Ned B. Smith

A novel, macrophage migration inhibitory factor suicide substrate inhibits motility and growth of lung cancer cells

Although chemokine and growth factor receptors are attractive and popular targets for cancer therapeutic intervention, structure-based targeting of the ligands themselves is generally not considered practical. New evidence indicates that a notable exception to this is macrophage migration inhibitory factor (MIF). MIF, an autocrine- and paracrine-acting cytokine/growth factor, plays a pivotal role in both the initiation and maintenance of neoplastic diseases. MIF possesses a nonphysiologic enzymatic activity that is evolutionarily well-conserved. Although small molecule antagonists of MIFs enzymatic active site have been reported to inhibit biological activities of MIF, universally high IC(50)s have limited their clinical appeal. Using a computational virtual screening strategy, we have identified a unique small molecule inhibitor that serves as a suicide substrate for MIF, resulting in the covalent modification of the catalytically active NH(2)-terminal proline. Our studies further reveal that this compound, 4-iodo-6-phenylpyrimidine (4-IPP), is approximately 5x to 10x times more potent in blocking MIF-dependent catalysis and lung adenocarcinoma cell migration and anchorage-independent growth than the prototypical MIF inhibitor, ISO-1. Finally, using an in silico combinatorial optimization strategy, we have identified four unique congeners of 4-IPP that exhibit MIF inhibitory activity at concentrations 10x to 20x lower than that of parental 4-IPP.

Docosahexaenoic acid reverses cyclosporin A-induced changes in membrane structure and function

The use of a fish oil vehicle for cyclosporin A (CsA) can decrease the toxic effects of CsA but the mechanism is unclear. Here we examine the mechanism by which docosahexaenoic acid (DHA), a fish oil-derived polyunsaturated fatty acid, can alter the toxic effects of CsA on mouse organ function, endothelial macromolecular permeability, and membrane bilayer function. Mice given CsA and fish oil showed increased liver toxicity, kidney toxicity, incorporation of DHA, and evidence of oxidized fatty acids compared to control animals. We hypothesized that the toxic effects of CsA were primarily a result of membrane perturbation, which could be decreased if DHA were not oxidized. The presence of CsA (10 mol%) alone increased dipalmitoylphosphatidylcholine membrane permeability by seven fold over control (no CsA, no DHA). However, if non-oxidized DHA (15 mol%) and CsA were added to the membrane, the permeability returned to control levels. Interestingly, if the DHA was oxidized, the antagonistic effect of DHA on CsA was completely lost. While CsA alone increased endothelial permeability to albumin, the combination of non-oxidized DHA and CsA had no effect on endothelial macromolecular permeability. However the combination of oxidized DHA and CsA was no different than the effects of CsA only. CsA increased the fluorescence anisotropy of DPH in the liquid crystalline state of DPPC, while DHA decreased fluorescence anisotropy. However the combination of CsA and DHA was no different than DHA alone. We conclude that non-oxidized DHA can reverse the membrane perturbing effects of CsA, and the increases in endothelial macromolecular permeability, which may explain how fish oil is capable of decreasing the toxicity of CsA.

Bone selective effect of an estradiol conjugate with a novel tetracycline-derived bone-targeting agent

In this study a novel bone-targeting agent containing elements of the tricarbonylmethane system of ring A of tetracycline was developed and was shown to bind to the mineral constituent of bone, hydroxyapatite. Conjugation of this bone-targeting agent to estradiol resulted in a bone-targeted estrogen (BTE(2)-A1) with an enhanced ability to bind to hydroxyapatite. In an ovariectomized rat model of osteoporosis a partial separation of the skeletal effects of estradiol from the uterine effects was observed following subcutaneous administration of BTE(2)-A1. This novel bone-targeting estradiol delivery system has the potential to improve the safety profile of estradiol in the treatment of osteoporosis.

Role of human CYP1A1 and NAT2 in 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine-induced mutagenicity and DNA adducts

2-Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) is carcinogenic in multiple organs and numerous species. Bioactivation of PhIP is initiated by PhIP N2-hydroxylation catalysed by cytochrome P450s. Following N-hydroxylation, O-acetylation catalysed by N-acetyltransferase 2 (NAT2) is considered a further possible activation pathway. Genetic polymorphisms in NAT2 may modify cancer risk following exposure. Nucleotide excision repair-deficient Chinese hamster ovary (CHO) cells stably transfected with human cytochrome P4501A1 (CYP1A1) and a single copy of either NAT2*4 (rapid acetylator) or NAT2*5B (slow acetylator) alleles were used to test the effect of CYP1A1 and NAT2 polymorphism on PhIP genotoxicity. Cells transfected with NAT2*4 had significantly higher levels of N-hydroxy-PhIP O-acetyltransferase (p = 0.0150) activity than cells transfected with NAT2*5B. Following PhIP treatment, CHO cell lines transfected with CYP1A1, CYP1A1/NAT2*4 and CYP1A1/NAT2*5B each showed concentration-dependent cytotoxicity and hypoxanthine phosphoribosyl transferase (hprt) mutagenesis not observed in untransfected CHO cells. dG-C8-PhIP was the primary DNA adduct formed and levels were dose dependent in transfected CHO cells in the order: CYP1A1 < CYP1A1 and NAT2*5B < CYP1A1 and NAT2*4, although levels did not differ significantly (p > 0.05) following one-way analysis of variance. These results strongly support activation of PhIP by CYP1A1 with little effect of human NAT2 genetic polymorphism on mutagenesis and DNA damage.

METHODS FOR AROMATIC AND HETEROCYCLIC AMINE CARCINOGEN-DNA ADDUCT ANALYSIS BY LIQUID CHROMATOGRAPHY-TANDEM MASS SPECTROMETRY

Xenobiotic-DNA adducts are used as biomarkers to assess the genotoxic effects of carcinogens. Rats were dosed with 4-aminobiphenyl (4-ABP), 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), or 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP). DNA was isolated from the colons of vehicle and carcinogen-treated rats and digested using different nucleases and alkaline phosphatase. Deoxyribonucleoside adducts were quantified by capillary liquid chromatography-tandem mass spectrometry (LC-MS/MS) using isotope dilution methods with deuterated internal standards. Major adducts were those bound to the C8 position of deoxyguanosine. 3′- and 5′- Exonucleases were the most efficient nucleases at isolating dG-C8-ABP adducts. However, bulky adducts such as dG-C8-MeIQx and dG-C8-PhIP were better isolated using nuclease P1 rather than a combination of micrococcal nuclease and spleen phosphodiesterase. The use of DNase I enhanced the detection of all three adducts. We describe LC-MS/MS methods for DNA adduct detection and support the testing of different nucleases that increase DNA digestion efficiency and make available more DNA adducts for detection.

Effect of N-acetyltransferase 2 polymorphism on tumor target tissue DNA adduct levels in rapid and slow acetylator congenic rats administered 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine or 2-amino-3,8-dimethylimidazo-[4,5-f]quinoxaline.

2-Amino-3,8-dimethylimidazo-[4,5-f]quinoxaline (MeIQx) and 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) are suspected human carcinogens generated in well done meats. After N-hydroxylation, they are O-acetylated by N-acetyltransferase 2 (NAT2) to electrophiles that form DNA adducts. dG-C8-MeIQx and dG-C8-PhIP adducts have been identified in human tissues. In the female rat, administration of PhIP leads to mammary and colon tumors, whereas MeIQx induces liver tumors. Both humans and rats exhibit NAT2 genetic polymorphism yielding rapid and slow acetylator phenotypes. Because O-acetylation is an activation pathway, we hypothesized that MeIQx- and PhIP-induced DNA damage would be greater in tumor target tissues and higher in rapid than slow NAT2 acetylators. Adult female rapid and slow acetylator rats congenic at the Nat2 locus received a single dose of 25 mg/kg MeIQx or 50 mg/kg PhIP by gavage, and tissue DNA was isolated after 24 h. Deoxyribonucleoside adducts were identified and quantified by capillary liquid chromatography-tandem mass spectrometry using isotope dilution methods with deuterated internal standards. Major adducts were those bound to the C8 position of deoxyguanosine. dG-C8-PhIP DNA adducts were highest in colon, lowest in liver and did not significantly differ between rapid and slow acetylator congenic rats in any tissue tested. In contrast, dG-C8-MeIQx adducts were highest in liver and significantly (p < 0.001) higher in rapid acetylator liver than in slow acetylator liver. Our results are consistent with the tumor target specificity of PhIP and MeIQx and with increased susceptibility to MeIQx-induced liver tumors in rapid NAT2 acetylators.

3-O-Phosphate ester conjugates of 17-β-O-{1-[2-carboxy-(2-hydroxy-4-methoxy-3-carboxamido)anilido]ethyl}1,3,5(10)-estratriene as novel bone-targeting agents

A series of 3-O-phosphorylated analogs (4-10) of a novel bone-targeting estradiol analog (3) were synthesized after a thorough study of the reaction of 3 with a selection of phosphoryl chlorides under a variety of reaction conditions. Evaluation of these novel phosphate analogs for affinity for hydroxyapatite revealed that they bind with equal or higher affinity when compared to the bone tissue accumulator, tetracycline.

Six-month, prospective, longitudinal, open-label caffeine and dextromethorphan phenotyping study in children with growth hormone deficiency receiving recombinant human growth hormone replacement.

BACKGROUND Recombinant human growth hormone (r-hGH) is increasingly being used in children. Although growth hormone (GH) may alter the clearance of concomitantly administered medications, its effects on individual drug-metabolizing enzymes in children have not been characterized. OBJECTIVE The goal of this study was to assess the activities of cytochrome P450 (CYP) 1A2, N-acetyltransferase 2, xanthine oxidase, and CYP2D6 in children with isolated idiopathic GH deficiency before and 3 and 6 months after initiation of r-hGH treatment. METHODS This 6-month, prospective, longitudinal, open-label phenotyping study was conducted at 4 academic tertiary care centers within the Pediatric Pharmacology Research Unit network. Prepubertal or early pubertal children (4-14 years) with short stature and isolated idiopathic GH deficiency were enrolled. Patients were given 4 ounces of a cola beverage and 0.5 mg/kg of dextromethorphan (DM) before and 3 and 6 months after initiation of r-hGH treatment. Urine was collected for 8 hours after probe substrate administration, and enzyme activity was assessed using validatedcaffeine/metaboliteandDM/metabolitemolar ratios. Patients with a DM/dextrorphan molar ratio > or =0.3 were classified as poor metabolizers, and those with a ratio <0.3 were classified as extensive metabolizers. Anthropometric and biochemical responses were assessed at each visit. Blood was also obtained for determination of serum insulinlike growth factor-1 (IGF-1) levels and CYP2D6 genotype. RESULTS Fourteen patients (mean [SD] age, 11.5 [2.6] years [age range, 4.5-14.6 years]; 11 males, 3 females; 100% white; median height and weight, 131.8 cm and 29.2 kg, respectively) completed the 3 study visits. However, data from 2 patients were excluded from analysis due to procedural violations. In all patients, growth velocity and serum IGF-1 concentrations were significantly higher (P < 0.001) after r-hGH treatment (mean doses, 0.32 and 0.33 mg/kg per week at 3 and 6 months, respectively). However, molar ratio values did not significantly change after initiation of r-hGH. CONCLUSIONS In this study population of children with isolated idiopathic GH deficiency, no significant differences in caffeine/metabolite and DM/metabolite molar ratios were observed after initiation of r-hGH treatment.

Reduced Activities of Cytochrome P450 1A2 and Xanthine Oxidase in Children With Growth Hormone Deficiency

Growth hormone (GH) regulates the sex‐specific expression of drug‐metabolizing enzymes (DMEs), 1 and it has been shown that the clearance of multi‐enzymatic substrates is altered following GH replacement. 2, 3 Baseline pharmacokinetic alterations have also been described, suggesting that GH deficiency itself alters DME activity. 3, 4, 5 However, the effects on individual enzymes have not been characterized. We therefore assessed the activity levels of DMEs that are likely to be affected in children with GH deficiency (a condition that affects 1 in 3,500 children) 6 and compared them with pediatric historical controls. 7

Abstract 2103: XB05: a promising small molecule for cancer therapy.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC XB05 is synthetic small molecule which was serendipitously discovered to have potent anticancer properties. XB05 displays selectivity for malignant cells over non-malignant cells. Human myeloid leukemia cells (U937) and human lung cancer cells (A549) treated with XB05 show an increase in oxidative stress, DNA damage and a mixed apoptotic and necrotic mechanism of cell death. We conducted a COMPARE analysis to search for correlations between XB05 response and gene expression using publicly available microarray studies of the National Cancer Institute 60 cell line panel. This analysis uncovered a correlation between cell death in response to XB05 and expression of the transcription factor, SOX9. We find that SOX9 is differentially expressed across a panel of human lung cancer cell lines. MTT proliferation and clonogenic cell survival assays suggest that SOX9 expression positively correlates with the sensitivity of lung cancer cells to XB05. Furthermore, we show that siRNA knockdown of SOX9 recapitulates the phenotype of XB05-induced cell death, and that SOX9 may be downregulated at the protein level by XB05 treatment. Overall, these data demonstrate potent activity of XB05 against human cancer cells via a novel mechanism of action, and a potential role for SOX9 as a biomarker for selection and monitoring of therapy with XB05. Citation Format: Francesca R. Salipur, Elsa M. Reyes Reyes, Bo Xu, Ned Smith, Jian Cai, Gerald B. Hammond, Paula J. Bates. XB05: a promising small molecule for cancer therapy. [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 2103. doi:10.1158/1538-7445.AM2013-2103