Adaphostin and other anticancer drugs quench the fluorescence of mitochondrial potential probes.

Fluorescent dyes are widely used to monitor changes in mitochondrial transmembrane potential (DeltaPsim). When MitoTracker Red CMXRos, tetramethylrhodamine methyl ester (TMRM), and 3,3'dihexyloxacarbocyanine iodide (DiOC6(3)) were utilized to examine the effects of the experimental anticancer drug adaphostin on intact cells or isolated mitochondria, decreased fluorescence was observed. In contrast, measurement of tetraphenylphosphonium uptake by the mitochondria using an ion-selective microelectrode failed to show any effect of adaphostin on DeltaPsim. Instead, further experiments demonstrated that adaphostin quenches the fluorescence of the mitochondrial dyes. Structure-activity analysis revealed that the adamantyl and p-aminobenzoic acid moieties of adaphostin are critical for this quenching. Anticancer drugs containing comparable structural motifs, including mitoxantrone, aminoflavone, and amsacrine, also quenched the mitochondrial probes. These results indicate the need for caution when mitochondrial dyes are utilized to examine the effects of xenobiotics on DeltaPsim and suggest that some previously reported direct effects of anticancer drugs on mitochondria might need re-evaluation.

The hollow fibre model in cancer drug screening: the NCI experience.

The in vivo hollow fibre model was developed by the National Cancer Institute (NCI) in the United States of America (USA) at a time when the number of potential anti-cancer drugs arising from in vitro screening efforts exceeded the available capacity for testing in traditional xenograft models. Updated analysis of the predictive value of the hollow fibre model continues to indicate that the greater the response in the hollow fibre assay, the more likely it is that activity will be seen in subsequent xenograft models. The original 12 cell line hollow fibre panel has been supplemented with histology-specific panels, and we begin here to analyse their utility in predicting activity in subsequent in vivo models. The key goal of using the hollow fibre model as a way to decrease the cost, both financial and in the number of animals used, to evaluate initial evidence of a compound's capacity to act across physiological barriers continues to be reinforced with our enlarging experience.

A potential role for imaging technology in anticancer efficacy evaluations.

The introduction of imaging methods suitable for rodents offers opportunities for new anticancer efficacy models. Traditional models do not provide the level of sensitivity afforded by these precise and quantitative techniques. Bioluminescent endpoints, now feasible because of sensitive charge-coupled device cameras, can be non-invasively detected in live animals. Currently, the most common luminescence endpoint is firefly luciferase, which, in the presence of O(2) and ATP, catalyses the cleavage of the substrate luciferin and results in the emission of a photon of light. In vivo implantation of tumour cells transfected with the luciferase gene allows sequential monitoring of tumour growth within the viscera by measuring these photon signals. Furthermore, tumour cell lines containing the luciferase gene transcribed from an inducible promoter offer opportunities to study molecular-target modulation without the need for ex vivo evaluations of serial tumour samples. In conjunction with this, transgenic mice bearing a luciferase reporter mechanism can be used to monitor the tumour microenvironment as well as to signal when transforming events occur. This technology has the potential to reshape the efficacy evaluations and drug-testing algorithms of the future.

Sensitivity of renal cell carcinoma to aminoflavone: role of CYP1A1.

PURPOSE: The aminoflavone analogue (AF) exhibits antitumor activity in vitro, particularly against neoplastic cells of renal origin. We identified cellular correlates of responsiveness to AF in continuous human tumor renal cell carcinoma lines and in tumor cell isolates, termed renal carcinoma cell strains, from patients with clear cell and papillary renal neoplasms. MATERIALS AND METHODS: In vitro antiproliferative activity of AF was evaluated using the sulforhodamine B protein dye assay. In vivo antitumor activity of the drug was determined in mice bearing xenografts. Covalent binding of AF/metabolite(s) was assessed following exposure of cells to AF for 16 hours. CYP1A1 and CYP1B1 mRNA and apoptosis were quantitated by reverse transcriptase-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. RESULTS: AF produced total growth inhibition in vitro in 3 of 6 human tumor renal cell lines at concentrations of 90 to 400 nM. In vivo treatment of mice bearing xenografts of the Caki-1 renal cell carcinoma, sensitive to AF in vitro, resulted in significant antitumor activity, including tumor-free survivors. Studies in 13 renal cell strains isolated from patients with clear cell (9) or papillary (4) renal cell carcinoma indicated that 3 of 4 papillary strains were sensitive to AF compared with 2 of 9 clear cell strains. AF sensitive renal cell lines and strains exhibited induction of CYP1A1 and CYP1B1 gene expression, increased covalent binding of AF metabolite(s) and apoptosis. CONCLUSIONS: AF has noteworthy antitumor activity against certain human tumor renal cell lines in vitro and in vivo, which correlates with drug metabolism to covalently binding metabolites after CYP1A1 and CYP1B1 gene expression. We hypothesize that it leads to apoptosis induction. AF sensitive renal cell strains are predominantly of the papillary histological type. These results are limited by the small numbers of cell lines and cell strains but they are suggestive of the need for further testing in larger collections of cell strains.

Target selection issues in drug discovery and development.

Targets for cancer treatments have been proposed which address oncogenes mutated in tumors (pathogenic targets). Yet additional opportunities may come from molecules expressed in the tissue of origin (ontogenic); allow drug handling (pharmacological) or address the tumor microenvironment. The key is to define an assay that defines a therapeutic index which can be achieved in modulated target function.

Geldanamycin analogs.

Benzoquinoid ansamycins can downregulate molecules which are chaperoned by heat shock protein (Hsp90), including numerous tyrosine kinases, steroid receptors, and cell cycle regulatory kinases. 17allylaminol7demethoxygeldanamycin (17AAG) has entered the clinic, but 17dimethyl-aminoethylaminogeldanamycin (17DMA) has emerged as an analog with potential preferential pharmaceutical features.

DNA damage and cell cycle arrest induced by 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) is attenuated in aryl hydrocarbon receptor deficient MCF-7 cells.

The fluorinated benzothiazole analogue 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) is a novel agent with potent and selective antitumour properties and, in the form of its L-lysylamide prodrug Phortress (NSC 710305), is a current candidate for early phase clinical studies. Previous findings have indicated that cytochrome P450 1A1 (CYP1A1) may play a role in the antitumour activity of molecules in the benzothiazole series including the nonfluorinated parent compound 2-(4-amino-3-methylphenyl)benzothiazole (DF 203, NSC 674495) (Kashiyama et al, 1999; Chua et al, 2000; Loaiza-Pérez et al, 2002). In this study, we assessed and verified that a fully functional aryl hydrocarbon receptor (AhR) signalling pathway is a necessary requisite for the induction of efficient cytotoxicity by 5F 203 in MCF-7 wild-type sensitive cells. Drug exposure caused MCF-7 sensitive cells to arrest in G(1) and S phase, and induced DNA adduct formation, in contrast to AhR-deficient AH(R100) variant MCF-7 cells. In sensitive MCF-7 cells, induction of CYP1A1 and CYP1B1 transcription (measured by luciferase reporter assay and real-time reverse transcriptase-polymerase chain reaction (RT-PCR)), and 7-ethoxyresorufin-O-deethylase (EROD) activity was demonstrated, following treatment with 5F 203. In contrast, in resistant AH(R100) cells, drug treatment did not affect CYP1A1 and CYP1B1 transcription and EROD activity. Furthermore, AH(R100) cells failed to produce either protein/DNA complexes on the xenobiotic responsive element (XRE) sequence of CYP1A1 promoter (measured by electrophoretic mobility shift assay) or DNA adducts. The data confirm that activation of the AhR signalling pathway is an important feature of the antitumour activity of 5F 203.

Developmental therapeutics program at the NCI: molecular target and drug discovery process.

As the drug discovery and developmental arm of the National Cancer Institute (NCI), the Developmental Therapeutics Program (DTP) plans, conducts and facilitates development of therapeutic agents for cancer and AIDS. DTP's goal is to turn 'molecules into medicine for the public health'. Areas of support by DTP are discovery, development and pathways to development for the intramural and the extramural community. The Developmental Therapeutics Program (DTP) operates a repository of synthetic and pure natural products, which are evaluated as potential anticancer agents. The repository derives from a historical database of greater than 600 000 compounds, which have been supplied to DTP from a variety of sources worldwide. The in vitro anti-cancer drug cell line screen established at DTP is unique in several respects. It has changed the NCI emphasis from a compound-oriented drug discovery effort to a disease-panel oriented exercise, emphasized human tumor cells derived from solid tumors, developed a high volume screening method that can adapt to processing of numerous chemical agents or natural source-derived extracts, that has minimized the use of animals, and saved on the amount of material required for the initial screening. The hollow fiber assay created at the DTP has demonstrated the ability to provide quantitative initial indices of in vivo drug efficacy, with minimum expenditures of time and materials and is currently being utilized as the initial in vivo experience for agents found to have reproducible activity in the in vitroanticancer drug screen. Drugs showing activity with unique mechanisms of actions are being further developed for treatment of hematopoietic neoplasms, prominent examples being flavopiridol, UCN-01 and depsipeptide among others.

In vitro evaluation of amino acid prodrugs of novel antitumour 2-(4-amino-3-methylphenyl)benzothiazoles.

Novel 2-(4-aminophenyl)benzothiazoles possess highly selective, potent antitumour properties in vitro and in vivo. They induce and are biotransformed by cytochrome P450 (CYP) 1A1 to putative active as well as inactive metabolites. Metabolic inactivation of the molecule has been thwarted by isosteric replacement of hydrogen with fluorine atoms at positions around the benzothiazole nucleus. The lipophilicity of these compounds presents limitations for drug formulation and bioavailability. To overcome this problem, water soluble prodrugs have been synthesised by conjugation of alanyl- and lysyl-amide hydrochloride salts to the exocyclic primary amine function of 2-(4-aminophenyl)benzothiazoles. The prodrugs retain selectivity with significant in vitro growth inhibitory potency against the same sensitive cell lines as their parent amine, but are inactive against cell lines inherently resistant to 2-(4-aminophenyl)benzothiazoles. Alanyl and lysyl prodrugs rapidly and quantitatively revert to their parent amine in sensitive and insensitive cell lines in vitro. Liberated parent compounds are sequestered and metabolised by sensitive cells only; similarly, CYP1A1 activity and protein expression are selectively induced in sensitive carcinoma cells. Amino acid prodrugs meet the criteria of aqueous solubility, chemical stability and quantitative reversion to parent molecule, and thus are suitable for in vivo preclinical evaluation.

Selected novel anticancer treatments targeting cell signaling proteins.

Empirical approaches to discovery of anticancer drugs and cancer treatment have made limited progress in the cure of cancer in the last several decades. Recent advances in technology and expanded knowledge of the molecular basis of tumorigenesis and metastasis have provided unique opportunities to design novel compounds that rationally target the abnormal molecular and biochemical signals leading to cancer. Several such novel agents have completed advanced stages in clinical development. The excellent clinical results achieved by some of these compounds are creating new paradigms in management of patients with neoplastic diseases. Clinical development of these agents also raises challenges to the traditional methods of drug evaluation and measurement of efficacy.

Genomic-scale measurement of mRNA turnover and the mechanisms of action of the anti-cancer drug flavopiridol.

BACKGROUND: Flavopiridol, a flavonoid currently in cancer clinical trials, inhibits cyclin-dependent kinases (CDKs) by competitively blocking their ATP-binding pocket. However, the mechanism of action of flavopiridol as an anti-cancer agent has not been fully elucidated. RESULTS: Using DNA microarrays, we found that flavopiridol inhibited gene expression broadly, in contrast to two other CDK inhibitors, roscovitine and 9-nitropaullone. The gene expression profile of flavopiridol closely resembled the profiles of two transcription inhibitors, actinomycin D and 5,6-dichloro-1-beta-D-ribofuranosyl-benzimidazole (DRB), suggesting that flavopiridol inhibits transcription globally. We were therefore able to use flavopiridol to measure mRNA turnover rates comprehensively and we found that different functional classes of genes had distinct distributions of mRNA turnover rates. In particular, genes encoding apoptosis regulators frequently had very short half-lives, as did several genes encoding key cell-cycle regulators. Strikingly, genes that were transcriptionally inducible were disproportionately represented in the class of genes with rapid mRNA turnover. CONCLUSIONS: The present genomic-scale measurement of mRNA turnover uncovered a regulatory logic that links gene function with mRNA half-life. The observation that transcriptionally inducible genes often have short mRNA half-lives demonstrates that cells have a coordinated strategy to rapidly modulate the mRNA levels of these genes. In addition, the present results suggest that flavopiridol may be more effective against types of cancer that are highly dependent on genes with unstable mRNAs.

N-(2-hydroxypropyl)methacrylamide copolymer-6-(3-aminopropyl)-ellipticine conjugates. Synthesis, in vitro, and preliminary in vivo evaluation.

Ellipticine derivatives have potential as anticancer drugs. Their clinical use has been limited, however, by poor solubility and host toxicity. As N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-anticancer conjugates are showing promise in early clinical trials, a series of novel HPMA copolymer conjugates have been prepared containing the 6-(3-aminopropyl)-ellipticine derivative (APE, NSC176328). Drug was linked to the polymer via GFLG or GG peptide side chains. To optimize biological behavior, HPMA copolymer-GFLG-APE conjugates with different drug loading (total APE: 2.3-7% w/w; free APE: <0.1% w/w) were synthesized. Conjugation of APE to HPMA copolymers considerably increased its aqueous solubility (>10-fold). HPMA copolymer-GG-APE did not liberate drug in the presence of isolated lysosomal enzymes (tritosomes), but HPMA copolymer-GFLG-APE released APE to a maximum of 60% after 5 h. The rate of drug release was influenced by drug loading; lower loading led to greater release. Whereas free APE (35 microg/mL) caused significant hemolysis (50% after 1 h), HPMA copolymer-APE conjugates were not hemolytic up to 300 microg/mL (APE-equiv). As would be expected from its cellular pharmacokinetics, HPMA copolymer-GFLG-APE was >75 times less cytotoxic than free drug (IC(50) approximately 0.4 microg/mL) against B16F10 melanoma in vitro. However, in vivo when tested in mice bearing s.c. B16F10 melanoma, HPMA copolymer-GFLG-APE (1-10 mg/kg single dose, APE-equiv) given i.p. was somewhat more active (highest T/C value of 143%) than free APE (1 mg/kg) (T/C =127%). HPMA copolymer-APE conjugates warrant further evaluation as potential anticancer agents.

Early development of cyclin dependent kinase modulators.

The protein kinase family presents remarkable opportunities for drug discovery and development targeting mainly to the ATP binding cleft. Cyclin-dependent kinases CDKs control the cell division in by controlling its sub phases. The regulation of CDKs is altered in a number of tumor types, and therefore CDKs are a particularly attractive target group of kinases with reference to proliferative disorders including cancer, but also extending to graft stenosis, and autoimmune disorders. Screening of chemical modulators of CDKs that modulate aberrant CDK activity might be beneficial for cancer therapy by directly inhibiting kinase activity, or influencing cell cycle "checkpoint" function, which is mediated through effects of exogenous cellular regulators of CDK activity. In this regard small molecule modulators such as flavopiridol and UCN-01 are in early clinical trials. Other more selective modulators of CDK function are being actively sought, and initial results with flavopiridol analogs, indirubins, paullones, and purine-based inhibitors will be considered.

Quantitative determination of perifosine, a novel alkylphosphocholine anticancer agent, in human plasma by reversed-phase liquid chromatography-electrospray mass spectrometry.

A sensitive and selective reversed-phase LC-ESI-MS method to quantitate perifosine in human plasma was developed and validated. Sample preparation utilized simple acetonitrile precipitation without an evaporation step. With a Develosil UG-30 column (10 x 4 mm I.D.), perifosine and the internal standard hexadecylphosphocholine were baseline separated at retention times of 2.2 and 1.1 min, respectively. The mobile phase consisted of eluent A, 95% 9 mM ammonium formate (pH 8) in acetonitrile-eluent B, 95% acetonitrile in 9 mM ammonium formate (pH 8) (A-B, 40:60, v/v), and the flow-rate was 0.5 ml/min. The detection utilized selected ion monitoring in the positive-mode at m/z 462.4 and 408.4 for the protonated molecular ions of perifosine and the internal standard, respectively. The lower limit of quantitation of perifosine was 4 ng/ml in human plasma, and good linearity was observed in the 4-2,000 ng/ml range fitted by linear regression with 1/x weight. The total LC-MS run time was 5 min. The validated LC-MS assay was applied to measure perifosine plasma concentrations from patients enrolled on a phase I clinical trial for pharmacokinetic/pharmacodynamic analyses.

Biochemical correlates of mTOR inhibition by the rapamycin ester CCI-779 and tumor growth inhibition.

The rapamycin ester, CCI-779, potently inhibits cell growth in vitro, inhibits tumor growth in vivo, and is currently in Phase I clinical trials. To further understand the relationship between plasma systemic exposure and inhibition of the target Ser/Thr kinase, mTOR/FRAP, two assays have been developed. The first assay involves determination of the 4E suppressor protein (4E-BP1) bound to eukaryotic initiation factor 4E (eIF4E), and the second is direct Western analysis of phosphorylation of residue Thr(70) of 4E-BP1. Under normal growth conditions in vitro, rapamycin caused rapid association of 4E-BP1 with eIF4E within 1 h in Rh30 and GC(3) human tumor cells. Association was persistent up to 16 h. In mice, administration of rapamycin (5 or 20 mg/kg) caused rapid association of 4E-BP1 with eIF4E within 4 h in both human colon adenocarcinoma GC(3) and rhabdomyosarcoma Rh30 xenografts. Using phospho-specific antibody against Thr(70) of 4E-BP1, rapid and persistent dephosphorylation within 30 min of exposure to rapamycin was detected in Rh18 rhabdomyosarcoma cells. Evaluation of CCI-779 against Rh18 xenografts showed this tumor to be growth inhibited at daily dose levels of > or =8.7 mg/kg. Because immunoblotting may be more suitable for assaying tumor biopsy tissue, a "blinded" comparison between the effect of CCI-779 on Thr(70) phosphorylation and growth inhibition of human tumor xenografts was undertaken. Mice were treated daily for 5 days with CCI-779 (20 mg/kg/day) or with drug vehicle, and tumor diameters were measured. Tumors were excised 1 h after the final administration and frozen, and phospho Thr(70) was determined by Western blot analysis. The correlation coefficient for decreases in Thr(70) phosphorylation and growth inhibition was high (r(2), 0.99). The results indicate that an assay of decreases in phosphorylation of Thr(70) of 4E-BP1 may be a useful surrogate for determining the inhibition of mTOR activity in tumor specimens.

Relationships between drug activity in NCI preclinical in vitro and in vivo models and early clinical trials.

An analysis of the activity of compounds tested in pre-clinical in vivo and in vitro assays by the National Cancer Institute's Developmental Therapeutics Program was performed. For 39 agents with both xenograft data and Phase II clinical trials results available, in vivo activity in a particular histology in a tumour model did not closely correlate with activity in the same human cancer histology, casting doubt on the correspondence of the pre-clinical models to clinical results. However, for compounds with in vivo activity in at least one-third of tested xenograft models, there was correlation with ultimate activity in at least some Phase II trials. Thus, an efficient means of predicting activity in vivo models remains desirable for compounds with anti-proliferative activity in vitro. For 564 compounds tested in the hollow fibre assay which were also tested against in vivo tumour models, the likelihood of finding xenograft activity in at least one-third of the in vivo models tested rose with increasing intraperitoneal hollow fibre activity, from 8% for all compounds tested to 20% in agents with evidence of response in more than 6 intraperitoneal fibres (P< 0.0001). Intraperitoneal hollow fibre activity was also found to be a better predictor of xenograft activity than either subcutaneous hollow fibre activity or intraperitoneal plus subcutaneous activity combined. Since hollow fibre activity was a useful indicator of potential in vivo response, correlates with hollow fibre activity were examined for 2304 compounds tested in both the NCI 60 cell line in vitro cancer drug screen and hollow fibre assay. A positive correlation was found for histologic selectivity between in vitro and hollow fibre responses. The most striking correlation was between potency in the 60 cell line screen and hollow fibre activity; 56% of compounds with mean 50% growth inhibition below 10(-7.5) M were active in more than 6 intraperitoneal fibres whereas only 4% of compounds with a potency of 10(-4) M achieved the same level of hollow fibre activity (P< 0.0001). Structural parameters of the drugs analysed included compound molecular weight and hydrogen-bonding factors, both of which were found to be predictive of hollow fibre activity.