Search

A novel series of benzhydroxamate esters derived from their precursor anthranilic acids have been prepared and have been identified as potent MEK inhibitors. 2-(2-Chloro-4-iodo-phenylamino)-N-cyclopropylmethoxy-3,4-difluoro-benzamide, CI-1040, was the first MEK inhibitor to demonstrate in vivo activity in preclinical animal models and subsequently became the first MEK inhibitor to enter clinical trial. CI-1040 suffered however from poor exposure due to its poor solubility and rapid clearance, and as a result, development of the compound was terminated. Optimization of the diphenylamine core and modification of the hydroxamate side chain for cell potency, solubility, and exposure with oral delivery resulted in the discovery of the clinical candidate N-(2,3-dihydroxy-propoxy)-3,4-difluoro-2-(2-fluoro-4-iodo-phenylamino)-benzamide PD 0325901.

Purpose: The median survival for patients diagnosed with glioblastoma multiforme, the most common type of brain tumor, is less than 1 year. Animal glioma models that are more predictive of therapeutic response in human patients than traditional models and that are genetically and histologically accurate are an unmet need. The nestin tv-a (Ntv-a) genetically engineered mouse spontaneously develops glioma when infected with ALV-A expressing platelet-derived growth factor, resulting in autocrine platelet-derived growth factor signaling. Experimental Design: In the Ntv-a genetically engineered mouse model, T2-weighted and T1-weighted, contrast-enhanced magnetic resonance images were correlated with histology, glioma grade (high or low), and survival. Magnetic resonance imaging (MRI) was therefore used to enroll mice with high-grade gliomas into a second study that tested efficacy of the current standard of care for glioma, temozolomide (100 mg/kg qdx5 i.p., n = 13). Results: The Ntv-a model generated a heterogeneous group of gliomas, some with high-grade growth rate and histologic characteristics and others with characteristics of lower-grade gliomas. We showed that MRI could be used to predict tumor grade and survival. Temozolomide treatment of high-grade tv-a gliomas provided a 14-day growth delay compared with vehicle controls. Diffusion MRI measurement of the apparent diffusion coefficient showed an early decrease in cellularity with temozolomide, similar to that observed in humans. Conclusions: The use of MRI in the Ntv-a model allows determination of glioma grade and survival prediction, distribution of mice with specific tumor types into preclinical trials, and efficacy determination both by tumor growth and early apparent diffusion coefficient response.

PD 0332991 is a highly specific inhibitor of cyclin-dependent kinase 4 (Cdk4) (IC50, 0.011 μmol/L) and Cdk6 (IC50, 0.016 μmol/L), having no activity against a panel of 36 additional protein kinases. It is a potent antiproliferative agent against retinoblastoma (Rb)-positive tumor cells in vitro, inducing an exclusive G1 arrest, with a concomitant reduction of phospho-Ser780/Ser795 on the Rb protein. Oral administration of PD 0332991 to mice bearing the Colo-205 human colon carcinoma produces marked tumor regression. Therapeutic doses of PD 0332991 cause elimination of phospho-Rb and the proliferative marker Ki-67 in tumor tissue and down-regulation of genes under the transcriptional control of E2F. The results indicate that inhibition of Cdk4/6 alone is sufficient to cause tumor regression and a net reduction in tumor burden in some tumors.

Purpose: Overexpression of the ErbB family of receptor tyrosine kinases has been associated with uncontrolled growth of many tumor types and, therefore, presents a promising molecular target for cancer therapy. CI-1033 is a small molecule tyrosine kinase inhibitor that differs from other 4-anilinoquinazolines by being a pan ErbB (instead of epidermal growth factor receptor-specific) irreversible (instead of reversible) inhibitor. Therefore, we investigated the antitumor effect of CI-1033 alone and in combination with ionizing radiation in vitro and in vivo. Experimental Design: We selected three human colon carcinoma cell-lines (LoVo, Caco-2, which express activated epidermal growth factor receptor and ErbB-2 family members, and SW620, which does not), and analyzed the effects of CI-1033 both in vitro and in vivo. For in vivo studies LoVo and Caco-2 cells were implanted s.c. in the flank of nude mice. After the tumor reached ∼100 mm3, treatment was initiated with 20 mg/kg of CI-1033 (orally once daily × 5 for 3 successive weeks), radiation treatment (a total of 30 Gy given in 2 Gy once daily × 5 for 3 successive weeks), or a combination of both CI-1033 and radiation treatment. Results: We found that exposure of LoVo and Caco-2, but not SW620 cells, to CI-1033 in the range of 1–3 μm could inhibit constitutive signaling by tyrosine kinases, arrest cell growth, inhibit cells in G1, stimulate expression of p53, and induce apoptosis. The inhibition of cell growth by CI-1033 seemed to produce only minimal radiosensitization in LoVo and Caco-2 cells. In contrast, the combination of CI-1033 and radiation produced significant (P < 0.0005 and P = 0.0002, respectively) and prolonged suppression of tumor growth in both the tumor types when compared with either treatment alone. Conclusions: These findings suggest that CI-1033 can increase the effectiveness of radiation therapy. The extent of suppression of tyrosine kinase activity by CI-1033, rather than the amount of activity in untreated cells, seemed to be more closely associated with the efficacy of combination treatment.

Triple negative breast cancer (TNBC) accounts for 15-20% of all breast cancers in the US. In general, patients have poorer prognosis and therapeutic intervention is more challenging due to insensitivity to hormonal and anti-HER2 therapies. Therapeutic options include chemotherapy and/or radiation (RT) treatment. In this work we utilized HCC70, a human TNBC cell line, grown subcutaneously in NSG mice. We investigated whether the combination of focal RT and paclitaxel would improve anti-tumor activity over paclitaxel alone. RT therapy has typically been delivered in low doses over long periods of time. More recently higher fractions of RT are being given in shorter time intervals. Utilizing the Small Animal Radiation Research Platform (SARRP; Xstrahl, Suwanee, GA) we compared a low dose (2.5Gy) fractionated regimen to a higher dose but less frequent regimen (8Gy, QDx3) either as monotherapy or in combination with paclitaxel (15mg/kg weekly). The total cumulative dose of radiation was 24-25Gy in both radiation regimens. Paclitaxel alone produced moderate activity with a 10 day tumor growth delay and no tumor regressions. Treatment with low dose RT resulted in tumor stasis for ≥68 days, but there were no tumor regressions or tumor free survivors (TFS). Treatment with 8Gy, produced a 75% incidence of confirmed partial regressions (PR) and a 25% incidence of complete regressions (CR) with no TFS. In this group we observed slightly more body weight loss (BWL), more frequent clinical signs, and more frequent need for food and water supplementation than with the fractionated RT regimen. Mean BWL was ~10% with a nadir around 8-10 days post first RT dose. BWL was recovered over the next 1-2 week period. The combination of low dose focal RT with paclitaxel produced activity similar to that with high dose radiation (50% PR, 40% CR, and 1 TFS). Treatment with paclitaxel plus 8Gy RT was highly efficacious, producing an 80% incidence of CRs, all of which remained tumor free at study day 108. The remaining mouse experienced tumor stasis in excess of 63 days. Thus, a higher dose of focal RT given in shorter intervals in combination with systemic paclitaxel resulted in the most efficacious therapeutic strategy but at the cost of increased BWL and clinical signs. Citation Format: Maryland Franklin, Thomas Dailey, Wilbur Leopold. Focal radiation enhances paclitaxel therapy in a mouse model of triple negative breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5194. doi:10.1158/1538-7445.AM2017-5194

The synthesis of a small series of 2-nitroimidazoles in which the β-amino alcohol side chain was amidated with a range of alkylating/acylating functionality is described. Synthetic methodologies were developed that generally provided for selective N-acyl versus N,O-bisacyl products. In vitro, target analogs showed minimal radiosensitization activity, with only a few exhibiting a sensitizer enhancement ratio (SER) >2.0 and C(1.6) values comparable to reference agents RB-6145 and RSU-1069. In an assay to determine potential to alkylate biomolecules, representative analogs showed

Progression through the G1 phase of the cell cycle requires phosphorylation of the retinoblastoma gene product (pRb) by the cyclin D-dependent kinases CDK4 and CDK6, whose activity can specifically be blocked by the CDK inhibitor p16(INK4A). Misregulation of the pRb/cyclin D/p16(INK4A) pathway is one of the most common events in human cancer and has lead to the suggestion that inhibition of cyclin D-dependent kinase activity may have therapeutic value as an anticancer treatment. Through screening of a chemical library, we initially identified the [2,3-d]pyridopyrimidines as inhibitors of CDK4. Chemical modification resulted in the identification of PD 0183812 as a potent and highly selective inhibitor of both CDK4 and CDK6 kinase activity, which is competitive with ATP. Flow cytometry experiments showed that of the cell lines tested, only those expressing pRb demonstrated a G1 arrest when treated with PD 0183812. This arrest correlated in terms of incubation time and potency with a loss of pRb phosphorylation and a block in proliferation, which was reversible. These results suggest a potential use of this chemical class of compounds as therapeutic agents in the treatment of tumors with functional pRb, possessing cell cycle aberrations in other members of the pRb/cyclin D/p16(INK4A) pathway.

Induction of heparin-binding epidermal growth factorlike growth factor (HB-EGF) mRNA in mouse skin organ culture was blocked by two pan-ErbB receptor tyrosine kinase (RTK) inhibitors but not by genetic ablation of ErbB1, suggesting involvement of multiple ErbB species in skin physiology. Human skin, cultured normal keratinocytes, and A431 skin carcinoma cells expressed ErbB1, ErbB2, and ErbB3, but not ErbB4. Skin and A431 cells expressed more ErbB3 than did keratinocytes. Despite strong expression of ErbB2 and ErbB3, heregulin was inactive in stimulating tyrosine phosphorylation in A431 cells. In contrast, it was highly active in MDA-MB-453 breast carcinoma cells. ErbB2 displayed punctate cytoplasmic staining in A431 and keratinocytes, compared to strong cell surface staining in MDA-MB-453. In skin, ErbB2 was cytoplasmic in basal keratinocytes, assuming a cell surface pattern in the upper suprabasal layers. In contrast, ErbB1 retained a cell surface distribution in all epidermal layers. Keratinocyte proliferation in culture was found to be ErbB1-RTK-dependent, using a selective inhibitor. These results suggest that in skin keratinocytes, ErbB2 transduces ligand-dependent differentiation signals, whereas ErbB1 transduces ligand-dependent proliferation/survival signals. Intracellular sequestration of ErbB2 may contribute to the malignant phenotype of A431 cells, by allowing them to respond to ErbB1dependent growth/survival signals, while evading ErbB2-dependent differentiation signals.

A series of 3-aryl-1,6-naphthyridine-2,7-diamines and related 2-ureas were prepared and evaluated as inhibitors of the FGF receptor-1 tyrosine kinase. Condensation of 4,6-diaminonicotinaldehyde and substituted phenylacetonitriles gave intermediate naphthyridine-2,7-diamines, and direct reaction of the monoanion of these (NaH/DMF) with alkyl or aryl isocyanates selectively gave the 2-ureas in varying yields (23-93%). For the preparation of more soluble 7-alkylamino-2-ureas, a number of protecting groups for the 2-amine were evaluated (phthaloyl, 4-methoxybenzyl) following selective blocking of the 7-amine (trityl), but these were not superior to the (required) 2-tert-Bu-urea group itself. Direct alkylation of the anion of the (unprotected) 7-amino group with excess 4-(3-chloropropyl)morpholine in DMF gave low (10%) yields of the desired product, but alkylation of the 7-acetamido anion, followed by mild alkaline hydrolysis, raised this to 64%. 3-Phenyl analogues were nonspecific inhibitors of isolated c-Src, FGFR, and PDGFR tyrosine kinases, whereas 3-(2,6-dichlorophenyl) analogues were most effective against c-Src and FGFR, and 3-(3,5-dimethoxyphenyl) derivatives showed high selectivity for FGFR alone. A water-soluble (7-morpholinylpropylamino) analogue retained high FGFR potency (IC(50) 31 nM) and selectivity. Pairwise comparison of the 1, 6-naphthyridines and the corresponding known pyrido[2,3-d]pyrimidine analogues showed little differences in potency or patterns of selectivity, suggesting that the 1-aza atom of the latter is not important for activity. A 7-acetamide derivative inhibited the growth of FGFR-expressing tumor cell lines and was particularly potent against HUVECs (IC(50) 4 nM). This compound was also a very potent inhibitor of HUVEC microcapillary formation (IC(50) 0.01 nM) and Matrigel invasion (IC(50) 7 nM) and showed significant in vivo antitumor effects in a highly vascularized mammary adenocarcinoma 16/c model at nontoxic doses. The compounds are worthy of further evaluation as antiangiogenesis agents.

Tumor metastasis is the main cause of mortality and treatment failure in cancer patients. It is a complex biological process regulated by alternations in expression of many genes. The p53 tumor suppressor gene has been shown to regulate expression of some metastasis-related genes. P53 transcriptionally activates expression of the genes encoding epidermal growth factor receptor, matrix metalloproteinase (MMP)-2, cathepsin D, and thrombospondin-1 but represses expression of the genes encoding basic fibroblast growth factor and multidrug resistance-1. Decreased expression of E-cadherin is associated with p53 alternations. Because these p53-regulatory genes either promote or inhibit tumor metastasis, the net effect of p53 expression on tumor metastasis depends upon the pattern of expression of these genes in a particular tumor. Because radiotherapy has been shown to increase tumor metastasis in both animal and human studies and because p53 is activated by radiation or DNA-damaging reagents, here we propose the working hypothesis that p53 may promote tumor metastasis upon induction by local radiotherapy or chemotherapy in some tumor types. For patients whose tumors contain wild-type p53, MMP inhibitors might be given with or before radiotherapy or chemotherapy to prevent an increase in tumor metastasis. Special caution should be taken with patients with cancers such as nasopharyngeal carcinoma in which p53 mutation is infrequent and radiotherapy is the main choice of treatment. To test our hypothesis, three studies are proposed and could serve as an initial step in understanding the complex biological process following radiation-induced p53 activation and its roles in regulation of tumor metastasis. Mol. Carcinog. 24:25–28, 1999. © 1999 Wiley-Liss, Inc.

A class of high-affinity inhibitors is disclosed that selectively target and irreversibly inactivate the epidermal growth factor receptor tyrosine kinase through specific, covalent modification of a cysteine residue present in the ATP binding pocket. A series of experiments employing MS, molecular modeling, site-directed mutagenesis, and 14 C-labeling studies in viable cells unequivocally demonstrate that these compounds selectively bind to the catalytic domain of the epidermal growth factor receptor with a 1:1 stoichiometry and alkylate Cys-773. While the compounds are essentially nonreactive in solution, they are subject to rapid nucleophilic attack by this particular amino acid when bound in the ATP pocket. The molecular orientation and positioning of the acrylamide group in these inhibitors in relation to Cys-773 entirely support these results as determined from docking experiments in a homology-built molecular model of the ATP site. Evidence is also presented to indicate that the compounds interact in an analogous fashion with erbB2 but have no activity against the other receptor tyrosine kinases or intracellular tyrosine kinases that were tested in this study. Finally, a direct comparison between 6-acrylamido-4-anilinoquinazoline and an equally potent but reversible analog shows that the irreversible inhibitor has far superior in vivo antitumor activity in a human epidermoid carcinoma xenograft model with no overt toxicity at therapeutically active doses. The activity profile for this compound is prototypical of a generation of tyrosine kinase inhibitors with great promise for therapeutic significance in the treatment of proliferative disease.

The synthesis of a small series of 2-nitroimidazoles in which the β-amino alcohol side chain was amidated with a range of alkylating/acylating functionality is described. Synthetic methodologies were developed that generally provided for selective N-acyl versus N,O-bisacyl products. In vitro, target analogs showed minimal radiosensitization activity, with only a few exhibiting a sensitizer enhancement ratio (SER) >2.0 and C(1.6) values comparable to reference agents RB-6145 and RSU-1069. In an assay to determine potential to alkylate biomolecules, representative analogs showed

CI-980, originally synthesized as a potential folate antagonist, is a tubulin-binding mitotic inhibitor currently in pediatric phase I and adult phase II clinical trials. Because of its extensive tissue distribution in animals and its favorable activity against multidrug resistant (MDR)-cells compared with other mitotic inhibitors, such as vincristine, we examined the membrane transport properties of CI-980. CI-980 accumulated rapidly in L1210 and CHO/K1 cells, reaching intracellular levels 40- and 8-fold higher, respectively, than those in the extracellular medium. Efflux was also quite rapid, but a small fraction of drug remained associated with the cells in drug-free medium. The uptake of CI-980 was not temperature or energy dependent, nor was it saturable up to an extracellular concentration of 100 microM. Inhibitors of nucleoside transport had no effect on CI-980 uptake. A cell line deficient in the transport of reduced folate was not resistant to CI-980, nor did it exhibit reduced CI-980 uptake. A 100-fold excess of the R-enantiomer inhibited CI-980 uptake by only 50%. These results are consistent with a model of CI-980 uptake involving passive diffusion followed by significant but largely reversible binding to intracellular or membrane components.

Each independently arising tumor is a separate and unique biologic entity with its own unique histologic appearance, biologic behavior, and drug response profile. Thus, in drug discovery, no single tumor has been a perfect predictor for any other tumor. For this reason, new agents are evaluated in a variety of tumor models which is known as breadth of activity testing. In recent years, human tumors implanted in athymic nude mice and SCID mice have also become available for breadth of activity testing. In studies carried out in these laboratories, it was found that 10 human tumors metastasized in the SCID mice, but failed to metastasize in nude mice. In addition, tumor growth and tumor takes were superior in the SCID mice. The strengths and weaknesses of xenograft model systems are discussed. For example, most human tumor xenograft models are excessively sensitive to alkylating agents as well as to a new class of DNA binders (XE840 and XP315). Using human tumor models that are the least sensitive t...

The laboratory mouse is one of the cornerstones of medical imaging research and has become integral to imaging technology development, image-based testing of medical hypotheses and new therapies against disease. Mouse imaging underlies one of the key advantages of medical imaging: translation between preclinical and clinical research. This chapter outlines the reasons behind the rapid expansion of translational medical imaging in the last two decades, and the key mouse imaging technologies and how they are used to generate clinically relevant disease-based end-points. Consideration of the most important aspects of mouse handling, preparation and biosecurity for imaging are given. The chapter ends with reference to future trends in mouse imaging and translational imaging in general.

In vitro screening of a number of 2-(aminoalkyl)-5-nitropyrazolo[3,4,5- kl]acridines has previously indicated (Sebolt, J.S.; et al. Cancer Res. 1987, 47, 4299-4304) that these compounds, in general, exhibit selective cytotoxicity against the human colon adenocarcinoma, HCT-8, cell line, relative to mouse leukemia L1210 cells. Comparative molecular field analysis (CoMFA) was applied to HCT-8 and L1210 growth inhibition assays (IC50s) of a series (44) of the pyrazoloacridine derivatives with the objective of predicting improved solid tumor selectivity. In the absence of crystallographic data, the 9-methoxy derivative (15), which is currently in clinical study, was selected as the template molecular model. Two different structural alignments were tested: an alignment of structures based on root mean square (RMS)-fitting of each structure to 15 was compared with an alternative strategy, steric and electrostatic alignment (SEAL). Somewhat better predictive cross-validation correlations (r2) were obtained with models based on RMS vis-à-vis SEAL alignment for both sets of assays. A large change in lattice spacing, e.g., 2 to 1 A, causes significant variations in the CoMFA results. A shift in the lattice of half of its spacing had a much smaller effect on the CoMFA data for a lattice of 1 A than one of 2 A. The relative contribution of steric and electrostatic fields to both models were about equal, underscoring the importance of both terms. Neither calculated log P nor HOMO and/or LUMO energies contribute to the model. Steric and electrostatic fields of the pyrazoloacridines are the sole relevant descriptors to the structure-activity (cross-validated and conventional) correlations obtained with the cytotoxic data for both the L1210 and HCT-8 cell lines. The cross-validated r2, derived from partial least-squares calculations, indicated considerable predictive capacity for growth inhibition of both the leukemia and solid-tumor data. Evidence for the predictive performance of the CoMFA-derived models is provided in the form of plots of actual vs predicted growth inhibition of L1210 and HCT-8 cells, respectively, by the pyrazoloacridines. The steric and electrostatic features of the QSAR are presented in the form of standard deviation coefficient contour maps of steric and electrostatic fields. The maps indicate that increases or decreases in steric bulk that would enhance growth inhibition of HCT-8 cells would likewise promote growth inhibition of L1210 cells. Contour maps generated to analyze the electrostatic field contributions of the pyrazoloacridines to growth inhibition provide an essentially similar set of results.(ABSTRACT TRUNCATED AT 400 WORDS)

As preclinical chemotherapists, we are often asked to identify experimental tumor models that can accurately predict for the drug response characteristics of all tumors of a given cellular subtype or molecular target. Unfortunately, it is impossible to give satisfactory answers to these inquiries. Because of the unique character of each independently arising tumor (whether spontaneous or induced), it does not take very long to realize that each tumor is a unique biologic entity with its own tumor growth behavior, histological appearance, drug response and molecular expression profiles. This is true whether the tumor is an experimental animal model or one originally derived from a patient. Further, many factors can influence the tumor growth and therapy response of experimental tumor models. Still, in vivo models are needed to adequately assess pharmacodynamics, toxicity and efficacy of any potential novel therapy. Presented herein is what we hope will be useful information regarding the transplant characteristics of tumor models, with some of the “pitfalls” to look out for when using any given tumor model for chemotherapy evaluations. Although most of the examples given use syngeneic models, the methodologies for assessing the predictive worth and maintaining model usefulness can be applied to almost any given transplantable tumor system (whether syngeneic or xenograft).

The antitumor activity of [SP-4-3-(R)]-[1,1-cyclobutanedicarboxylato-(2-)](2 methyl-1,4-butanediamine-N,N')platinum (CI-973) was characterized in a number of preclinical model systems. CI-973 retained substantial activity in cisplatin resistant murine leukemia cell lines, in vitro and in vivo; in L1210 leukemia resistant to cisplatin in vivo, CI-973 retained as much activity as was found in animals bearing sensitive L1210 leukemia. When compared in five solid murine tumors in vivo, both CI-973 and cisplatin were approximately equivalent in activity. In one human colon tumor and one human non-small cell lung carcinoma tested as xenografts in immunodeficient mice, cisplatin and CI-973 were ineffective. In two other human non-small cell lung carcinomas tested in the same fashion, cisplatin did possess activity. CI-973 has entered phase I clinical trials.

One of the greatest challenges in developing therapeutic regimens is the inability to rapidly and objectively assess tumor response due to treatment. Moreover, tumor response to therapeutic intervention in many cases is transient, and progressive alterations within the tumor may mask the effectiveness of an initially successful therapy. The ability to detect these changes as they occur would allow timely initiation of alternative approaches, maximizing therapeutic outcome. We investigated the ability of diffusion magnetic resonance imaging (MRI) to provide a sensitive measure of tumor response throughout the course of treatment, possibly identifying changes in sensitivity to the therapy. Orthotopic 9L gliomas were subjected to two separate therapeutic regimens, with one group receiving a single 5-day cycle (1ω) of low-dose 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and a second group receiving two cycles at the same dose, bisected with 2 days of rest (2ω). Apparent diffusion coefficient maps were acquired before and throughout treatment to observe changes in water mobility, and these observations were correlated to standard measures of therapeutic response and outcome. Our results showed that diffusion MRI was indeed able to detect the emergence of a drug-resistant tumor subpopulation subsequent to an initially successful cycle of BCNU therapy, leading to minimal gains from a second cycle. These diffusion MRI findings were highly correlated with tumor growth delay, animal survival, and ex vivo growth inhibition assays showing emerging resistance in excised tumors. Overall, this study highlights the ability of diffusion MRI to provide sensitive dynamic assessment of therapy-induced response, allowing early opportunities for optimization of therapeutic protocols. (Cancer Res 2006; 66(9): 4687-92)

CI-1033 (N-[4-[N-(3-chloro-4-fluorophenyl)amino-7-[3-(4-morpholynyl)propoxy]quinazolin-6-yl]acrylamide, PD 0183805-mesylate salt) was identified as a potent, selective inhibitor of erbB family tyrosine kinases, which are overexpressed in a number of solid tumors and have been shown to be involved in tumor progression. Because objective response of clinical patients to erbB-targeted therapies like CI-1033 has been observed only in a subset of cancer patients that exhibit the intended molecular targets, much emphasis has been placed on the identification of biomarkers of antitumor efficacy. Vascular endothelial growth factor (VEGF) and interleukin-8 (IL-8) were considered as potential biomarkers for CI-1033 due to ease of detection in patient plasma and showed roles in angiogenesis and cancer progression and positive regulation by the erbB receptor family. In the present studies, mice bearing established xenografts (A431 epidermoid carcinoma, H125 non–small cell lung carcinoma, SF767 glioblastoma, and MDA-MB-468 mammary carcinoma) were treated with efficacious and subefficacious doses of CI-1033, and plasma levels and xenograft gene expression of VEGF and IL-8 were evaluated. Oral administration of CI-1033 to tumor-bearing mice at efficacious doses resulted in markedly decreased levels of VEGF and/or IL-8 plasma levels and tumor mRNA levels relative to vehicle-treated control mice in xenograft models that exhibited evaluable levels of these markers. In contrast, subefficacious doses of CI-1033 did not significantly affect VEGF or IL-8 levels in any of the xenograft models. These studies indicate that plasma VEGF and IL-8 may have use as biomarkers of antitumor efficacy for epidermal growth factor receptor/erbB–targeted therapies such as CI-1033 and suggest that further clinical study of these markers in cancer patients are warranted.

A small molecule called PD 153035 inhibited the epidermal growth factor (EGF) receptor tyrosine kinase with a 5-pM inhibition constant. The inhibitor was specific for the EGF receptor tyrosine kinase and inhibited other purified tyrosine kinases only at micromolar or higher concentrations. PD 153035 rapidly suppressed autophosphorylation of the EGF receptor at low nanomolar concentrations in fibroblasts or in human epidermoid carcinoma cells and selectively blocked EGF-mediated cellular processes including mitogenesis, early gene expression, and oncogenic transformation. PD 153035 demonstrates an increase in potency over that of other tyrosine kinase inhibitors of four to five orders of magnitude for inhibition of isolated EGF receptor tyrosine kinase and three to four orders of magnitude for inhibition of cellular phosphorylation.

The chapter focuses on five major groups of targets that have been the object of particularly vigorous drug-discovery research over the past decade, which include the erbB family of growth factor receptors, the ras/MAPK pathway components, src family kinases, the AKT family, and the nuclear hormone receptor family. The erbB receptor family comprises four distinct membrane glycoproteins consisting of epidermal growth factor (EGF) receptors erbB1, erbB2, erbB3, and erbB4. They are widely expressed in epithelial, mesenchymal, and neuronal tissues, and are structurally similar. They are composed of an extracellular ligand-binding domain, a single transmembrane region, an intracellular domain possessing protein tyrosine kinase activity, and a C-terminal tail that contains specific tyrosine containing sequences, which, upon phosphorylation, become binding sites for src homology region 2 (SH2)–containing signaling proteins. The receptors also contain other phosphorylation sites that are believed to have a role in the regulation and function of the protein. Another family of tyrosine kinases that may contain appropriate targets for therapeutic intervention in cancer is the nonreceptor, cytoplasmic c-Src kinase, and other members of that kinase family. The amino acid sequence of this kinase family is composed of three major structural domains numbered from the C terminus. The first domain is the Src Homology1, or SH1, which encompasses the catalytic domain of the kinase. The SH2 domain, which contains tyrosine phosphorylation sites, and the SH3 domain containing proline-rich regions are likely to be involved in regulatory functions via intra and intermolecular interactions. Akt is a critical mediator of cell survival, and activated Akt protects cells from apoptosis induced by ultraviolet radiation, ionizing radiation, GF withdrawal, detachment from ECM, and cell cycle irregularities.

BACKGROUND: Intraocular melanomas in the West represents 70% of all primary eye cancers with a mortality rate greater than 50%. Current treatment options include enucleation, plaque radiotherapy, and proton beam radiotherapy. While enucleation is a highly invasive surgical procedure that removes the eye, the side effects of the two different radiotherapies include cataracts, retinopathy, cystoid macular edema, and secondary retinal detachment. Due to the relatively protected nature of the eye within the orbit, most topical treatment applications are precluded and systemic treatments increases the chance and incidence of off-target side effects. Up and coming targeted therapies that may not be constrained by such limitations require a non-invasive, imaging-relevant model in order to evaluate their efficacy. Therefore, the aim of this study was to characterize the imaging of orthotopic luc-enabled OCM-1 human choroidal melanoma tumors using bioluminescence imaging (BLI) and magnetic resonance imaging (MRI) for future use in anti-cancer drug efficacy testing. MATERIALS & METHODS: Human choroidal melanoma cells were modified to express luciferase. 8-12 week old female NIH-Foxn1rnu rats were implanted in the suprachoroidal space with OCM-1-luc tumor spheroids on Day 0. BLI and MRI imaging was performed every 5-7 days starting at Day 7 after implantation of the OCM-1-luc spheroids until animals reached a moribund state to monitor disease progression. For BLI, animals were injected with 150mg/kg luciferin, anesthetized with isoflurane in air and imaged at 10 minutes following luciferin administration. Anatomical MRI was performed using a gradient-echo pulse sequence on a 7T MRI system. After euthanasia, eyes were removed and preserved in formalin for histological analysis. RESULTS & CONCLUSIONS: Animals tolerated the ocular tumor implant well. As a result of these efforts, we have successfully characterized the growth of orthotopic luc-enabled OCM-1 choroidal melanomas using both bioluminescence and magnetic resonance imaging. This provides a simple approach with which to monitor tumor growth and treatment response in a highly efficient way which should allow a high daily animal throughput. These promising results will serve as a solid foundation with which dose routes and levels of anti-cancer drugs can be optimized and their efficacy evaluated with noninvasive imaging. Citation Format: John L. Chunta, Meridith Baugher, Deanne Lister, Erin Trachet, Kevin P. Guley, Chris Bull, Scott Wise, Wilbur R. Leopold, Patrick McConville. Orthotopic human choroidal melanoma model characterization with bioluminescence and magnetic resonance imaging for therapeutic efficacy evaluation. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3929. doi:10.1158/1538-7445.AM2014-3929

RB 6145, the ring-opened analog of RSU 1069, and PD 130908, the desoxy ring-opened analog of RSU 1069, were compared to RSU 1069 for their emetic potential in dogs. When RB 6145 and PD 130908 were administered intravenously at doses ranging from 20% to 50% of the mouse equivalent maximum tolerated dose (MTD), both analogs were less emetic than RSU 1069 on a molar basis. Furthermore, the 5HT 3 antagonist ondansetron prevented emesis at doses as high as 75% of the MTD. The reactivity, and hence the emetic liability of these compounds, is thought to be mediated by formation of the corresponding aziridine intermediate. In mouse plasma, both analogs rapidly converted to two products, the reactive aziridine and a stable oxazolidinone species formed upon reaction with bicarbonate in the blood. A positive correlation exists between the amounts of aziridine formed by these analogs and their emetic potential.

The mitogen-activated protein kinase pathway is thought to be essential in cellular growth and differentiation. Here we report the discovery of a highly potent and selective inhibitor of the upstream kinase MEK that is orally active. Tumor growth was inhibited as much as 80% in mice with colon carcinomas of both mouse and human origin after treatment with this inhibitor. Efficacy was achieved with a wide range of doses with no signs of toxicity, and correlated with a reduction in the levels of activated mitogen-activated protein kinase in excised tumors. These data indicate that MEK inhibitors represent a promising, noncytotoxic approach to the clinical management of colon cancer.

Glioblastoma multiforme (GBM) is the most common and most aggressive form of malignant primary brain tumors, affecting nearly 35,000 people in the United States. Most preclinical studies in glioma utilize survival as the primary endpoint to study, which provides limited information about disease progression, tumor burden response to treatment (a primary clinical end point). We have characterized two human glioma cell lines, Gli36 and LN827 that were modified to express luciferase in order to enable in vivo monitoring of disease progression and response to treatment using bioluminescence imaging (BLI). Anatomical magnetic resonance imaging (MRI) was also performed to directly correlate bioluminescence signal with tumor volume. Both models exhibited >90% tumor take-rate and responded to treatment with temozolomide, a clinical standard of care. Analysis of lifespan, tumor volume doubling times, and tumor growth delay all indicated that BLI is a reliable indicator of disease progression and response to treatment. BLI-based endpoints also showed good correlation with MR-based endpoints. These results support the use of in vivo imaging in these modified cell lines for longitudinal monitoring of tumor progression and response to therapy. Imaging was not only a reliable method for quantifying tumor burden, but enabled clinically relevant end points that could not be accessed through non-imaging means or lifespan determination alone. Further utility can be driven in these models through the use of other functional imaging end points. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B15. Citation Format: Deanne Lister, Mary Anne Meade, Tracey Woolliscroft, Deepa Balagurunathan, Erin Trachet, Wilbur Leopold, Patrick McConville. Increased clinical relevance of orthotopic glioma models through bioluminescence and MR imaging. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B15.

Background: Multimodality imaging presents a unique opportunity to image the efficacy of candidate compounds against various models of cancer. The challenge is in integrating the complimentary data that these modalities can provide. The purpose of this study was to evaluate the feasibility of quintuple modality imaging in order to optimize workflows for maximal efficacy testing of candidate compounds using PET, CT, MRI, Bioluminescence imaging (BLI), and Fluorescence Molecular Tomography (FMT). Materials and Methods: Female SCID-Beige mice were implanted IV with 5×10∧6 MM1s-luc cells and female NIH III mice were implanted with 5×10∧6 5TGM-1-luc cells. The presence and extent of disease was confirmed with BLI concurrently or before multimodality imaging was initiated 28-35 days post-implant. The day prior to imaging, mice were injected IV with 2nmol of Integrisense 750 (PerkinElmer) to allow for 24 hours of uptake prior to FMT imaging. The next day, animals were anesthetized with 2.0% isoflurane, were injected with 200µCi 18F-FDG, and were immediately positioned in an FMT cassette for imaging. Whole body FMT data was acquired with excitation/emission at 755nm/775nm. MicroCT data was then acquired by placing the FMT cassette into a custom Animal Handling System (AHS) dock that coupled the FMT cassette to an integrated bed holder (ASI Instruments) for the remaining modalities. Whole body CT images were acquired at 512 projections, 75kVp, and 220µA. A ten minute static PET data set was then acquired with a Seimen's Inveon system after a 1 hour tracer uptake period, and reconstructed with a 3D-OSEM algorithm. MicroPET imaging was followed by a series of T1-weighted, gadolinium-enhanced, and T2-weighted anatomical MR images using an Agilent 7 Tesla MRI. Results: Animals were maintained under anesthesia for a total of approximately 2 hours in order to acquire data from all the modalities. Data was able to be co-registered in various forms in order to verify the location and extent of disease, as well as the usefulness of 18F-FDG PET and Integrisense to serve as biomarker probes. Conclusions: Animals tolerated the length of anesthesia well. Initial difficulties were found in performing the BLI on the same day as the remaining modalities, as anesthesia administration to animals inside the FMT cassette while acquiring BLI perturbed animal positioning. When BLI was acquired the day prior to multimodality imaging, the reliability of positioning was increased, permitting more facile fusion of the various modalities. The feasibility of this imaging was only made possible through the use of the AHS bed and dock that permitted reproducible imaging across the different platforms. In order to further optimize workflows, multiple AHS bed systems should be employed so that data can be acquired on the various modalities in parallel, instead of sequentially, thereby greatly increasing animal throughputs. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B142. Citation Format: John L. Chunta, Deanne Lister, Chris Bull, Deepa Balagurunathan, Erin Trachet, Chris Chiodo, Scott Wise, Wilbur R. Leopold, Patrick McConville. Evaluating the feasibility and throughput of quintuple modality imaging in disseminated models of cancer with PET, CT, MRI, bioluminescence, and fluorescence molecular tomography. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B142.

Multiple myeloma is a cancer of the plasma cells that is characterized by multiple localized lesions in the marrow, particularly of the spine, skull, and pelvis, although soft tissue lesions also occur. It is the second most common blood cancer, affecting approximately 45,000 people in the US. Most preclinical modeling of myeloma employs SC xenografts that mimic the less common plasmacytoma form of the disease. Systemic (IV) implants are also used, but studies typically are based on a single survival endpoint, limiting knowledge about the progression and response of the disease under treatment. In order to more quantitatively monitor disseminated disease progression and response to treatment, we have characterized two human (JJN3 and MM1S) and one murine (5TGM1) myeloma models that have been modified to express luciferase. All models were characterized by 100% tumor take rate and focal dissemination of the disease to the spine and skull that mimic clinical experience. These models showed individual and reproducible patterns of spread to other sites, and differed in their sensitivities to standards of care. Analysis of tumor doubling times, tumor titrations, luminescence-based growth delay, and survival all indicated that the bioluminescence signal was a reliable quantitative indicator of viable tumor burden, even under treatment with clinical standard of care agents. Luciferase labeling and the tight correlation between luminescence signal and viable tumor burden raises the possibility of differential real-time tracking of tumor progression and response at individual sites. Citation Format: Meridith Baugher, Jenifer Baranski, Deepa Balagurunathan, Christopher Bull, Noah Winchell, Darren Shaw, Erin Trachet, Deanne Lister, Patrick McConville, Wilbur R. Leopold. Characterization of imaging-enhanced models of disseminated multiple myeloma. [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 3859. doi:10.1158/1538-7445.AM2013-3859

The specificity of protein kinases has been shown to be influenced by residues near the phosphoaccepting amino acid. To examine the determinants for platelet-derived growth factor receptor (PDGFR) tyrosine kinase specificity, a peptide library with three degenerate positions N-terminal to tyrosine was constructed. After reaction with PDGFR, the most abundant phosphopeptides were isolated by immunoaffinity chromatography on a column containing monoclonal anti-phosphotyrosine antibody. Further separation of bound phosphopeptides with reverse-phase HPLC led to the identification of three optimal substrates for PDGFR: Ala-Ala-Asn-Ile-Thr-Tyr-Ala-Ala-Arg-Arg-Gly, Ala-Ala-Asn-Arg-Thr-Tyr-Ala-Ala-Arg-Arg-Gly and Ala-Ala-Leu-Ile-Thr-Tyr-Ala-Ala-Arg-Arg-Gly, where underlined residues are in the degenerate positions of the peptide library. Kinetic analyses of the three individual peptides (synthesized separately) showed these peptides to be among the best reported substrates for PDGFR. Our results expand the range of amino acid residues that have been shown to serve as recognition elements for receptor tyrosine kinases.

Treatment with ionizing radiation is a major modality for the treatment of cancer. Between 50 and 60 percent of all cancer patients receive treatment with ionizing radiation at some time during the course of their disease (1). Thus, approximately 600,000 new patients will receive radiotherapy this year in the United States alone. Despite the proven efficacy of radiotherapy in the treatment of certain tumor types, nearly one half of the patients treated with radiotherapy will die with at least microscopic recurrence of tumor at the irradiated site (2).

Purpose: ALB-109564 is a novel semi-synthetic Vinca alkaloid which is currently in a Phase I clinical trial. The antitumor activity of ALB-109564 was compared to vinorelbine in human tumor xenograft studies when dosed ip and iv. Methods: The maximum tolerated dose (MTD) of ALB-109564 and vinorelbine was determined in athymic nude mice using both ip and iv dosing on q4dx4 and q7dx4 schedules. Athymic nude mice (n = 10) were implanted sc with a number of human tumor cell lines. Following establishment of measurable tumors, mice received equivalent therapeutic doses, based on their MTDs, of vinorelbine and ALB-109564. Vinorelbine and ALB-109564 were compared by dosing ip (q4dx4) in a panel of five xenografts (H460 NSCLC, Colo205 colon cancer, PC3 prostate cancer, H69 SCLC, and MX-1 breast cancer). Additionally, a comparison of ALB-109564 and vinorelbine was run dosing iv on a q7dx4 schedule in two xenograft models (PC3 and H460). Results: It was discovered that ALB-109564 and vinorelbine were better tolerated when dosed iv rather than ip, allowing higher dose levels of both agents. For example, the MTD for ALB-109564 using the q4dx4 schedule was 6 mpk ip compared with 14 mpk iv. ALB-109564 demonstrated antitumor activity superior to vinorelbine when dosed at their respective MTDs ip. Statistically significant tumor growth delays (TGDs) were observed with ALB-109564 in four of the five xenografts studied while vinorelbine did not significantly delay tumor growth in any of the models when dosing ip. A comparison of the two agents dosed iv in the PC3 and H460 xenograft models showed that both agents induced significant TGDs in both models with ALB-109564 showing superior efficacy in the H460 study (35.7 days TGD vs. 15.7 days at respective MTDs). Conclusions: Preclinical evaluation of tubulin inhibitor ALB-109564 (12′-methylthiovinblastine dihydrochloride) demonstrated in vivo oncolytic activity against several human tumor cell types in xenograft models greater than that seen with vinorelbine. These results demonstrate that minor structural modifications of vinca alkaloids can have a large impact on activity. Based on these results a Phase I clinical study with ALB-109564 was initiated in August 2008 and is ongoing through dose-escalation. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C232.

A series of compounds related to alpha-(1-aziridinylmethyl)-2-nitro-1H-imidazole-1-ethanol (RSU 1069, 1) were synthesized and evaluated as selective hypoxic cell cytotoxic agents and as radiosensitizers. The aziridine moiety was replaced with a number of other potential alkylating groups including cycloalkylaziridines and azetidines. The data indicated that modification of the aziridine of 1 resulted in a substantial decrease in the ability of the compounds to selectively kill hypoxic cells. However, these modifications did not affect the compounds' in vitro radiosensitizing activity since many of the derivatives were as potent as 1. All of the compounds that were evaluated in vivo were less toxic than 1, and several members of this series had significant activity. The best compound was trans-alpha-(((4-bromotetrahydro-2H-pyran-3-yl) amino)methyl)-2-nitro-1H-imidazole-1-ethanol (18), which, due to its activity and log P value, is a candidate for additional in vivo studies.

1. 1. APP is activated by adenosine kinase to its 5′-phosphate (APP-MP). 2. 2. APP-MP inhibits PRPP synthetase, and depletes cellular PRPP and purine and pyrimidine nucleotides. 3. 3. APP inhibits synthesis of DNA and RNA, and blocks cells in G1 phase of the cell cycle. 4. 4. APP retains full activity against MDR cells. 5. 5. APP is equally active against quiescent and proliferating CHO cells. 6. 6. APP has only weak activity against L1210 leukemia in vivo, but has substantial activity against mammary carcinoma 16/c. 7. 7. In vitro, APP has a relatively high ratio of solid tumor: leukemia activity.

3-Deazaguanine (dezaguanine, USAN; CI-908) is a new antipurine antimetabolite which is entering Phase I studies in the USA. This compound differs from guanine only in the substitution of a carbon for the 3-nitrogen of guanine. Dezaguanine has an unusual spectrum of activity against experimental rodent tumors; its activity against transplantable rodent leukemias is only modest, but it has significant activity against transplantable rodent solid tumors, particularly mammary adenocarcinomas. Mammary adenocarcinoma models against which this compound is active include slow and fast-growing tumors, hormone sensitive and hormone insensitive tumors, and the subrenal capsule implanted human breast cancer xenograft, MX-1. Dezaguanine must be converted to its nucleotides to be active. Dezaguanine nucleotides inhibit synthesis of guanine nucleotides, and can be incorporated into nucleic acids in place of guanine nucleotides; incorporation into DNA may be particularly important in the cytotoxicity of this compound. Addition of certain purines or purine nucleosides can prevent dezaguanine cytotoxicity in vitro. Preclinical studies suggest that dezaguanine does not undergo deamination to 3-deazaxanthine, and is not metabolized by xanthine oxidase. Therefore, this compound may not be subject to metabolic inactivation in vivo, and active metabolites may have a prolonged half-life. This concept is supported by the prolonged half-life of radiolabelled dezaguanine in rats. Finally, dezaguanine can cross the blood-brain barrier. In summary, the novel biochemical and experimental antitumor properties of dezaguanine indicate that this compound could have better activity against some human solid tumors than currently used purine antimetabolites.(ABSTRACT TRUNCATED AT 250 WORDS)

The antibiotic acetomycin was active in vitro against HCT-8 human colon adenocarcinoma cells (IC50, 1.5 microgram/ml) and L1210 murine leukemia cells (IC50, 2.2 micrograms/ml). Acetomycin also had marked activity in the human tumor stem cell assay, with a 33% overall response rate (less than or equal to 30% survival) against 49 primary tumors. However, acetomycin was inactive in four in vivo tumor assay systems (L1210 and P388 leukemias, B16 melanoma and the MX-1 mammary xenograft system). This lack of in vivo activity may result from metabolic inactivation of acetomycin.

1. 1. CI-920 is a structurally novel, phosphate-containing polyene lactone antitumor agent isolated from a previously undescribed subspecies of Streptomyces pulveraceus cultured from a Brazilian soil sample. 2. 2. CI-920 was active against murine leukemia P388, and highly active and curative against L1210 leukemia in vivo. CI-920 was less active or inactive against the murine solid tumors tested. 3. 3. Daily administration for five to nine days was more effective against L1210 leukemia than a single dose or doses every four days. Given three times daily for five days, CI-920 was more toxic and less active. CI-920 had similar activity intravenously and intraperitoneally. Oral administration was inactive and nontoxic. Subcutaneous treatment was less effective and more toxic. 4. 4. Structure-activity relationship studies showed that the phosphate group was essential for antitumor activity in vivo and in vitro. Hydrolyzing the lactone ring also resulted in loss of antitumor activity, as did acetylation of the 6-hydroxyl group. Hydroxylation at the 5-position of the lactone ring resulted in partial retention of antitumor activity, but in greater toxicity to mice. Removal of the 13-hydroxyl group resulted in retention of high antitumor activity with approximately three-fold improvement in dose-potency. 5. 5. CI-920 is not cytotoxic to prokaryotic cells. 6. 6. CI-920 causes inhibition of biosynthesis of RNA and DNA in intact L1210 cells. Protein synthesis is also inhibited at higher drug concentrations. The inhibition of nucleic acid synthesis is not an antimetabolite effect, since pools of ribonucleoside triphosphates and deoxyribonucleoside triphosphates are not depleted. 7. 7. CI-920 does not cause DNA strand breakage, as measured by alkaline elution, and is not mutagenic in the Ames test at concentrations up to 200 μg/ml. 8. 8. CI-920 does not cause direct inhibition of RNA polymerase or DNA polymerase in permeabilized cells. It is possible that CI-920 must be metabolically activated within the target cells; alternatively it may interact with a component of chromatin other than DNA or the polymerases. 9. 9. Flow cytometry studies showed that growth-inhibitory levels of CI-920 caused accumulation of cells in the G2+M region. Higher drug concentrations caused an S-phase block. 10. 10. CI-920 is an inhibitor and irreversible inactivator of reduced folate membrane transport, and appears to enter cells by this receptor. L1210 cells selected for resistance to CI-920 are cross-resistant to methotrexate, and deficient in reduced folate transport. The requirement of CI-920 for carrier-mediated transport may explain its limited antitumor spectrum, since it is inactive against cells with low levels of the reduced folate receptor. 11. 11. CI-920 is highly active in the human tumor stem cell colony assay, particularly in tumor types that are sensitive to methotrexate. 12. 12. CI-920 is highly water-soluble. When formulated with four equivalents of sodium ascorbate, under nitrogen, CI-920 has satisfactory stability. 13. 13. Based upon its novel structure, unusual biochemical mechanism, and transport properties, its experimental antileukemic activity and activity in the human tumor clonogenic assay, it is planned to initiate clinical trials of CI-920.

Chromophore modification of the anthracenediones related to mitoxantrone in an attempt to provide agents with diminished or no cardiotoxicity has resulted in a novel class of DNA binders, the anthrapyrazoles. Their synthesis was carried out by a two-stage condensation sequence starting from requisite 1,4- or 1,5-dichloro-9,10-anthracenedione precursors. Reaction with a monoalkylhydrazine gave a chloroanthrapyrazole intermediate whose subsequent condensation with primary or secondary alkylamines provided the target "two-armed" anthrapyrazoles. A-ring 7,10-dihydroxy anthrapyrazoles were derived from amine condensation with intermediate 5-chloro-7,10-dihydroxyanthrapyrazoles or, alternatively, from intermediate 5-chloro-7,10-bis(benzyloxy)anthrapyrazoles followed by hydrogenolysis of the benzyl protecting groups to provide the target compounds. Potent in vitro activity was demonstrated against murine L1210 leukemia in vitro (IC50 = 10(-7)-10(-8) M) as well as against P388 leukemia in vivo over a wide range of structural variants. In general, activity against the P388 line was maximized by basic side chains at N-2 and C-5, two to three carbon spacers between proximal and distal nitrogens of the side chain, and A-ring hydroxylation. Besides having curative activity against the P388 line, the more active compounds were curative against murine B-16 melanoma in vivo. On the basis of their exceptional in vivo anticancer activity, A-ring dihydroxy compounds 71 and 74 reported in this study have been selected for development toward clinical trials.

A complex of novel and exceptionally potent antibiotics has been evaluated for antitumor activity in vitro and in vivo and characterized with regard to their ability to cause DNA strand scission. The major component, PD 114,759, was quite active against all in vitro tumor systems including the human tumors, MCF-7 breast, HCT-8 colon, and A549 lung and the murine tumors M16/c mammary, Lewis lung, Pan 02 pancreas and L1210 leukemia. ID50 values ranged from 2–57 pg/ml. In vivo this agent produced significant increases of host life spans in mice bearing L1210 leukemia, B16 melanoma and the M5076 sarcoma. Further, it inhibited growth of subcutaneous implants of the Ridgway osteogenic sarcoma by 80% and growth of the MX-1 human mammary xenograft by 90–95%. PD 114, 759, however, had no activity against the colon adenocarcinoma 11a or mammary adenocarcinoma 16c. Chinese hamster ovary cells exposed for 24 hours to concentrations of PD 114,759 ranging from 18 to 37 pg/ml accumulated in the S and G2 + M phases of the cell cycle with a corresponding decrease in G1. Higher concentrations of drug apparently stopped any progression through the cell cycle. PD 114,759 caused significant DNA single strand breaks in L1210 cells exposed for 1 hour to drug concentrations as low as 20 pg/ml and the frequency of these lesions increased in proportion to the drug concentration. A portion of these DNA breaks appeared to be associated with protein. In contrast, no double strand DNA breaks were detected at the highest drug concentration tested (100 pg/ml).

Reaction of the nitrothioxanthen-9-ones (I) with the aminoalkylhydrazine (II) gives the 5-nitrobenzothiopyranoindazoles (III) which are reduced, forming the corresponding amino compounds (IV).

Proline analogues such as cis-4-hydroxy-L-proline (CHP) and L-azetidine-2-carboxylic acid (A2C) were tested for their antitumor activity in tissue culture and in vivo. In culture, CHP specifically inhibited those tumor cells that synthesized basement-membrane collagen. CHP appeared to selectively inhibit collagen biosynthesis with only a slight effect on protein synthesis. Culturing cells on type IV collagen matrix did not alter the antiproliferative effect of CHP. The inhibition of 450.1 mouse mammary tumor cells was fully reversible when cultures were incubated for 6 or 12 hours with 25 micrograms CHP/ml but was irreversible after 24 hours of exposure. Of the proline analogues tested against 450.1 tumor cells, A2C and CHP were the most potent inhibitors of cell growth. These two compounds were therefore tested in vivo using 3 transplantable tumors, all of which synthesized basement-membrane collagen. CHP and A2C were given twice daily to mice for 7 to 10 days at doses ranging from 50 mg/kg (body wt) to 600 mg/kg (body wt) per injection. Both CHP and A2C were completely inactive against the 450.1 mammary tumor and the EHS sarcoma. Both compounds also caused considerable liver toxicity. Against CD8F1 mammary tumors, treatment with maximum tolerated doses of CHP and A2C resulted in a slight but insignificant inhibition of tumor growth. While our studies confirmed previous findings that CHP specifically inhibited those tumor cells that synthesized basement-membrane collagen, CHP and A2C did not appear to be efficacious antitumor agents.

Trimetrexate is a novel lipophilic folate antagonist that causes growth inhibition, inhibition of nucleic acid biosynthesis, and cytotoxicity at nanomolar concentrations in tissue cultures. The potency of trimetrexate cytotoxicity against most cell lines is greater than that of methotrexate. Trimetrexate has antitumor activity in vivo in several murine leukemia and solid tumor systems, including tumors in which methotrexate is inactive. Antitumor activity was seen following oral, intravenous, or intraperitoneal administration. Trimetrexate causes a pronounced and early depression in incorporation of deoxyuridine into DNA. In tumor cell lines resistant to methotrexate because of a drug transport defect, trimetrexate retains activity. In many such cases the methotrexate-resistant tumors show collateral sensitivity to trimetrexate. In methotrexate-resistant cells with impaired drug transport, trimetrexate sensitivity was even more pronounced when cells were grown in folate-free medium supplemented with physiological levels of tetrahydrofolate cofactor. In the human tumor stem cell colony assay, trimetrexate, at concentrations achievable in vivo, gave activity against many human tumors, including samples that were unresponsive to methotrexate. Trimetrexate crosses the blood-brain barrier, and at very high doses may cause neurotoxicity. At conventional doses the primary toxic effects in mice are gastrointestinal. This toxicity is reversible at therapeutic doses. Unlike earlier lipophilic antifolates, trimetrexate has rapid plasma clearance (t1/2 in mice of 45 minutes). Trimetrexate is a tight-binding competitive inhibitor of dihydrofolate reductase. The Ki,slope for inhibition of the human enzyme was 4 X 10(-11) M. A dose-dependent decrease in cellular purine ribonucleotide pools is given by trimetrexate. Pyrimidine ribonucleotide pools tend to increase in treated cells. Trimetrexate caused a marked depression of cellular pools of dTTP and dGTP, and a lesser depression in dATP. Cytotoxicity of trimetrexate in vitro was prevented by leucovorin. Leucovorin also protected mice from trimetrexate toxicity. Thymidine protected cells from lethal effects of low concentrations of trimetrexate, but not from high concentrations. The combination of thymidine and hypoxanthine completely protected cells from low and high concentrations of trimetrexate. A new, stable and highly water-soluble formulation of trimetrexate has been developed. Because of the interesting biochemical and pharmacological properties of trimetrexate, and its experimental antitumor activity, clinical trials are planned.

2-β- D -Ribofuranosyl-4-selenazolecarboxamide (selenazofurin, CI-935), the selenium analog of tiazofurin (CI-909), was 3- to 10-fold more cytotoxic to murine or human tumor cells in vitro than tiazofurin and was also more active against P388 mouse leukemia in vivo. In vitro cytotoxicity could be reversed by guanosine or guanine but not by other purine nucleosides or bases. Three human tumor cell lines selected for selenazofurin or tiazofurin resistance showed crossresistance between selenazofurin and tiazofurin. Treatment with tiazofurin, selenazofurin, or mycophenolic acid decreased guanylate pools and caused an accumulation of IMP in WIL2 human lymphoma cells. The decrease in guanylate pools was accompanied by inhibition of RNA and DNA synthesis. The NAD analogs of tiazofurin and selenazofurin were inhibitors of L1210 IMP dehydrogenase (IMP:NAD oxidoreductase, EC 1.2.1.14), and both showed uncompetitive inhibition with respect to NAD having Kii values of 5.7 × 10−8M and 3.3 × 10−8M respectively.