Importance of the stress kinase p38alpha in mediating the direct cytotoxic effects of the thalidomide analogue, CPS49, in cancer cells and endothelial cells.

PURPOSE: Thalidomide has gained renewed interest as a cancer therapeutic due to its potential antiangiogenic effects. The thalidomide analogues CPS11 and CPS49 are active in preclinical angiogenesis assays and xenograft model systems, but the biochemical basis for these observations is unclear. EXPERIMENTAL DESIGN: To address this question, we assessed the toxicity of these thalidomide analogues in cancer cells, endothelial cells, and genetically modified cells using assays that measure apoptotic and nonapoptotic cell death. Phosphospecific and native antibodies were used in immunoblotting and immunohistochemical experiments to assess the activation states of kinases that control cellular survival in vitro and in vivo. RESULTS: CPS49 predominantly induced nonapoptotic cell death in lung cancer cells, prostate cancer cells, and endothelial cells in a dose-dependent manner, whereas CPS11 was not cytotoxic. CPS49 did not inhibit kinases that promote survival, such as Akt or extracellular signal-regulated kinase, but rather rapidly activated the stress kinase p38 pathway in both cancer cells and endothelial cells. CPS49 activated p38 in tumor xenografts. Using p38alpha-/- cells or an inhibitor of p38, we show that the presence and activation of p38alpha is important for cytotoxicity in all cell types examined. CONCLUSIONS: Our studies identify a unifying mechanism of action for cytotoxicity of the tetraflourinated thalidomide analogue, CPS49, and suggest that activation of p38 could serve as a biomarker in clinical trials with CPS49.

Thalidomide metabolism and hydrolysis: mechanisms and implications.

Despite its controversial past, thalidomide is currently under investigation for the treatment of several disease types, ranging from inflammatory conditions to cancer. The mechanism of action of thalidomide is complex and not yet fully understood, but there is some evidence to suggest that metabolism may play a role. Consequently, there has been a considerable effort to characterize the metabolism of thalidomide in recent years. Thalidomide undergoes biotransformation by non-enzymatic hydrolysis and enzyme-mediated hydroxylation to form a multitude of metabolites. Metabolite identification and reaction phenotyping studies have been performed and will be discussed in this review in addition to interspecies differences in thalidomide metabolism.

Effect of common CYP3A4 and CYP3A5 variants on the pharmacokinetics of the cytochrome P450 3A phenotyping probe midazolam in cancer patients.

PURPOSE: To evaluate the effect of naturally occurring variants in genes encoding the cytochrome P450 (CYP) isoforms CYP3A4 and CYP3A5 in patients with cancer receiving midazolam as a phenotyping probe. EXPERIMENTAL DESIGN: Five variants in CYP3A4 and CYP3A5 were evaluated in 58 patients (21 women and 37 men) receiving a short i.v. bolus of midazolam (dose, 0.0145 or 0.025 mg/kg). Midazolam concentrations in plasma were determined using liquid chromatography-mass spectrometry, and pharmacokinetic variables were calculated using noncompartmental analysis. Genomic DNA was characterized for the variants by PCR-RFLP, and all genotypes were confirmed by direct nucleotide sequencing. RESULTS: The mean clearance of midazolam was 24.4 +/- 9.12 L/h, and phenotypic CYP3A activity varied about 4-fold in this population (range, 10.8-44.3 L/h). There were six carriers of the CYP3A4*1B allele (allele frequency, 0.061). No variant alleles for CYP3A4*17, CYP3A4*18A, or CYP3A5*6 were identified. Forty-eight of the 58 patients were homozygous variant for CYP3A5*3C, eight were heterozygous, and two were homozygous wild type (allele frequency, 0.897). No associations were noted between any of the studied genotypes and the phenotypic measures (P > or = 0.16). Likewise, a common variant in exon 26 in the gene encoding P-glycoprotein [i.e., ABCB1 (MDR1) 3435C>T] that was previously reported to be linked to CYP3A4 mRNA levels was unrelated to any of the studied phenotypic measures (P > or = 0.49). CONCLUSIONS: The studied genetic variants in CYP3A4 and CYP3A5 are unlikely to have an important functional significance to phenotypic CYP3A activity in patients with cancer.

Evaluation of biologic end points and pharmacokinetics in patients with metastatic breast cancer after treatment with erlotinib, an epidermal growth factor receptor tyrosine kinase inhibitor.

PURPOSE: To evaluate changes in epidermal growth factor receptor (EGFR) phosphorylation and its downstream signaling in tumor and surrogate tissue biopsies in patients with metastatic breast cancer treated with erlotinib, an EGFR tyrosine kinase inhibitor, and to assess relationships between biomarkers in tumor and normal tissues and between biomarkers and pharmacokinetics. PATIENTS AND METHODS: Eighteen patients were treated orally with 150 mg/d of erlotinib. Ki67, EGFR, phosphorylated EGFR (pEGFR), phosphorylated mitogen-activated protein kinase (pMAPK), and phosphorylated AKT (pAKT) in 15 paired tumor, skin, and buccal mucosa biopsies (at baseline and after 1 month of therapy) were examined by immunohistochemistry and analyzed quantitatively. Pharmacokinetic sampling was also obtained. RESULTS: The stratum corneum layer and Ki67 in keratinocytes of the epidermis in 15 paired skin biopsies significantly decreased after treatment (P = .0005 and P = .0003, respectively). No significant change in Ki67 was detected in 15 tumors, and no responses were observed. One was EGFR-positive and displayed heterogeneous expression of the receptor, and 14 were EGFR-negative. In the EGFR-positive tumor, pEGFR, pMAPK, and pAKT were reduced after treatment. Paradoxically, pEGFR was increased in EGFR-negative tumors post-treatment (P = .001). Although markers were reduced in surrogate and tumor tissues in the patient with EGFR-positive tumor, no apparent associations were observed in patients with EGFR-negative tumor. CONCLUSION: Erlotinib has inhibitory biologic effects on normal surrogate tissues and on an EGFR-positive tumor. The lack of reduced tumor proliferation may be attributed to the heterogeneous expression of receptor in the EGFR-positive patient and absence of target in this cohort of heavily pretreated patients.

Mechanisms of resistance to anticancer drugs: the role of the polymorphic ABC transporters ABCB1 and ABCG2.

ATP-binding cassette (ABC) genes play a role in the resistance of malignant cells to anticancer agents. The ABC gene products, including ABCB1 (P-glycoprotein) and ABCG2 (breast cancer-resistance protein [BCRP], mitoxantrone-resistance protein [MXR], or ABC transporter in placenta [ABCP]), are also known to influence oral absorption and disposition of a wide variety of drugs. As a result, the expression levels of these proteins in humans have important consequences for an individual's susceptibility to certain drug-induced side effects, interactions, and treatment efficacy. Naturally occurring variants in ABC transporter genes have been identified that might affect the function and expression of the protein. This review focuses on recent advances in the pharmacogenetics of the ABC transporters ABCB1 and ABCG2, and discusses potential implications of genetic variants for the chemotherapeutic treatment of cancer.

Comparative molecular field analysis and comparative molecular similarity indices analysis of thalidomide analogues as angiogenesis inhibitors.

Thalidomide, 2-(2,6-dioxo-3-piperidinyl)-1H-isoindole-1,3(2H)-dione, has been shown to inhibit angiogenesis, the formation of new blood vessels from existing vasculature. As a result, there is renewed interest in this drug as a potential therapy for solid tumors. Thalidomide forms a number of metabolites and has been shown to require metabolic activation for antiangiogenic activity. A series of 39 compounds, based upon the structure of some of these metabolites, was synthesized and tested for their ability to inhibit microvessel growth in the rat aortic ring assay. The results of this testing have been used as the basis for a three-dimensional quantitative structure-activity relationship (3D-QSAR) study, utilizing comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) procedures. The best resulting CoMFA and CoMSIA models have conventional r(2) values of 0.924 and 0.996, respectively. The cross-validated q(2) values are 0.666 and 0.635, respectively. These models offer insight into the structural requirements for activity of thalidomide analogues as angiogenesis inhibitors, since there is only speculative knowledge of the target. Additionally, it appears as though there is more than one active site or mechanism of action.

Liquid-chromatographic determination of erlotinib (OSI-774), an epidermal growth factor receptor tyrosine kinase inhibitor.

A high-performance liquid-chromatographic (HPLC) assay with UV detection has been developed for the quantitative determination of erlotinib (OSI-774) in human plasma. Quantitative extraction was achieved by a single-solvent extraction involving a mixture of acetonitrile and n-butyl chloride (1:4, v/v). Erlotinib and the internal standard hydrochloride salt (OSI-597) were separated on a column packed with Nova-Pak C18 material and a mobile phase composed of acetonitrile and water, pH 2.0 (60:40, v/v). The column effluent was monitored with dual UV detection at wavelengths of 348 nm (erlotinib) and 383 nm (OSI-597). The calibration graph was linear in the range of 100-4500 ng/ml, with values for accuracy and precision ranging from 87.9 to 96.2% and 2.13 to 5.10%, respectively, for three different sets of quality control samples. The developed method was successfully applied to study the pharmacokinetics of erlotinib in a cancer patient at the recommended daily dose of 150 mg.

Determination of midazolam in human plasma by liquid chromatography with mass-spectrometric detection.

A liquid chromatographic assay with mass-spectrometric detection was developed for the quantitative determination of the cytochrome p450 3A phenotyping probe midazolam in human plasma. Sample pretreatment involved a one-step extraction of 600 microl aliquots with ethyl acetate. Midazolam and the internal standard, lorazepam, were separated on a column (150 mm x 4.6mm, i.d.) packed with 5 microm Zorbax Eclipse XDB-C8 material, using a mobile phase composed of methanol and 10mM aqueous ammonium acetate (60:40, v/v). Column effluents were analyzed using mass-spectrometry with an atmospheric pressure chemical ionization source. Calibration curves were linear in the concentration range of 1.00-200 ng/ml. The accuracy and precision ranged from 92.8 to 112% and 0.056 to 13.4%, respectively, for four different concentrations of quality control samples analyzed in triplicate on eight separate occasions. The developed method was subsequently applied to study the pharmacokinetics of midazolam in a group of 35 human subjects at a single dose of 25 microg/kg.

Determination of CC-5013, an analogue of thalidomide, in human plasma by liquid chromatography-mass spectrometry.

A high-performance liquid chromatographic assay with MS detection has been developed for the quantitative determination of the anti-angiogenic agent CC-5013 in human plasma. Sample pretreatment involved liquid-liquid extraction with acetonitrile/1-chlorobutane (4:1, v/v) solution containing the internal standard, umbelliferone. Separation of the compounds of interest was achieved on a column packed with Waters C18 Nova-Pak material (4 microm particle size; 300 mm x 3.9 mm internal diameter) using acetonitrile, de-ionized water, and glacial acetic acid in ratios of 20:80:0.1 (v/v/v) (pH 3.5) delivered at an isocratic flow rate of 1.00 ml/min. Simultaneous MS detection was performed at m/z 260.3 (CC-5013) and m/z 163.1 (umbelliferone). The calibration curve was fit to a linear response-concentration data over a range of 5-1000 ng/ml using a weighting factor of 1/x. Values for accuracy and precision, obtained from four quality controls analyzed on three different days in replicates of five, ranged from 98 to 106% and from 5.5 to 15.5%, respectively. The method was successfully applied to study the pharmacokinetics of CC-5013 in a cancer patient receiving the drug as single daily dose.

Synthesis of racemic cis-5-hydroxy-3-phthalimidoglutarimide. A metabolite of thalidomide isolated from human plasma.

[reaction: see text] A synthesis of the glutarimide-derived metabolite of thalidomide, 5'-hydroxythalidomide (2), is described. The synthesis employed the lactone derivative of N-benzyloxycarbonyl (CBZ)-protected 4-hydroxyglutamic acid 12, which is prepared by a de novo route from diethyl acetamidomalonate. The reaction of 12 with 4-methoxybenzylamine gave the corresponding isoglutamine, which then provided the key CBZ-protected N-PMB-glutarimide 14 after dehydration. Deprotection of both the CBZ and PMB groups followed by phthalimidation and deacetylation of the 3-amino-5-acetoxyglutarimide 16 afforded 2.

Determination of 2-methoxyestradiol in human plasma, using liquid chromatography/tandem mass spectrometry.

A liquid chromatography/tandem mass spectrometric (LC/MS/MS) assay was developed for the quantitative determination of 2-methoxyestradiol (2ME2) in human plasma. Sample pretreatment involved liquid-liquid extraction with ethyl acetate of 0.3-mL aliquots of plasma spiked with the internal standard, deuterated 2ME2 (2ME2-d5). Separation was achieved on a Zorbax Eclipse C18 column (2.1 x 50 mm, i.d., 5 microm) at room temperature using a gradient elution with methanol and water at a flow rate of 0.25 mL/min. Detection was performed using atmospheric pressure chemical ionization MS/MS by monitoring the ion transitions from m/z 303.1 --> 136.8 (2ME2) and m/z 308.1 --> 138.8 (2ME2-d5). Calibration curves were linear in the concentration range of 1-100 ng/mL. The accuracy and precision values, obtained from three different sets of quality control samples analyzed in quintuplicate on four separate occasions, ranged from 105-108% and from 3.62-5.68%, respectively. This assay was subsequently used for the determination of 2ME2 concentration in plasma of a patient with cancer after a single oral administration of 2ME2 at a dose of 2200 mg.

New directions in cancer research 2003: technological advances in biology, drug resistance, and molecular pharmacology.

The 94th Annual Meeting of the American Association for Cancer Research (AACR) was held from July 11 to 14, 2003 in Washington, DC, and provided an overview of the latest developments in the field of cancer. This report provides highlights of presentations on array-based and RNA-interference technologies to study cancer biology and molecular pharmacology of anticancer drugs, mechanisms and modulation of drug resistance patterns, recent developments in the treatment of prostate cancer, and the medicinal chemistry of established and novel anticancer drugs.

Prediction of irinotecan pharmacokinetics by use of cytochrome P450 3A4 phenotyping probes.

BACKGROUND: Irinotecan is a topoisomerase I inhibitor that has been approved for use as a first- and second-line treatment for colorectal cancer. The response to irinotecan is variable, possibly because of interindividual variation in the expression of the enzymes that metabolize irinotecan, including cytochrome P450 3A4 (CYP3A4) and uridine diphosphate glucuronosyltransferase 1A1 (UGT1A1). We prospectively explored the relationships between CYP3A phenotype, as assessed by erythromycin metabolism and midazolam clearance, and the metabolism of irinotecan and its active metabolite SN-38. METHODS: Of the 30 white cancer patients, 27 received at least two treatments with irinotecan administered as one 90-minute infusion (dose, 600 mg) with 3 weeks between treatments, and three received only one treatment. Before the first and second treatments, patients underwent an erythromycin breath test and a midazolam clearance test as phenotyping probes for CYP3A4. Erythromycin metabolism was assessed as the area under the curve for the flux of radioactivity in exhaled CO2 within 40 minutes after administration of [N-methyl-14C]erythromycin. Midazolam and irinotecan were measured by high-performance liquid chromatography. Genomic DNA was isolated from blood and screened for genetic variants in CYP3A4 and UGT1A1. All statistical tests were two-sided. RESULTS: CYP3A4 activity varied sevenfold (range = 0.223%-1.53% of dose) among patients, whereas midazolam clearance varied fourfold (range = 262-1012 mL/min), although intraindividual variation was small. Erythromycin metabolism was not statistically significantly associated with irinotecan clearance (P = .090), whereas midazolam clearance was highly correlated with irinotecan clearance (r = .745, P<.001). In addition, the presence of a UGT1A1 variant with a (TA)7 repeat in the promoter (UGT1A1*28) was associated with increased exposure to SN-38 (435 ng x h/mL, 95% confidence interval [CI] = 339 to 531 ng x h/mL in patients who are homozygous for wild-type UGT1A1; 631 ng x h/mL, 95% CI = 499 to 762 ng . h/mL in heterozygous patients; and 1343 ng x h/mL, 95% CI = 0 to 4181 ng x h/mL in patients who are homozygous for UGT1A1*28) (P = .006). CONCLUSION: CYP3A4 phenotype, as assessed by midazolam clearance, is statistically significantly associated with irinotecan pharmacokinetics. Evaluation of midazolam clearance combined with UGT1A1*28 genotyping may assist with optimization of irinotecan chemotherapy.