Alcohol Conjugates Research Articles

Design, Synthesis, and Anti-bacterial Activity of Novel Deoxycholic Acid- Amino Alcohol Conjugates.

Numerous synthetic bile acid derivatives have been recognized for their various biological activities. Among these, bile acid amides have emerged as an attractive antibacterial agent. We herein illustrate the synthesis and antibacterial evaluation of deoxycholic acidamino alcohols conjugates. Design and Synthesis of novel deoxycholic acid-amino alcohol conjugates to investigate their antibacterial activity against E. coli and S. aureus. Novel deoxycholic acid-amino alcohol conjugates were synthesized, from conjugation of deoxycholic acid-NHS ester with amino alcohols. Various amino alcohols moieties were appended to the C24 position of deoxycholic acid to yield deoxycholic acid-amino alcohol conjugates. All the synthesized compounds were characterized by 1H NMR, 13C NMR, IR and massspectroscopy. The entire synthesized deoxycholic acid-amino alcohol conjugates were evaluated for their antibacterial activity against E. coli and S. aureus using the broth dilution method. The outcome illustrated that some of the novel deoxycholic acid-amino alcohol conjugates exhibited enhanced anti-bacterial activities. Amongst them, deoxycholic acid-amino alcohol conjugate containing (-R)-2-aminocyclohexanol (1) demonstrated promising efficacy against both strains S. aureus ATCC 25923 (MIC 15 μg/mL) and E. coli ATCC 25922 (MIC 45 μg/mL) and was identified as a lead molecule. Numbers of novel deoxycholic acid-amino alcohol conjugates were synthesized and their antimicrobial activities provided useful information that the potency was strongly depending on the structures of deoxycholic acid-amino alcohol conjugates.

Pharmacokinetic properties of the temozolomide perillyl alcohol conjugate (NEO212) in mice.

BackgroundNEO212 is a novel small-molecule anticancer agent that was generated by covalent conjugation of the natural monoterpene perillyl alcohol (POH) to the alkylating agent temozolomide (TMZ). It is undergoing preclinical development as a therapeutic for brain-localized malignancies. The aim of this study was to characterize metabolism and pharmacokinetic (PK) properties of NEO212 in preclinical models.MethodsWe used mass spectrometry (MS) and modified high-performance liquid chromatography to identify and quantitate NEO212 and its metabolites in cultured glioblastoma cells, in mouse plasma, brain, and excreta after oral gavage.ResultsOur methods allowed identification and quantitation of NEO212, POH, TMZ, as well as primary metabolites 5-aminoimidazole-4-carboxamide (AIC) and perillic acid (PA). Intracellular concentrations of TMZ were greater after treatment of U251TR cells with NEO212 than after treatment with TMZ. The half-life of NEO212 in mouse plasma was 94 min. In mice harboring syngeneic GL261 brain tumors, the amount of NEO212 was greater in the tumor-bearing hemisphere than in the contralateral normal hemisphere. The brain:plasma ratio of NEO212 was greater than that of TMZ. Excretion of unaltered NEO212 was through feces, whereas its AIC metabolite was excreted via urine.ConclusionsNEO212 preferentially concentrates in brain tumor tissue over normal brain tissue, and compared to TMZ has a higher brain:plasma ratio, altogether revealing favorable features to encourage its further development as a brain-targeted therapeutic. Its breakdown into well-characterized, long-lived metabolites, in particular AIC and PA, will provide useful equivalents for PK studies during further drug development and clinical trials with NEO212.

NEO212 induces mitochondrial apoptosis and impairs autophagy flux in ovarian cancer

BackgroundTemozolomide-perillyl alcohol conjugate (NEO212), a novel temozolomide (TMZ) analog, was previously reported to exert its anti-cancer effect in non-small cell lung cancer (NSCLC), and human nasopharyngeal carcinoma (NPC), etc.. In the current study, we intend to illuminate the potential anticancer property and the underly mechanisms of NEO212 in ovarian cancer cells.MethodsThe cytotoxicity of NEO212 was detected by MTT, colony formation analysis and xenograft model. The proteins involved in cell proliferation, DNA damage, autophagy and lysosomal function were detected by western blots; mitochondria, lysosome and autophagosome were visualized by TEM and/or immunofluorescence; Apoptosis, cell cycle analysis and mitochondrial transmembrane potential were detected by flow cytometry. TFEB translocation was detected by immunofluorescence and western blot.ResultsNEO212 has the potential anticancer property in ovarian cancer cells, as evidence from cell proliferation inhibition, G2/M arrest, DNA damage, xenograft, mitochondrial dysfunction and apoptosis. Importantly, we observed that although it induced significant accumulation of autophagosomes, NEO212 quenched GFP-LC3 degradation, down-regulated a series of lysosome related gene expression and blocked the autophagic flux, which significantly facilitated it induced apoptosis and was largely because it inhibited the nuclear translocation of transcription factor EB (EB).ConclusionsNEO212 inhibited TFEB translocation, and impaired the lysosomal function, implying NEO212 might avoid from autophagy mediated chemo-resistance, thus proposing NEO212 as a potential therapeutic candidate for ovarian cancer.

The Rolipram-Perillyl Alcohol Conjugate (NEO214) Is A Mediator of Cell Death through the Death Receptor Pathway.

Glioblastoma (GBM) is a highly aggressive primary brain tumor with a poor prognosis. Treatment with temozolomide, standard of care for gliomas, usually results in drug resistance and tumor recurrence. Therefore, there is a great need for drugs that target GBM. NEO214 was generated by covalently linking rolipram to perillyl alcohol (POH) via a carbamate bond to form the rolipram-perillyl alcohol conjugate. We show here that NEO214 is effective against both temozolomide-sensitive and temozolomide-resistant glioma cells. Furthermore, NEO214 is effective for different mechanisms of temozolomide resistance: overexpression of MGMT (O6-methylguanine methyl-transferase); deficiency in specific mismatch repair proteins; and overexpression of base excision repair (BER) proteins. NEO214-induced cytotoxicity involves apoptosis triggered by endoplasmic reticulum (ER) stress, as well as activating the Death Receptor 5 (DR5)/TNF-related apoptosis-inducing ligand (TRAIL/Apo2L) pathway. In vitro studies show that glioma cells treated with NEO214 express DR5 and exhibit cell death in the presence of recombinant TRAIL, a growth factor constitutively produced by astrocytes. Our in vitro 3D coculture data show that induction of DR5 in glioma cells with NEO214 and TRAIL cause tumor cell death very effectively and specifically for glioma cells. In vivo studies show that NEO214 has antitumor efficacy in orthotropic syngeneic rodent tumor models. Furthermore, NEO214 has therapeutic potential especially for brain tumors because this drug can cross the blood-brain barrier (BBB), and is effective in the TRAIL-rich astrocyte microenvironment. NEO214 is a strong candidate for use in the treatment of GBMs.

Temozolomide-Perillyl alcohol conjugate impairs Mitophagy flux by inducing lysosomal dysfunction in non-small cell lung Cancer cells and sensitizes them to irradiation

BackgroundTemozolomide-perillyl alcohol conjugate (TMZ-POH), a novel Temozolomide (TMZ) analog developed based on the conjugation of TMZ and perillyl alcohol (POH), displayed strong anticancer potency in multiple cancer types. In this study, we aimed to clarify the relationship between TMZ-POH and autophagy, and explore the underlying mechanisms involved in.MethodsThe proteins involved in autophagy, mitochondrial fission, lysosomal function and membrane traffic were detected by western blots; Autophagosome, mitochondria and lysosome were visualized by transmission electron microscope (TEM) and immunostaining; Apoptosis analysis and fluorescence probe detection were applied by flow cytometry.ResultsTMZ-POH blocked mitophagy flux although the number of autophagosomes which colocalized with mitochondria in the cells was increased via inducing lysosomal dysfunction as evidence from impaired lysosomal acidification, maturation and hampered autophagosome- lysosome fusion, which largely depended on its downregulation on the small GTPase RAB7A via mevalonate pathway. More importantly, our data demonstrated TMZ-POH sensitized cancer cell to irradiation induced apoptosis.ConclusionsTemozolomide-perillyl alcohol conjugate impairs mitophagy flux by inducing lysosomal dysfunction in Non-Small Cell Lung Cancer (NSCLC) cells and sensitizes them to irradiation, thereby proposing TMZ-POH as a potential radiosensitizer.

Abstract 4673: Selective delivery of tumor specific antigen to dendritic cells by mannose-labeled PLGA nanoparticles to induce immune response for cancer immunotherapy

Abstract Objective: To overcome limitation of ex vivo manipulation against dendritic cells (DC) vaccines, we developed mannose-labeled poly(d,l-lactide-co-glycolide) nanoparticles (MN-PLGA-NPs). The MN-PLGA-NPs enabled selective delivery of tumor-specific antigen to mannose receptor overexpressed DCs without ex vivo manipulation. Methods: Conjugation of mannose (MN) and polyvinyl alcohol (PVA) was analyzed by H-NMR. Size and zeta potential of the MN-PLGA-NPs were examined by dynamic light scattering using an electrophoretic light scattering photometer. Selective delivery efficiency of MN-PLGA-NPs to DCs was examined by confocal microscope and flow cytometry. Migration of DCs containing MN-PLGA-NPs was assessed flow cytometry. Therapeutic efficacy of MN-PLGA(OVA+poly I:C)-NPs was examined in EG7 tumor-bearing mouse models. Biological effect and activated CD8+ T cells in tumor tissue were confirmed by immunohistochemistry assay. Results: Conjugation of MN and PVA was confirmed that the peaks for the CH2 of methylene group in PVA was observed at 1.48 ppm and 1.59 ppm, and CH of tetrahydropyran group in MN was observed at 3.40 ppm, 3.49 ppm, 3.76 ppm, and 5.41 ppm. Size and zeta potential of MN-PLGA-NPs were 200 nm formed spherical shape and -10 mV, respectively. Loading efficiency of OVA or poly I:C into MN-PLGA-NPs was 90 % or 45 %, individually. Intracellular uptake of MN-PLGA-NPs in DCs was increased through selective binding between MN and MN-receptor compared to PLGA-NPs. Additionally, DCs containing MN-PLGA-NPs exhibited efficient migration to popliteal lymph node. Therapeutic efficacy of MN-PLGA-NPs showed significant inhibition of tumor growth in EG7 tumor-bearing mice compared to the control (p < 0.001) and PLGA-NPs (p < 0.05), and showed 100% of survival up to 60 days. Additionally, MN-PLGA-NPs treatment group showed a greater number of activated CD8+ T cells in tumor tissue compared to the other groups (p < 0.001), and prevention effect of tumor growth resulted in effectively suppressed compared to the control (p < 0.001) or PLGA-NPs (p < 0.001). Conclusion: MN-PLGA-NPs is an attractive system that can directly activate DC in vivo without ex vivo manipulation, which overcome the limitation of existing DC vaccine. Keyword: dendritic cells, selective delivery, PLGA nanoparticle, cancer immunotherapy. Citation Format: Yeongseon Byeon, Ji Eun Won, Ga Hee Kim, Min Gi Kim, Yeong Min Park, Hee Dong HAN. Selective delivery of tumor specific antigen to dendritic cells by mannose-labeled PLGA nanoparticles to induce immune response for cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4673.

Temozolomide\u2013perillyl alcohol conjugate\xa0downregulates O6-methylguanin DNA methltransferase via inducing ubiquitination-dependent proteolysis in non-small cell lung cancer

The DNA repair enzyme O6-methylguanin-DNA-methltransferase (MGMT) is able to remove products of alkylating agent such as O6-meG and emerges as a central determinant of cancer resistance to temozolomide (TMZ). Temozolomide–perillyl alcohol conjugate (TMZ–POH), a novel TMZ analog developed based on the conjugation of TMZ and POH, displayed strong anticancer potency in multiple cancer types, but seemed not to experience the chemoresistance even in cells with high MGMT expression unlike TMZ and other alkylating agents. In this study, we demonstrated TMZ–POH inhibited MGMT dependent on proteasomal pathway and this inhibition is a significant factor in its toxic effect in the non-small cell lung cancer (NSCLC) cells.

Induction of Pro-Apoptotic Endoplasmic Reticulum Stress in Multiple Myeloma Cells by NEO214, Perillyl Alcohol Conjugated to Rolipram.

Despite the introduction of new therapies for multiple myeloma (MM), many patients are still dying from this disease and novel treatments are urgently needed. We have designed a novel hybrid molecule, called NEO214, that was generated by covalent conjugation of the natural monoterpene perillyl alcohol (POH), an inducer of endoplasmic reticulum (ER) stress, to rolipram (Rp), an inhibitor of phosphodiesterase-4 (PDE4). Its potential anticancer effects were investigated in a panel of MM cell lines. We found that NEO214 effectively killed MM cells in vitro with a potency that was over an order of magnitude stronger than that of its individual components, either alone or in combination. The cytotoxic mechanism of NEO214 involved severe ER stress and prolonged induction of CCAAT/enhancer-binding protein homologous protein (CHOP), a key pro-apoptotic component of the ER stress response. These effects were prevented by salubrinal, a pharmacologic inhibitor of ER stress, and by CHOP gene knockout. Conversely, combination of NEO214 with bortezomib, a drug in clinical use for patients with MM, resulted in synergistic enhancement of MM cell death. Combination with the adenylate cyclase stimulant forskolin did not enhance NEO214 impact, indicating that cyclic adenosine 3′,5′-monophosphate (AMP) pathways might play a lesser role. Our study introduces the novel agent NEO214 as a potent inducer of ER stress with significant anti-MM activity in vitro. It should be further investigated as a potential MM therapy aimed at exploiting this tumor’s distinct sensitivity to ER stress.

Synthesis and Characterization of Complexes of Remodulated Polyvinyl Alcohol Conjugates

Synthesis and Characterization of Complexes of Remodulated Polyvinyl Alcohol Conjugates

ChemInform Abstract: Organocatalytic Asymmetric Nucleophilic Addition to o‐Quinone Methides by Alcohols.

AbstractThe first catalytic asymmetric intermolecular alcohol conjugate addition to o‐quinone methides is described.

Temozolomide-perillyl alcohol conjugate induced reactive oxygen species accumulation contributes to its cytotoxicity against non-small cell lung cancer.

Temozolomide-perillyl alcohol conjugate (TMZ − POH), a novel temozolomide analog, was reported to play a cytotoxic role in triple-negative breast cancer and TMZ-resistant gliomas. In a current study we had demonstrated how TMZ − POH also exhibited its cytotoxicity against non-small cell lung cancer (NSCLC), the most common type of lung cancer, as evidence from cell/tumor proliferation inhibition, G2/M arrest, DNA damage and mitochondrial apoptosis. Importantly, TMZ − POH’s cytotoxicity is closely related to reactive oxygen species (ROS) accumulation because it can be reversed by two ROS scavengers, catalase (CAT) and N-acetyl-L-cysteine (NAC). TMZ − POH induces mitochondrial transmembrane potential (MTP) decrease and ROS accumulation, in turn activates mitogen-activated protein kinase (MAPKs) signaling and mitochondrial apoptosis, and then exerts its cytotoxicity, thus proposing TMZ − POH as a potential therapeutic candidate for NSCLC.

Organocatalytic Asymmetric Nucleophilic Addition to o-Quinone Methides by Alcohols.

The first catalytic asymmetric intermolecular alcohol conjugate addition to o-quinone methides (o-QMs) is disclosed. Due to reversible C-O bond formation and low nucleophilicity of alcohols, catalytic asymmetric oxa-Michael additions with simple alcohol nucleophiles to establish acyclic oxygenated carbon stereocenters remain scarce. The present reaction represents a rare example of this type. With a suitable chiral acid catalyst, the in situ formation of o-QMs and subsequent conjugate addition proceeded with high efficiency and enantioselectivity. The chiral ether products are versatile precursors to other chiral molecules.

Abstract A22: NEO212: A novel temozolomide-perillyl alcohol conjugate exhibits superior activity against temozolomide resistant gliomas and intracranial triple negative breast cancer metastasis

Abstract Although the alkylating agent temozolomide (TMZ) has become the standard of care in the treatment of malignant gliomas, its overall efficacy is still limited by development of drug resistance, limited blood brain barrier (BBB) penetrance, and myelotoxicity. Recently, we have synthesized a TMZ analog where the monoterpene perillyl alcohol (POH) is covalently linked to TMZ via a carbamide bond. This new compound (NEO212) has been tested in two situations: TMZ resistant gliomas and breast cancer intracranial metastasis. Glioma cell lines (U251TR, LN229TR2, T98G) determined to be TMZ resistant based on base excision repair (BER) pathway, mismatch repair (MMR) deficiency, or overexpression of O6 methyl-guanine-DNA methyltransferase (MGMT) were evaluated in-vitro. In vivo experiments were performed using intracranial implant of luciferase labeled TMZ resistant cell lines. Triple negative breast cancer lines were tested, and implanted intracranially for in-vivo model. NEO212 was administered in both cases using subcutaneous injection using 10 day treatment, 7 day rest cycles did not have significant toxicity on normal astrocytes and brain endothelial cells. NEO212 induced cell death appeared to be independent of the mechanism of DNA repair resistance. In vivo experiments using intracranial implant of luciferase labeled TMZ resistant cell lines demonstrated that NEO212 crossed the BBB, reduced intracranial growth, and increased animal survival significantly compared to TMZ, without significant toxicity (including bone marrow). NEO212 was also effective in TMZ resistant breast cancer lines. In an intracranial mouse tumor model with triple-negative breast cancer, NEO212 revealed considerably greater therapeutic efficacy than TMZ, where a single cycle of treatment (10 days) extended median survival benefit from 6 days (in the case of TMZ) to 28 days, with good tolerance. NEO212 appears to be a promising well tolerated new agent with similar mechanism of cytotoxicity to TMZ. Its increased potency is most likely multi-factorial: 1) increased DNA damage, involving a broader scope of DNA repair mechanisms. 2) linkage with POH, resulting in longer biological half-life and greater opportunity for placement of cytotoxic DNA lesions. 3) increased lipophilicity, allowing for better penetration of the BBB, and possibly cell membrane. Preliminary results demonstrate that it can safely be administered systemically; more detailed toxicity studies are currently underway. Citation Format: Thomas C. Chen, Axel H. Schonthal, Weijun Wang, Heeyon Cho, Florence H. Hofman. NEO212: A novel temozolomide-perillyl alcohol conjugate exhibits superior activity against temozolomide resistant gliomas and intracranial triple negative breast cancer metastasis. [abstract]. In: Proceedings of the AACR Special Conference: Advances in Brain Cancer Research; May 27-30, 2015; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2015;75(23 Suppl):Abstract nr A22.

Metabolism of (Z)-(1R,3R)-Profluthrin in Rats.

When [benzyl-α-(14)C]-labeled (Z)-(1R,3R)-profluthrin (2,3,5,6-tetrafluoro-4-methylbenzyl (Z)-(1R,3R)-2,2-dimethyl-3-(prop-1-enyl) cyclopropanecarboxylate, a newly developed pyrethroid) was administered orally to rats at 1 mg/kg, around 70% was absorbed, metabolized, and mainly excreted into urine within 48 h. Radioactivity in plasma reached Cmax at 6-8 h, and decreased (half-life; 37-52 h). A similar tendency was observed also in tissues. Absorption rate was slightly lower at high dose, while kinetics and distribution did not change. Eight metabolites were detected in urine and one in feces. Most of the (14)C in feces was unabsorbed (Z)-(1R,3R)-profluthrin. The main metabolic reactions were ester cleavage, hydroxylation of the methyl group on the C4-position of the benzene ring, and its glucuronidation or oxidation to carboxylic acid. Oxidation of the geminal dimethyl on the cyclopropane-C2 to carboxylic acid, oxidation followed by hydration of the propenyl double bond, and ω-oxidation to carboxylic acid and mercapturic acid conjugation of the benzyl alcohol were observed as minor reactions.

Effect of Solvent Dielectric Constant and Acidity on the OH Vibration Frequency in Hydrogen-Bonded Complexes of Fluorinated Ethanols.

Infrared spectroscopy measurements were used to characterize the OH stretching vibrations in a series of similarly structured fluoroethanols, RCH2OH (R = CH3, CH2F, CHF2, CF3), a series which exhibits a systematic increase in the molecule acidity with increasing number of F atoms. This study, which expands our earlier efforts, was carried out in non-hydrogen-bonding solvents comprising molecules with and without a permanent dipole moment, with the former solvents being classified as polar solvents and the latter designated as nonpolar. The hydrogen bond interaction in donor-acceptor complexes formed in solution between the fluorinated ethanol H-donors and the H-acceptor base DMSO was investigated in relation to the solvent dielectric and to the differences ΔPA of the gas phase proton affinities (PAs) of the conjugate base of the fluorinated alcohols and DMSO. We have observed that νOH decreases as the acidity of the alcohol increases (ΔPA decreases) and that νOH varies inversely with ε, exhibiting different slopes for nonpolar and polar solvents. These 1/ε slopes tend to vary linearly with ΔPA, increasing with increasing acidity. These experimental findings, including the ΔPA trends, are described with our recently published two-state Valence Bond-based theory for acid-base H-bonded complexes. Lastly, the correlation of the alcohol's conjugate base PAs with Taft σ* values of the fluorinated ethyl groups CH(n)F(3-n)CH2- provides a connection of the inductive effects for these groups with the acidity parameter ΔPA associated with the H-bonded complexes.

A Novel Temozolomide–Perillyl Alcohol Conjugate Exhibits Superior Activity against Breast Cancer Cells In Vitro and Intracranial Triple-Negative Tumor Growth In Vivo

There is no effective therapy for breast cancer that has spread to the brain. A major roadblock is the blood-brain barrier (BBB), which prevents the usual breast cancer drugs from effectively reaching intracranial metastases. The alkylating agent temozolomide (TMZ) is able to penetrate the BBB and has become the gold standard for chemotherapeutic treatment of glioblastoma. However, when it was tested in clinical trials for activity against brain metastases of breast cancer, the results were mixed and ranged from "encouraging activity" to "no objective responses." In an effort to generate an agent with greater activity against intracranial breast metastases, we synthesized a TMZ analog where the natural product perillyl alcohol (POH) was covalently linked to TMZ's amide functionality. The resulting novel compound, called TMZ-POH (T-P), displayed greatly increased anticancer activity in a variety of breast cancer cell lines, inclusive of TMZ-resistant ones. It caused DNA damage and cell death much more efficiently than its parental compound TMZ, because linkage with POH increased its biologic half-life and thus provided greater opportunity for placement of cytotoxic DNA lesions. In an intracranial mouse tumor model with triple-negative breast cancer, T-P revealed considerably greater therapeutic efficacy than TMZ, where a single cycle of treatment extended median survival benefit from 6 days (in the case of TMZ) to 28 days. At the same time, T-P seemed to be well tolerated by the animals. Thus, T-P may have potential as a novel therapy for brain-targeted breast cancer metastases.

RGD-fatty alcohol-modified docetaxel liposomes improve tumor selectivity in vivo

The tripeptide arginine-glycine-aspartate (RGD) was conjugated with various fatty alcohols to obtain RGDOCnH2n+1 (n=8, 10, 12, 14, 16, 18), which were incorporated into the bilayer of docetaxel liposomes to improve their tumor specificity. The fatty alcohols were accepted as linking groups to insert the tetrapeptide RGDX (X=amino acid) into the bilayer of liposomes. RGDX was previously shown to be a potent ligand to target tumor cell-surface integrin receptors, whereas RGD was not shown to have this ability. We hypothesized that RGD-fatty alcohol conjugates lacking the fourth amine X can guide liposomes to tumors without reducing their binding affinity to integrins. Antitumor activity, pharmacokinetics and biodistribution studies were evaluated in mice inoculated with S180 sarcoma. Compared with unmodified liposomes, RGD-fatty alcohol-modified liposomes successfully delivered significantly more docetaxel to tumors, which led to significant tumor weight loss and increased tumor docetaxel concentrations accompanied by reduced liver accumulation. Improved affinity of RGD-fatty alcohols to integrins was also confirmed on A375 cell model. Further comparisons among the tumor-targeting capacities of liposomes containing RGD-fatty alcohols, RGDF-fatty alcohols and RGDV-fatty acids demonstrated that RGD-fatty alcohols were as effective as the other two tetrapeptide derivatives. Therefore, a simplified tumor-targeting delivery system using RGD-fatty alcohols was developed.

Abstract 3740: Temozolomide-perillyl alcohol conjugate is cytotoxic for glioma cancer stem cells.

Abstract Glioblastoma multiforme (GBM), the most malignant glioma, is a highly invasive tumor which is usually fatal within 2 years. The chemotherapeutic agent of choice is temozolomide (TMZ); however glioma patients inevitably become resistant to TMZ and tumors recur. Tumor recurrence has been associated with highly invasive, chemoresistant cancer stem cells (CSC) which is resistant to TMZ. We have constructed a new chemical entity by conjugating TMZ to perillyl alcohol (POH) via a carbamate bond. This compound, TMZ-POH, contains POH, a naturally occurring monoterpene that reduces tumor cell migration and has anti-tumor properties against gliomas as well as other cancers. In the studies presented here we demonstrate that TMZ-POH is cytotoxic to CSCs without affecting normal stem cells. The mechanism of TMZ-POH induced cytotoxicity is mediated in part through the DNA damage response pathway and apoptosis. Furthermore, the TMZ-POH conjugate is 5 to 10 fold more effective in inducing cell death compared to TMZ, POH or the mixture of these agents. Our data show that TMZ-POH is a promising novel compound directed against cancer stem cells and therefore important for the treatment of drug resistant, recurrent gliomas. Citation Format: Niyati Jhaveri, Fabienne Agasse, Florence M. Hofman, Thomas C. Chen. Temozolomide-perillyl alcohol conjugate is cytotoxic for glioma cancer stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3740. doi:10.1158/1538-7445.AM2013-3740

Abstract C74: Temozolomide-perillyl alcohol conjugate is cytotoxic for glioma cancer stem cells

Abstract Glioblastoma multiforme (GBM), the most malignant glioma, is a highly invasive tumor which is usually fatal within 2 years. The chemotherapeutic agent of choice is temozolomide (TMZ); however glioma patients inevitably become resistant to TMZ and tumors recur. Tumor recurrence has been associated with highly invasive, chemoresistant cancer stem cells (CSC) which is resistant to TMZ. We have constructed a new chemical entity by conjugating TMZ to perillyl alcohol (POH) via a carbamate bond. This compound, TMZ-POH, contains POH, a naturally occurring monoterpene that reduces tumor cell migration and has anti-tumor properties against gliomas as well as other cancers. In the studies presented here we demonstrate that TMZ-POH is cytotoxic to CSCs without affecting normal stem cells. The mechanism of TMZ-POH induced cytotoxicity is mediated in part through the DNA damage response pathway and apoptosis. Furthermore, the TMZ-POH conjugate is 5 to 10 fold more effective in inducing cell death compared to TMZ, POH or the mixture of these agents. Our data show that TMZ-POH is a promising novel compound directed against cancer stem cells and therefore important for the treatment of drug resistant, recurrent gliomas. Citation Format: Niyati Jhaveri, Fabienne Agasse, Florence M. Hofman, Thomas C. Chen. Temozolomide-perillyl alcohol conjugate is cytotoxic for glioma cancer stem cells. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Invasion and Metastasis; Jan 20-23, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;73(3 Suppl):Abstract nr C74.

Mutagenicity of 5-Hydroxymethylfurfural in V79 Cells Expressing Human SULT1A1: Identification and Mass Spectrometric Quantification of DNA Adducts Formed

5-Hydroxymethylfurfural (HMF), a heterocyclic product of the Maillard reaction, is a ubiquitous food contaminant. It has demonstrated hepatocarcinogenic activity in female mice. This effect may originate from sulfo conjugation of the benzylic alcohol yielding 5-sulfooxymethylfurfural (SMF), which is prone to react with DNA via nucleophilic substitution. Indeed, we showed that HMF induces gene mutations in Chinese hamster V79 cells engineered for the expression of human (h) sulfotransferase (SULT)1A1 but not in parental V79 cells. In order to identify potential DNA adducts, we incubated DNA samples with SMF or HMF in aqueous solution. Modified DNA was digested and surveyed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for adducts that may be formed by nucleosides either via nucleophilic substitution at the electrophilic carbon atom of SMF or via imine formation with the aldehyde group present in HMF and SMF. The most abundant adducts formed from SMF, N(6)-((2-formylfuran-5-yl)methyl)-2'-deoxyadenosine (N(6)-FFM-dAdo) and N(2)-((2-formylfuran-5-yl)methyl)-2'-deoxyguanosine (N(2)-FFM-dGuo), were synthesized, purified, and characterized by (1)H NMR. Imine adducts were only detected when DNA was incubated with very high levels of HMF following reduction of the imines to corresponding secondary amines by NaBH(3)CN. Sensitive techniques based on LC-MS/MS multiple reaction monitoring for the quantification of the adducts in DNA samples were devised using isotope-labeled [(15)N(5)]N(6)-FFM-dAdo and [(13)C(10),(15)N(5)]N(2)-FFM-dGuo as internal standards. Both 5-methylfurfuryl adducts were detected in DNA from V79-hSULT1A1 treated with HMF but not in DNA from V79 control cells. Considering the lack of other known mutagenic metabolites, we hypothesize that the hepatocarcinogenic potential of HMF originates from the formation of mutagenic SMF.