Sherman F. Stinson

Cyclin-dependent kinases: initial approaches to exploit a novel therapeutic target.

Cyclin-dependent kinases (CDKs) have been recognized as key regulators of cell cycle progression. Alteration and deregulation of CDK activity are pathogenic hallmarks of neoplasia. Therefore, inhibitors or modulators would be of interest to explore as novel therapeutic agents in cancer, as well as other hyperproliferative disorders. Flavopiridol is a semisynthetic flavonoid that emerged from an empirical screening program as a potent antiproliferative agent that mechanistic studies demonstrated to directly inhibit CDKs 1, 2, and 4 as a competitive ATP site antagonist. Initial clinical trials have shown that concentrations that inhibit cell proliferation and CDK activity in vitro can be safely achieved in humans, and additional clinical trials will establish its clinical potential. To address the need for additional chemotypes that may serve as lead structures for drugs that would not have the toxicities associated with flavopiridol, compounds with a similar pattern of cell growth inhibitory activity in the National Cancer Institutes in vitro anticancer drug screen have been recognized by the computer-assisted pattern recognition algorithm COMPARE and then screened for anti-CDK activity in a biochemical screen. The benzodiazepine derivative NSC 664704 (7,12-dihydro-indolo[3,2-d][1]benzazepin-6(5H)-one) was revealed by that approach as a moderately potent (IC50 0.4 microM) inhibitor of CDK2, which in initial experiments shows evidence of causing cell cycle redistribution in living cells. NSC 664704 is, therefore, a candidate for further structural optimization, guided in part by understanding of the ATP-binding site in CDK2. This approach represents one way of combining empirical screening information with structure-based design to derive novel candidate therapeutic agents directed against an important cellular target.

17-(allylamino)-17-demethoxygeldanamycin activity in human melanoma models

17-(Allylamino)-17-demethoxygeldanamycin (17-AAG) is a semisynthetic antitumor agent, which has entered phase I/II clinical trials. Melanoma cell lines in the NCI in vitro screen (mean GI50=84 nM) were relatively sensitive to the agent, which was therefore tested in vivo in four s.c. growing human melanoma xenografts (MEXF 276, 989, 462 and 514) in athymic mice. 17-AAG markedly inhibited tumor growth at doses of 80 (maximum tolerated dose) and 60 mg/kg/day in a qd×5 (h: 0, 6; i.p.) schedule in two of four xenograft models. Cell lines derived from the 17-AAG-sensitive MEXF 276 and -resistant MEXF 514 melanomas, MEXF 276L and 514L, were chosen to study the effects of 17-AAG on its target Hsp90 as well as the Hsp90 ‘client’ protein c-Raf-1 in vitro. Cells were exposed to drug concentrations which just cause total growth inhibition (total growth inhibition=375 nM in MEXF 276L and 10 μM in MEXF 514L). Pharmacokinetic determinations confirmed that 17-AAG concentrations producing growth inhibition invitro are readily achievable in vivo at the MTD (AUC0–∞ 1068 μM·min). Whilst 17-AAG treatment did not affect Hsp90 expression in the relatively resistant MEXF 514L cells, it caused a rapid transient decline in the markedly sensitive MEXF 276L cell line. In contrast, Hsp72 expression increased. Following Hsp90 depletion at 2–8 h in MEXF 276L cells, down-regulation of c-Raf-1 was seen starting at 16 h after drug addition. In MEXF 276 xenograft tissues treated with effective dose levels, loss of Hsp90 was seen and was associated with occurrence of apoptotic figures. The apoptotic index rose from 9% after 48 h, greater than 12% at 72 h to 45% at 10 days. These data support the hypothesis that in some melanoma models, a very good response (e.g. with tumor regressions) to 17-AAG may be associated with modulation of Hsp90 expression. The expression of this target should be followed in clinical studies with 17-AAG.

Identification of cytosolic aldehyde dehydrogenase 1 from non-small cell lung carcinomas as a flavopiridol-binding protein

The synthetic flavone flavopiridol can be cytostatic or cytotoxic to mammalian cells, depending on the concentration of the drug and the duration of exposure. It has been shown to inhibit the cyclin‐dependent kinase (CDK) family of cell cycle regulatory enzymes. However, the existence of additional potential targets for drug action remains a matter of interest to define. To identify cellular targets, flavopiridol was immobilized. CDKs, particularly CDK 4, bound weakly to immobilized flavopiridol when ATP was absent but not in its presence. Two proteins with molecular weights of 40 kDa and 120 kDa had high affinities to the immobilized flavopiridol independent of the presence of ATP. They were present in all cell lines analyzed: cervical (HeLa), prostate and non‐small cell lung carcinoma (NSCLC) cell lines. A 60‐kDa protein, which was present only in NSCLC cells and bound similarly well to immobilized flavopiridol, was identified as cytosolic aldehyde dehydrogenase class 1 (ALDH‐1). The level of this protein correlated with the resistance of NSCLC cell lines to cytotoxicity caused by 500 nM flavopiridol but not higher flavopiridol concentrations. Despite binding to ALDH‐1, there was no inhibition of dehydrogenase activity by flavopiridol concentrations as high as 20 μM and flavopiridol was not metabolized by ALDH‐1. The results suggest that high cellular levels of ALDH‐1 may reduce cytotoxicity of flavopiridol and contribute to relative resistance to the drug. This is the first report that flavopiridol binds to proteins other than CDKs.

Clinical Pharmacology of Flavopiridol following a 72-Hour Continuous Infusion

Background Flavopiridol, a novel flavone derivative, inhibits cyclin-dependent kinase-1. We initiated a Phase I trial in patients with refractory solid tumors to determine the maximum tolerated dose and characterize the adverse effect profile. Objective To characterize the clinical pharmacology of flavopiridol. Methods Serial plasma samples were collected and analyzed by HPLC using electrochemical detection. The pharmacokinetics were analyzed by noncompartmental analysis. Enterohepatic recirculation was studied by analyzing fecal samples, with an attempt to correlate cholecystokinin and post-infusional peak concentrations. The plasma protein binding was studied using equilibrium dialysis. Results Seventy-six patients were treated with flavopiridol at 13 dose levels for a total of 504 cycles of treatment. The average steady-state concentration was 26.5 and 253 nM at 4 and 122.5 mg/m2, respectively. The clearance ranged from 49.9 to 2943 mL/min, with nonlinearity at doses >50 mg/m2/d. A post-infusional increase in plasma flavopiridol concentrations was noted in a subset of patients and generally occurred between 3 and 24 hours after the end of infusion. Flavopiridol was found in fecal matter, suggesting enterohepatic recirculation. There was nonsaturable plasma protein binding of flavopiridol (fu = 6%). Conclusions The dose-limiting toxicity for the Phase I trial of flavopiridol was secretory diarrhea. We failed to identify a clear relationship between dose or concentration and diarrhea. At 50 and 78 mg/m2/d, the mean steady-state plasma concentrations were 278 and 390 nM. These concentrations were well above those noted for in vitro antiproliferative activity. Nonlinear elimination was observed at doses above 50 mg/m2/d, and postinfusional peaks appear to be related to enterohepatic recirculation.

FLUORINATED 2-(4-AMINO-3-METHYLPHENYL)BENZOTHIAZOLES INDUCE CYP1A1 EXPRESSION, BECOME METABOLIZED, AND BIND TO MACROMOLECULES IN SENSITIVE HUMAN CANCER CELLS

Fluorinated 2-(4-amino-3-methylphenyl)benzothiazoles possess potent antiproliferative activity against certain cancer cells, similar to the unfluorinated 2-(4-amino-3-methylphenyl)benzothiazole (DF 203, NSC 674495). In “sensitive” cancer cells, DF 203 is metabolized by, can induce expression of, and binds covalently to CYP1A1. Metabolism appears to be essential for its antiproliferative activity through DNA adduct formation. However, a biphasic dose-response relationship compromises its straightforward development as a chemotherapeutic agent. We investigated whether fluorinated benzothiazoles inhibit cancer cell growth without the biphasic dose-response, and whether the fluorinated benzothiazoles are also metabolized into reactive species, with binding to macromolecules in sensitive cancer cells. One fluorinated benzothiazole, 2-(4-amino-methylphenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) did exhibit potent, antiproliferative activity without a biphasic dose-response. The fluorinated benzothiazoles were also metabolized only in cells, which subsequently showed evidence of cell death. We used microsomes from genetically engineered human B-lymphoblastoid cells expressing cytochromes P450 (CYP1A1, CYP1A2, or CYP1B1) to clarify the basis for fluorinated benzothiazole metabolism. 5F 203 induced CYP1A1 and CYP1B1 mRNA expression in sensitive breast and renal cancer cells, whereas 5F 203 induced CYP1A1 mRNA but not CYP1B1 mRNA expression in sensitive ovarian cancer cells. 5F 203 did not induce CYP1A1 or CYP1B1 mRNA expression in any “resistant” cancer cells. The fluorinated benzothiazoles induced CYP1A1 protein expression exclusively in sensitive cells. [14C]5F 203 bound substantially to subcellular fractions in sensitive cells but only minimally in resistant cells. These data are concordant with the antiproliferative activity of fluorinated benzothiazoles deriving from their ability to become metabolized and bind to macromolecules within sensitive cells.

Selective toxicity of the tricyclic thiophene NSC 652287 in renal carcinoma cell lines: differential accumulation and metabolism.

The tricyclic compound 2,5-bis(5-hydroxymethyl-2-thienyl)furan (NSC 652287) has shown a highly selective pattern of differential cytotoxic activity in the tumor cell lines comprising the National Cancer Institute (NCI) Anticancer Drug Screen. The mechanism underlying the selective cytotoxicity is unknown. We hypothesized that differential sensitivity to the compound observed in several renal tumor cell lines could be the result of selective accumulation or differential metabolism of this agent. We demonstrated here that the capacity of certain renal cell lines to accumulate and retain the compound, determined by accumulation of [14C]NSC 652287-derived radioactivity and by flow cytometric determination of unlabeled compound, paralleled the sensitivity of the renal cell lines to growth inhibition by NSC 652287: A-498 > TK-10 >> ACHN approximately/= to UO-31. The ability of the cell lines to metabolize [14C]NSC 652287 to a reactive species capable of binding covalently to cellular macromolecules also directly correlated with sensitivity to the compound. Different patterns of metabolites were generated by relatively more drug-sensitive cell lines in comparison with drug-resistant cell lines. The metabolizing capacity for NSC 652287 was localized primarily to the cytosolic (S100) fraction. The rate of metabolism in the cytosolic fraction from the most sensitive renal cell line, A-498, was faster than that observed in the cytosolic fractions from the other, less sensitive cell lines. The data support the hypothesis that both selective cellular accumulation and the capacity to metabolize NSC 652287 to a reactive species by certain renal carcinoma cell types are the basis for the differential cytotoxicity of this compound class.

Anti-tumor drug candidate 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole induces single-strand breaks and DNA-protein cross-links in sensitive MCF-7 breast cancer cells

Purpose: The fluorinated benzothiazole analogue 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F 203, NSC 703786) exhibits selective and potent anticancer activity, and its lysylamide prodrug (Phortress, NSC 710305) recently entered Phase I clinical trials in the United Kingdom. Only cancer cells sensitive to the anti-proliferative effects of 5F 203 deplete this drug candidate from nutrient media. 5F 203 induces cell cycle arrest, cytochrome P450 1A1 (CYP 1A1) mRNA and protein expression, and is metabolized into reactive electrophilic species that can covalently bind to DNA and form adducts in sensitive (i.e., MCF-7) but not in resistant (i.e., MDA-MB-435) breast cancer cells. Methods: In this present study, we investigated additional anticancer effects of 5F 203 in MCF-7 cells. In addition, we sought to determine if cells deficient in the xeroderma pigmentosum D gene, a gene critical in DNA repair, would show greater sensitivity to the cytotoxic effects of 5F 203 than those complemented with XPD. Results: Alkaline Elution revealed that 5F 203 induced single-strand breaks and DNA-protein cross-links in sensitive MCF-7 cells. In contrast, we detected no double-strand breaks or protein-associated strand breaks typically associated with topoisomerase I (top1) or topoisomerase II (top2) inhibition. In addition, 5F 203 was unable to trap top1- or top2-DNA cleavage complexes in MCF-7 cells. 5F 203 induced cell cycle arrest in MCF-7 cells following DNA damage after brief exposures. Cells deficient in the nucleotide excision repair xeroderma pigmentosum group D (XPD) gene displayed sensitivity to 5F 203 while cells complemented with XPD displayed resistance to 5F 203. Conclusion: These data suggest that the anti-cancer activity of 5F 203 depends upon targets other than top1 or top2 and on the ability of this benzothiazole to form single-strand breaks and DNA-protein cross-links in cancer cells.

Respiratory pathology in rats and mice after inhalation of 1,2-dibromo-3-chloropropane or 1,2 dibromoethane for 13 weeks

Seventy F344 rats and 144 B6C3F1 mice were subdivided into seven groups. Three groups were each exposed via inhalation to 1, 5, or 25 ppm of 1,2-dibromo-3-chloropropane (DBCP) for 6 h per day, 5 days per week for 13 weeks. Three additional groups were each similarly exposed to 3, 15, or 75 ppm of 1,2-Dibromoethane (EDB). The remaining group was exposed to room air under the same conditions. At 13 weeks, rats and mice showed severe necrosis and atrophy of the olfactory epithelium in the nasal cavity after inhalation of 5 or 25 ppm DBCP and 75 ppm EDB. Lower concentrations induced squamous cell metaplasia, hyperplasia and cytomegaly of the epithelium of the respiratory nasal turbinals. Squamous metaplasia, hyperplasia and cytomegaly of the epithelium was also seen in larynx, trachea, bronchi and bronchioles. Other compound related toxic lesions in rats were seen in the liver, kidney and testes.

Characteristics of proliferative lesions in the nasal cavities of mice following chronic inhalation of 1,2-dibromoethane.

Groups of 50 male and 50 female B6C3F1 mice inhaled 10 or 40 ppm 1,2-dibromoethane 6 h/day, 5 days/week, for 103 (10 ppm) or 90 (40 ppm) weeks. Focal epithelial hyperplasia was found in 1 (low dose group) and 10 (high dose group) males and 3 (low dose group) and 11 (high dose group) females. Squamous papillomas or adenomas were seen in 3 males and 7 females in the high dose groups. Squamous, adeno-, or mixed carcinomas were present in 7 females in the high dose group. One poorly differentiated sarcoma and 2 hemangiosarcomas were observed in females in the low and high dose groups, respectively.

Determination of flavopiridol (L86 8275; NSC 649890) in human plasma by reversed-phase liquid chromatography with electrochemical detection

Purpose: Flavopiridol is a flavone which inhibits several cyclin-dependent kinases, and exhibits potent growth-inhibitory activity against a number of human tumor cell lines both in vitro, and when grown as xenografts in mice. It is currently being evaluated in a phase I clinical trial at the National Cancer Institute. The objective of this project was to develop and validate an analytical method for the assay of flavopiridol in human plasma, with sufficient sensitivity to permit the plasma pharmacokinetics of flavopiridol to be studied during clinical trials. Methods: Flavopiridol was isolated from human plasma samples by extraction with t-butylmethyl ether following alkalinization with borate buffer (pH 8.0). The extract was evaporated, the residue was dissolved in mobile phase, and analyzed by reversed-phase high-pressure liquid chromatography. Chromatography was accomplished with a polymer-based C18 column eluted with a mobile phase consisting of methanol-phosphate buffer, pH 11.0 (53:47 v/v). Electrochemical detection (ECD) was employed. Results: Flavopiridol was recovered from human plasma with an efficiency of 85–87%. Calibration curves were linear over the concentration range 10–500 nM (4.4–219 ng/ml). Plasma standard concentrations were measured with an accuracy and precision ranging from 3.2% to 10%. Regression analysis of flavopiridol concentrations of 15 clinical trial plasma samples ranging in concentration from approximately 50 to 4000 μM quantitated by both ECD and mass spectrometry showed close agreement. The equation of the regression line was y = 1.02x + 8 with a correlation coefficient of 0.969. Continuous infusion of flavopiridol in four patients for 72 h at a rate of 50 mg/m2 per day, resulted in mean steady-state plasma concentrations of from 200 to 300 nM. Levels declined in a biexponential manner following termination of the infusion, falling to approximately 10 nM after 48 h. Conclusions: An analytical method for the assay of flavopiridol in human plasma was developed with sensitivity to at least 10 nM. The assay is accurate, precise and specific, and is suitable for determination of plasma flavopiridol concentrations for pharmacokinetic studies during clinical trials.