AN446) acids, which release the active acids upon metabolic degradation, were studied examining their differential effects on the viability, HDAC inhibitory activity and the DNA damage response (DDR), in glioblastoma cell and normal human astrocytes (NHAs). In xenografts of glioblastoma, AN7 or AN446 given or the combination of each of them with Dox augmented the anticancer activity of Dox and protected the heart from its toxicity. In order to determine the processes underlying these opposing effects, the changes induced by these treatments on the epigenetic landscape, the DDR, and fibrosis were compared in tumors and hearts of glioblastoma xenografts. The potency of AN7 and AN446 as HDAC inhibitors was correlated with their effects on the viability of the cancer and non-cancer cells. The prodrugs affected the epigenetic landscape and the DDR in a tissue-specific and context-dependent manner. Findings suggest that the selectivity of the prodrugs could be attributed to their different effects on histone modification patterns in normal vs. transformed tissues. Further studies are warranted to substantiate the potential of AN446 as a new anticancer drug for glioblastoma patients.

Effect of Histone Deacetylase Inhibitor, Butyroyloxymethyl-Diethyl Phosphate (AN-7), on Corneal Neovascularization in a Mouse Model.

PURPOSE: To examine whether butyroyloxymethyl-diethyl phosphate (AN-7), a histone deacetylase inhibitor, inhibits chemically induced corneal neovascularization (NV) in mice. METHODS: Corneal NV was induced in the right eye of male C57BL mice by application of a mixture of 75% silver nitrate and 25% potassium nitrate to the corneal center. Immediately thereafter, the mice were randomized into 2 groups, receiving an intraperitoneal injection of AN-7 or saline, which served as control. Corneal NV was evaluated at constant time intervals from the corneal injury by corneal photographs and the area of corneal NV was measured. Centricity and density of the corneal vascularization were graded. Corneal flat mounts blood vessels staining and histological studies were performed on day 10. Unpaired t-test was used for group comparisons. RESULTS: The corneal neovascular area was statistically significantly reduced by AN-7 treatment on days 10 and 14 postinjury and compared with the untreated group. The centricity and density of the corneal NV between treated and untreated groups showed no significant difference at any time point. CONCLUSIONS: Systemic treatment with AN-7 had a significant inhibitory effect on chemical burn-induced corneal NV in mice. These results suggest that AN-7 should be further evaluated for its therapeutic potential for the treatment of corneal NV.

Bi-functional prodrugs of 5-aminolevulinic acid and butyric acid increase erythropoiesis in anemic mice in an erythropoietin-independent manner.

Anemia is a major cause of morbidity and mortality worldwide resulting from a wide variety of pathological conditions. In severe cases it is treated by blood transfusions or injection of erythroid stimulating agents, e.g., erythropoietin (Epo), which can be associated with serious adverse effects. Therefore, there is a need to develop new treatment modalities. We recently reported that treatment of erythroleukemic cells with the novel the bi-functional prodrugs of 5-aminolevulinic acid (ALA) and butyric acid (BA), AN233 and AN908, enhanced hemoglobin (Hb) synthesis to a substantially higher level than did ALA and BA individually or their mixture. Herein, we describe that these prodrugs when given orally to mice induced histone deacetylase inhibition in the kidneys, bone marrow and spleen, thus, indicating good penetrability to the tissues. In mice where anemia was chemically induced, treatment with the prodrugs increased the Hb, the number of red blood cells (RBCs) and the percentage of reticulocytes to normal levels. The prodrugs had no adverse effects even after repeated treatment at 100-200mg/kg for 50days. The lack of increased levels of Epo in the blood of mice that were treated with the prodrugs suggests that AN233 and AN908 affected the Hb and RBC levels in an Epo-independent manner. Taken together with our previous studies, we propose that the prodrugs increase globin expression by BA inhibition of histone deacetylase and elevation heme synthesis by ALA. These results support an Epo-independent approach for treating anemia with these prodrugs.

The Therapeutic Potential of AN-7, a Novel Histone Deacetylase Inhibitor, for Treatment of Mycosis Fungoides/Sezary Syndrome Alone or with Doxorubicin.

The 2 histone deacetylase inhibitors (HDACIs) approved for the treatment of cutaneous T-cell lymphoma (CTCL) including mycosis fungoides/sezary syndrome (MF/SS), suberoylanilide hydroxamic acid (SAHA) and romidepsin, are associated with low rates of overall response and high rates of adverse effects. Data regarding combination treatments with HDACIs is sparse. Butyroyloxymethyl diethylphosphate (AN-7) is a novel HDACI, which was found to have selective anticancer activity in several cell lines and animal models. The aim of this study was to compare the anticancer effects of AN-7 and SAHA, either alone or combined with doxorubicin, on MF/SS cell lines and peripheral blood lymphocytes (PBL) from patients with Sezary syndrome (SPBL). MyLa cells, Hut78 cells, SPBL, and PBL from healthy normal individuals (NPBL) were exposed to the test drugs, and the findings were analyzed by a viability assay, an apoptosis assay, and Western blot. AN-7 was more selectively toxic to MyLa cells, Hut78 cells, and SPBL (relative to NPBL) than SAHA and also acted more rapidly. Both drugs induced apoptosis in MF/SS cell lines, SAHA had a greater effect on MyLa cell line, while AN-7 induced greater apoptosis in SPBL; both caused an accumulation of acetylated histone H3, but AN-7 was associated with earlier kinetics; and both caused a downregulation of the HDAC1 protein in MF/SS cell lines. AN-7 acted synergistically with doxorubicin in both MF/SS cell lines and SPBL, and antagonistically with doxorubicin in NPBL. By contrast, SAHA acted antagonistically with doxorubicin on MF/SS cell lines, SPBL, and NPBL, leaving <50% viable cells. In conclusion, AN-7 holds promise as a therapeutic agent in MF/SS and has several advantages over SAHA. Our data provide a rationale for combining AN-7, but not SAHA, with doxorubicin to induce the cell death in MF/SS.

Potential Therapeutic Advantages of Doxorubicin when Activated by Formaldehyde to Function as a DNA Adduct-Forming Agent.

Doxorubicin has been in use as a key anticancer drug for forty years, either as a single agent or in combination chemotherapy. It functions primarily by interfering with topoisomerase II activity but in the presence of formaldehyde, it forms adducts with DNA, mainly with the exocyclic amine of guanine at GpC sites and these adducts are more cytotoxic than topoisomerase II induced damage. High levels of adducts form spontaneously from the endogenous level of formaldehyde in tumour cells (1,300 adducts per cell after a 4 hr treatment with doxorubicin), but substantially higher levels form with the addition of exogenous sources of formaldehyde, such as formaldehyde releasing prodrugs. The enhanced cytotoxicity of adducts has been confirmed in mouse models, with adduct-forming conditions resulting in much improved inhibition of tumour growth, as well as cardioprotection. Doxorubicin cardiotoxicity has been attributed to topoisomerase II poisoning, and the cardioprotection is consistent with a mechanism switch from topoisomerase II poisoning to covalent adduct formation. Although the adducts have a half-life of less than one day, a population remains as essentially permanent lesions. The capacity of doxorubicin to form adducts offers a range of potential advantages over the conventional use of doxorubicin (as a topoisomerase II poison), including: enhanced cell kill; tumour-selective activation, hence tumour-selective cell kill; decreased cardiotoxicity; decreased resistance to prolonged doxorubicin treatment. There is therefore enormous potential to improve clinical responses to doxorubicin by using conditions which favour the formation of doxorubicin-DNA adducts.

Synthesis and in vitro evaluation of anti-inflammatory activity of ester and amine derivatives of indoline in RAW 264.7 and peritoneal macrophages.

UNLABELLED: A prolonged increase in pro-inflammatory cytokines, TNF-α and IL-6 occurs in inflammatory diseases. Although existing therapies like steroids and TNF-α antagonists are effective they may cause serious adverse effects. We describe the preparation and evaluation for anti-inflammatory activity of 11 novel derivatives of indoline carbamates with a propionic ester, 2-aminoethyl, 3-aminopropyl 2-(dimethylamino)ethyl or 3-(dimethylamino)propyl group in positions 3 or 1. Compounds 25, 26 and 29 were previously shown to inhibit acetylcholinesterase with IC50s ranging from 0.4 to 55µM and to prevent cytotoxicity induced by reactive oxygen species in a concentration range of 100pM-1µM. Compounds 25, 26, 29, 9, 10, 17 and 18, reduced NO, TNF-α and IL-6 at concentrations of 1-10pM in LPS-activated RAW-264.7 and mouse peritoneal macrophages. The reduction in cytokines by compound 25 was associated with an increase in IκBα degradation and a decrease in the phosphorylation of p38 but not that of ERK. CONCLUSION: Indoline derivatives substituted at position 3 with chains carrying ester or amino groups may have potential for the treatment of chronic inflammatory and neurodegenerative diseases.

A novel valproic acid prodrug as an anticancer agent that enhances doxorubicin anticancer activity and protects normal cells against its toxicity in vitro and in vivo.

The poor survival of patients with malignant gliomas, underscores the need to develop effective treatment modalities for this devastating disease. Epigenetic agents used in combination with chemotherapy provide a promising approach to evoke synergistic cytotoxicity in glioblastomas. Previously we have described the cytotoxic synergy between a butyric acid prodrug and radiation in glioblastoma cell lines and the potentiation of radiation efficacy in glioma xenografts. Herein, we describe and compare the activities of AN446 (valproyl ester-valpramide of acyclovir) a novel histone deacetylase inhibitor (HDACI) to the previously described AN7 a HDACI prodrug of butyric acid. In various cancer cell lines, AN446 was a ~2-5-fold more potent anticancer agent HDACI than AN7. While AN446 augmented the anticancer efficacy of doxorubicin (Dox) it also reduced the Dox toxicity in non-cancerous cells. The interaction between AN446 and Dox in U251 and in 4T1 cell lines was synergistic in inducing cytotoxicity. We examined the concomitant physical and molecular changes in the tumor and heart of glioblastoma xenografts treated with AN446, AN7, Dox and the combination of the prodrugs with Dox. A weekly dose of 4 mg/kg Dox, caused toxicity in mice whereas AN446 (25mg/kg) or AN7 (50mg/kg) administered thrice weekly, did not. When Dox was administered with AN446 or AN7, the prodrugs ameliorated the decline in body weight, prolonged the time to failure and increased anticancer efficacy. Thus, the combination of Dox with AN446 or AN7 could add safety and efficacy to future treatment protocols for treating glioblastoma and other cancers.

The hydroxyl epimer of doxorubicin controls the rate of formation of cytotoxic anthracycline-DNA adducts.

Epirubicin was developed as a semi-synthetic anthracycline derivative to circumvent the cardiotoxic limitations associated with the use of doxorubicin in the clinic. Anthracycline compounds have been demonstrated to form covalent drug-DNA adducts utilising endogenous and exogenous sources of formaldehyde; however, previous investigations of the formation of epirubicin-DNA adducts provide conflicting evidence for adduct formation. This work provides evidence that epirubicin acts to form drug-DNA adducts at physiologically relevant concentrations and demonstrates that the rate of formation of epirubicin-DNA adducts is slower than that observed for other anthracycline compounds, explaining why they are only detectable under defined experimental conditions. Formation of covalent epirubicin-DNA adducts improves the apoptotic profile of epirubicin and provides opportunities to overcome drug resistance and cardiotoxic limitations.

Activation of DNA damage response pathways as a consequence of anthracycline-DNA adduct formation.

The cytotoxicity of doxorubicin, a clinically used anti-neoplastic drug, can be enhanced by formaldehyde (either endogenous or exogenous) to promote the formation of doxorubicin-DNA adducts. Formaldehyde supplies the carbon required for the covalent linkage of doxorubicin to one strand of DNA, with hydrogen bonds stabilising the doxorubicin mono-adduct to the other strand of DNA, to act much like an interstrand crosslink. Interstrand crosslinks present a major challenge for cellular repair processes, requiring the activation of numerous DNA damage response proteins for resolution of the resulting DNA intermediates and damage. This work investigates DNA damage response proteins activated by doxorubicin-DNA adducts. Although p53 was phosphorylated at Serine 15 in response to adducts, long term growth inhibition of mammalian cells was not affected by p53 status. Using siRNA technology and kinase inhibitors we observed enhanced cellular sensitivity to doxorubicin-DNA adducts when the activity of the signalling protein kinases ATM and ATR were lost. Cells synchronised using a double thymidine block were sensitised to adduct-initiated cell death upon ATR knockdown, but relatively unaffected by ATM knockdown. Loss of ATR was associated with abrogation of a drug-induced G(2)/M block and induction of mitotic catastrophe, while loss of ATM was associated with drug-induced apoptosis in non-synchronised cells. These proteins may therefore be potential drug targets to achieve synergistic cytotoxic responses to doxorubicin-DNA adduct forming therapies. The analysis of these protein kinases with respect to cell cycle progression indicates that ATR is required for G(2)/M checkpoint responses while ATM appears to function in G(1) mediated responses to anthracycline adducts.

Disparate impact of butyroyloxymethyl diethylphosphate (AN-7), a histone deacetylase inhibitor, and doxorubicin in mice bearing a mammary tumor.

The histone deacetylase inhibitor (HDACI) butyroyloxymethyl diethylphosphate (AN-7) synergizes the cytotoxic effect of doxorubicin (Dox) and anti-HER2 on mammary carcinoma cells while protecting normal cells against their insults. This study investigated the concomitant changes occurring in heart tissue and tumors of mice bearing a subcutaneous 4T1 mammary tumor following treatment with AN-7, Dox, or their combination. Dox or AN-7 alone led to inhibition of both tumor growth and lung metastases, whereas their combination significantly increased their anticancer efficacy and attenuated Dox- toxicity. Molecular analysis revealed that treatment with Dox, AN-7, and to a greater degree, AN-7 together with Dox increased tumor levels of γH2AX, the marker for DNA double-strand breaks and decreased the expression of Rad51, a protein needed for DNA repair. These events culminated in increased apoptosis, manifested by the appearance of cytochrome-c in the cytosol. In the myocardium, Dox-induced cardiomyopathy was associated with an increase in γH2AX expression and a reduction in Rad51 and MRE11 expression and increased apoptosis. The addition of AN-7 to the Dox treatment protected the heart from Dox insults as was manifested by a decrease in γH2AX levels, an increase in Rad51 and MRE11 expression, and a diminution of cytochrome-c release. Tumor fibrosis was high in untreated mice but diminished in Dox- and AN-7-treated mice and was almost abrogated in AN-7+Dox-treated mice. By contrast, in the myocardium, Dox alone induced a dramatic increase in fibrosis, and AN7+Dox attenuated it. The high expression levels of c-Kit, Ki-67, c-Myc, lo-FGF, and VEGF in 4T1 tumors were significantly reduced by Dox or AN-7 and further attenuated by AN-7+Dox. In the myocardium, Dox suppressed these markers, whereas AN-7+Dox restored their expression. In conclusion, the combination of AN-7 and Dox results in two beneficial effects, improved anticancer efficacy and cardioprotection.