Assessment of epidermal growth factor receptor (EGFR, ErbB1) and HER2 (ErbB2) protein expression levels and response to lapatinib (Tykerb, GW572016) in an expanded panel of human normal and tumour cell lines.

OBJECTIVE: Lapatinib (Tykerb, GW572016), a potent inhibitor of the catalytic activities of epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) (ErbB2), inhibits population growth of selected EGFR and HER2 overexpressing cell lines. Previous studies with a small number of cell lines suggest a correlation between overexpression of EGFR and/or HER2 and sensitivity to growth inhibition by lapatinib; however, the precise determinants of lapatinib selectivity for tumour and/or other cells remain unclear. MATERIALS AND METHODS: To clarify the determinants of its selectivity in cultured cells, lapatinib-induced cell population growth inhibition and relative EGFR and HER2 protein expression were quantified in 61 different human tumour cell lines from 12 tumour types, two oncogene transformed human cell lines and two normal human cell cultures. Using statistical tools to analyse the data, a model describing the relationship between lapatinib IC(50) (the response variable) and EGFR and HER2 expression and tissue type (explanatory variables) was derived. CONCLUSION: The results suggest that simultaneous consideration of EGFR and HER2 expression, as well as tissue type yields the best determinant of lapatinib selectivity in cultured cells.

Antitumour efficacy of VEGFR2 tyrosine kinase inhibitor correlates with expression of VEGF and its receptor VEGFR2 in tumour models.

During the development of indazolylpyrimidines as novel and potent inhibitors of vascular endothelial growth factor (VEGF) receptor-2 (VEGFR2) tyrosine kinase, we observed that some human tumour xenografts are more sensitive to VEGFR2 kinase inhibitors than others. A better understanding of the basis for this differential response may help to identify a predictive marker that would greatly aid in the identification of a suitable patient population for treatment. One representative compound from the indazolylpyrimidine series is GW654652 that inhibited all three VEGFRs with similar potency. The inhibition of VEGFR2 kinase by GW654652 was about 150 to >8800 more potent than the inhibition of eight other kinases tested. GW654652 inhibited VEGF- and bFGF-induced proliferation in endothelial cells with an IC(50) of 110 and 1980 nM, respectively, and has good pharmacokinetic profile in mouse and dog. We investigated the association between VEGF and VEGFR2 expression and the antitumour efficacy of GW654652, in various xenograft models. Statistically significant associations were observed between the antitumour efficacy of GW654652 in xenografts and VEGF protein (P=0.005) and VEGFR2 expression (P=0.041). The oral dose of GW654652 producing 50% inhibition of tumour growth (ED(50)) decreased with increasing levels of VEGF (r=-0.94); and, in contrast, the ED(50) increased with the increased expression of VEGFR2 (r=0.82). These results are consistent with the observed inverse correlation between VEGF and VEGFR2 expression in tumours. These findings support the hypothesis that VEGF and VEGFR2 expression by tumours may predict the therapeutic outcome of VEGFR kinase inhibitors.

The characterization of novel, dual ErbB-2/EGFR, tyrosine kinase inhibitors: potential therapy for cancer.

The type I receptor tyrosine kinases constitute a family of transmembrane proteins involved in various aspects of cell growth and survival and have been implicated in the initiation and progression of several types of human malignancies. The best characterized of these proteins are the epidermal growth factor receptor (EGFR) and ErbB-2 (HER-2/neu). We have developed potent quinazoline and pyrido-[3,4-d]-pyrimidine small molecules that are dual inhibitors of ErbB-2 and EGFR. The compounds demonstrate potent in vitro inhibition of the ErbB-2 and EGFR kinase domains with IC(50)s <80 nM. Growth of ErbB-2- and EGFR-expressing tumor cell lines is inhibited at concentrations <0.5 microM. Selectivity for tumor cell growth inhibition versus normal human fibroblast growth inhibition ranges from 10- to >75-fold. Tumor growth in mouse s.c. xenograft models of the BT474 and HN5 cell lines is inhibited in a dose-responsive manner using oral doses of 10 and 30 mg/kg twice per day. In addition, the tested compounds caused a reduction of ErbB-2 and EGFR autophosphorylation in tumor fragments from these xenograft models. These data indicate that these compounds have potential use as therapy in the broad population of cancer patients overexpressing ErbB-2 and/or EGFR.

The effects of the novel, reversible epidermal growth factor receptor/ErbB-2 tyrosine kinase inhibitor, GW2016, on the growth of human normal and tumor-derived cell lines in vitro and in vivo.

The epidermal growth factor receptor (EGFR) and ErbB-2 transmembrane tyrosine kinases are currently being targeted by various mechanisms in the treatment of cancer. GW2016 is a potent inhibitor of the ErbB-2 and EGFR tyrosine kinase domains with IC50 values against purified EGFR and ErbB-2 of 10.2 and 9.8 nM, respectively. This report describes the efficacy in cell growth assays of GW2016 on human tumor cell lines overexpressing either EGFR or ErbB-2: HN5 (head and neck), A-431 (vulva), BT474 (breast), CaLu-3 (lung), and N87 (gastric). Normal human foreskin fibroblasts, nontumorigenic epithelial cells (HB4a), and nonoverexpressing tumor cells (MCF-7 and T47D) were tested as negative controls. After 3 days of compound exposure, average IC50 values for growth inhibition in the EGFR- and ErbB-2-overexpressing tumor cell lines were < 0.16 microM. The average selectivity for the tumor cells versus the human foreskin fibroblast cell line was 100-fold. Inhibition of EGFR and ErbB-2 receptor autophosphorylation and phosphorylation of the downstream modulator, AKT, was verified by Western blot analysis in the BT474 and HN5 cell lines. As a measure of cytotoxicity versus growth arrest, the HN5 and BT474 cells were assessed in an outgrowth assay after a transient exposure to GW2016. The cells were treated for 3 days in five concentrations of GW2016, and cell growth was monitored for an additional 12 days after removal of the compound. In each of these tumor cell lines, concentrations of GW2016 were reached where outgrowth did not occur. Furthermore, growth arrest and cell death were observed in parallel experiments, as determined by bromodeoxyuridine incorporation and propidium iodide staining. GW2016 treatment inhibited tumor xenograft growth of the HN5 and BT474 cells in a dose-responsive manner at 30 and 100 mg/kg orally, twice daily, with complete inhibition of tumor growth at the higher dose. Together, these results indicate that GW2016 achieves excellent potency on tumor cells with selectivity for tumor versus normal cells and suggest that GW2016 has value as a therapy for patients with tumors overexpressing either EGFR or ErbB-2.

Antibody-directed enzyme prodrug therapy with the T268G mutant of human carboxypeptidase A1: in vitro and in vivo studies with prodrugs of methotrexate and the thymidylate synthase inhibitors GW1031 and GW1843.

Antibody-directed enzyme prodrug therapy (ADEPT) is a technique to increase antitumor selectivity in cancer chemotherapy. Our approach to this technology has been to design a mutant of human carboxypeptidase A (hCPA1-T268G) which is capable of hydrolyzing in vivo stable prodrugs of MTX and targeting this enzyme to tumors on an Ep-CAM1-specific antibody, ING1. Through the use of this >99% human enzyme which is capable of catalyzing a completely nonhuman reaction, we hope to increase ADEPT selectivity while decreasing overall immunogenicity of the enzyme-antibody conjugate. In the current report, prodrugs of the thymidylate synthase inhibitors GW1031 and GW1843 and the dihydrofolate reductase inhibitor methotrexate were studied for their wild-type and mutant hCPA enzyme hydrolysis, their in vivo stability, and their use in therapy. Prodrugs with high kcat/Km ratios for mutated versus wild-type hCPA1 were examined in vitro for their stability in human pancreatic juice, and in vivo for their stability in mouse plasma and tissues. In addition, targeting and in vivo enzyme activity studies were performed with an ING1 antibody conjugate of the mutant enzyme (ING1-hCPA1-T268G). Finally, in vivo therapy studies were performed with LS174T tumors to demonstrate proof of principle. Results indicate that prodrugs can be synthesized that are selective and efficient substrates of hCPA1-T268G and not substrates of the endogenous CPA activities; this leads to excellent in vivo stability for these compounds. In vivo conjugate targeting studies showed that the antibody-enzyme conjugate was targeted to the tumor and enzyme was initially active in vivo at the site. Unfortunately therapeutic studies did not demonstrate tumor reduction. Experiments to determine reasons for the lack of antitumor activity showed that the enzyme activity decreased as a result of enzyme instability. The results offer encouragement for additional novel mutant enzyme improvements and additional in vivo studies on this unique approach to ADEPT.

Effect of purine synthesis inhibition on WiDr spheroids in vitro or on WiDr or colon 38 tumors in vivo. Complete growth inhibition but not regression.

Clinical responses for anticancer agents are based upon tumor regression. We have investigated the potential of glycineamide ribonucleotide transformylase (GAR TFase) inhibitors to produce regressions in multiple preclinical models of colon carcinoma. The growth of multicellular tumor spheroids of WiDr human colon carcinoma was inhibited by the GAR TFase inhibitors 5-deazaacyclotetrahydrofolate (5-DACTHF), its 2'-fluoro, 3'-fluoro, 10-deaza, and 10-thia analogs as well as 5,10-dideazatetrahydrofolate, but none of the compounds caused spheroid regressions. By contrast, complete spheroid disruption was observed with exposure to etoposide, m-AMSA (amsacrine), piritrexim, or 2-desamino-2-methyl-10-propargyl-5,8-dideazafolate (DMPDDF). Light microscopy of the spheroids treated with either 5-DACTHF or DMPDDF suggested that the reason for the difference is extensive cell kill throughout the spheroid in the presence of DMPDDF compared with little or no kill, over that found in controls, with 5-DACTHF. Treatment of spheroids with 5-DACTHF in the presence of 1 microM hypoxanthine resulted in no significant reversal of growth inhibition; 50% reversal required 10 microM hypoxanthine. The spheroid studies were extended to in vivo studies examining the effects of 5-DACTHF on established WiDr and colon 38 tumors. The results showed that, in contrast to melphalan, which produced cures and tumor regressions, 5-DACTHF produced reversible growth inhibition with no significant regression of tumors. The results predict that clinical response, typically measured by tumor regression, may be rare following single agent therapy with inhibitors of de novo purine biosynthesis.

Synthesis and biological evaluation of N alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-ornithine.

A novel folic acid analogue, N alpha-(5-deaza-5,6,7,8-tetrahydropteroyl)-L-ornithine, 3, was prepared via a multistep synthetic sequence. The key steps involved the conversion of 5-deazapteroic acid to its N10-formyl derivative followed by catalytic hydrogenation of the pyridine ring and subsequent heating in dilute sodium hydroxide to afford the new 5-deaza-5,6,7,8-tetrahydropteroic acid. After trifluoroacetylation, this compound was coupled to N delta-(tert-butyl-oxycarbonyl)-L-ornithine using conventional peptide bond forming conditions. Deprotection first in base and then in acid gave the title compound. Compound 3 was an effective inhibitor of hog liver folylpolyglutamate synthetase (Kis, estimated = 64 nM), and was shown to retard the formation of polyglutamates of a structurally related folic acid analogue in HCT-8 cells in vitro.

In vivo antitumor activity and metabolism of a series of 5-deazaacyclotetrahydrofolate (5-DACTHF) analogues.

This study compares the antitumor activity and metabolism of the purine de novo biosynthesis inhibitor 5-deazaacyclotetrahydrofolate and a series of analogues. All compounds have similar IC50 values for inhibition of MCF-7 cell growth, activity of glycineamide ribonucleotide transformylase, and methotrexate uptake by MOLT-4 cells, the latter a measure of cellular uptake potential. Only 5-deazaacyclotetrahydrofolate and the 2'-fluoro and 3'-fluoro analogues demonstrated significant inhibition of colon 38 adenocarcinoma or HCT-116 colon carcinoma growth in vivo. This correlated with the Km of these compounds for folylpolyglutamate synthetase. 5-Deazaacyclotetrahydrofolate and 2'-fluoro-5-deazaacyclotetrahydrofolate which displayed the strongest antitumor activity were detectable in colon 38 tumor tissue 24 hr after dosing and were present nearly exclusively as the polyglutamated species. These results indicate that polyglutamation represents a critical step in the in vivo antitumor activity of these compounds.

A radiometric method for objectively screening large numbers of compounds against Pneumocystis carinii in vitro.

A relatively simple method is reported for accurately quantitating the incorporation of [3H]para aminobenzoic acid (pABA) into the folates of Pneumocystis carinii cultured in vitro, and the subsequent development of a highly sensitive and reproducible 96-well microtitre plate drug screening system. Incorporation of [3H]pABA under optimized conditions has been utilized as a selective indicator of the in vitro viability of P. carinii against which the inhibitory effects of potential drugs were quantified. The anti-Pneumocystis agents pentamidine, sulfamethoxazole, 566C80 and piritrexim gave median inhibitory concentration values of 7.3, 0.1, 1.4 and approximately 100 microM, respectively in this assay. The results suggest that this 96-well plate P. carinii [3H]pABA-incorporation system is suitable as a rapid high throughput primary in vitro screen for detecting compounds with anti-Pneumocystis activity.

Microculture screening assay for primary in vitro evaluation of drugs against Pneumocystis carinii.

Pneumocystis carinii inoculated into 96-well filtration plate assemblies was shown to synthesize radiolabeled folates de novo from [para-3H]aminobenzoic acid ([3H]pABA). At the end of each incubation with [3H]pABA, a vacuum manifold was used to remove the medium and wash P. carinii. The membrane at the base of each well was dried and punched out, and the level of 3H retained was determined by direct scintillation counting. High-pressure liquid chromatography analysis of duplicate filters confirmed that direct counting of 3H retained on membranes (after correction for unmetabolized [3H]pABA) was an accurate reflection of total [3H]pABA incorporation by P. carinii. Greater than 95% of the 3H recovered was shown to be present as polyglutamated species. After digestion with rat plasma folic acid gamma-glutamyl hydrolase, para-aminobenzoylglutamate, N10-formyltetrahydrofolate, and tetrahydrofolate were identified as the major 3H-labeled components. para-Aminobenzoylglutamate was presumed to have arisen from folylpolyglutamates synthesized by P. carinii and was therefore included in the calculation of total [3H]pABA incorporation. P. carinii incorporation of [3H]pABA under optimal conditions was used as a selective measure of in vitro viability against which the inhibitory effects of some antipneumocystis agents (pentamidine, sulfamethoxazole, 566C80, and piritrexim) were quantitated. The concentrations of pentamidine, sulfamethoxazole, 566C80, and piritrexim required for 50% inhibition in this assay were 7.3, 0.1, 1.4, and approximately 100 microM, respectively. The results suggest that this 96-well [3H]pABA incorporation assay has considerable potential for objective in vitro drug screening against P. carinii.

Preparation of a chiral matrix. Resolution of (6R,S)-N5-formyltetrahydrofolate.

Combination chemotherapy involving (6R,S)-N5-formyltetrahydrofolate and 5-fluorouracil has raised considerable speculation concerning the effects of the unnatural (6R) diastereomer. The inability to obtain quantities of the individual diastereomers has greatly limited work in this area. Commercially available chiral columns, suitable for diastereomer analysis, are inadequate for preparative work. We report here on the use of epoxide-activated media in the construction of a bovine serum albumin-based high-performance liquid chromatography matrix capable of resolving the diastereomers of (6R,S)-N5-formyltetrahydrofolate in milligram quantities. Similar columns based upon alternative protein matrices may prove useful for the resolution of additional materials.

Activity of an NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase in normal tissue, neoplastic cells, and oncogene-transformed cells.

As an extension of the previously reported observation concerning the existence of NAD-dependent 5,10-methylenetrahydrofolate dehydrogenase in transformed cells a variety of tissues and cell lines have been assayed for this activity. This activity was found in all assayed transformed cells. Results with rat liver derived epithelial (RLE) cells transformed with a series of oncogenes (v-raf, v-raf/v-myc (J2), v-myc (J5), and v-Ha-ras (pRNR16)) indicated that expression of activity correlates with the extent of transformation and was independent of the oncogene used for transformation. Compared to previously reported values for normal tissue, surprisingly high levels of the NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase were found in the rat adrenal cortex. This activity was not seen in mouse or bovine adrenal. Enzymatic activity was also detected in mouse bone marrow and was strain dependent. The levels of activity in mouse bone marrow were lower than previously reported. The NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase activity in rat adrenal and RLE cells may represent tools for studying the regulation of expression of this activity.

Direct demonstration of the active salvage of preformed purines by murine tumors.

The purine de novo biosynthetic pathway has become a target for chemotherapeutic agents and because of the possible contribution of the salvage of extracellular purines to cellular purine pools an examination of the ability of mouse tumors in vivo to exploit the salvage pathways was undertaken. Our data reveal that circulating radiolabeled preformed purines are rapidly and actively salvaged in both normal liver and in two different types of model tumors. The salvaged purines were found to be distributed between both acid soluble cytoplasmic purines and acid insoluble nucleic acid associated purine species. The ability to salvage adenine, the most abundant circulating purine in C57BL/6 mice, was highest in normal liver with the two different model tumors demonstrating lower specific activities of salvaged acid soluble purines. The amount of radiolabel incorporated into acid insoluble nucleic acid was dependent upon the tumor type. Because of the active salvage observed in these tumors, the mechanism by which de novo purine biosynthesis inhibitors serve as effective chemotherapeutic agents may be more complex than simple biosynthetic inhibition.

In vivo and in vitro metabolism of 5-deazaacyclotetrahydrofolate, an acyclic tetrahydrofolate analogue.

5-Deazaacyclotetrahydrofolate is a cytotoxic tetrahydrofolate analogue which inhibits glycinamide ribonucleotide transformylase (Kelley et al., J. Med. Chem., 33: 561-567, 1990). Cultured mouse L-cells and human MCF-7 and MOLT-4 cells concentrated the drug several hundred-fold after 24 h of continuous exposure to a cytotoxic level (100-200 nM) of radiolabeled drug. High performance liquid chromatography analysis revealed that each cell type metabolized greater than or equal to 80% of the internalized drug to polyglutamated forms, which are more potent glycinamide ribonucleotide transformylase inhibitors. In L-cells, 45% of the polyglutamated metabolites were also N-formylated. The pharmacokinetics and distribution of [14C]-deazaacyclotetrahydrofolate were studied in C57BL/6 male mice. Its plasma half-life was 2.15 h. Radiolabel was concentrated to well above plasma level in the kidney, pancreas, and liver. Metabolism was examined in tumor-bearing and in normal mice. Twenty-four h after a single i.p. injection (50 mg/kg), drug equivalents were 0.6 nmol/g (83% polyglutamated) in colon-38 adenocarcinoma carried s.c., 2.4 nmol/g (100% polyglutamated) in ascitic P388 cells, and 3.7 nmol/g (76% polyglutamated and approximately 20% formylated) in mouse liver. Elimination was mostly in the urine as unmetabolized drug. Feces contained 5-deazaacyclotetrahydropteroate (parent compound less glutamate). In conclusion, 5-deazaacyclotetrahydrofolate was shown to be concentrated to well above the extracellular level and metabolized to more active polyglutamated forms by transformed cells grown in culture and in mice.

Vitamin C biosynthesis in prosimians: evidence for the anthropoid affinity of Tarsius.

This report examines the taxonomic distribution of the in vitro biosynthesis of ascorbic acid in the Prosimii (Order: Primates). Liver and kidney samples of 15 prosimian taxa, including Tarsius bancanus, were quantitatively tested for the enzyme L-gulono-1,4-lactone oxidase. Liver samples from all taxa except Tarsius had substantial levels of the enzyme. Furthermore, unlike other eutherian mammals, kidney tissue from members of the family Lemuridae showed low but consistent levels of enzyme activity. The result for Tarsius, by fitting with the pattern exhibited by the monkeys, apes, and man, adds significant independent evidence for this animal's relatively close genetic relationship with the Anthropoidea.

Isolation and characterization of beta- and gamma-caseins from horse milk.

Three groups of casein components were isolated from horse milk. Group I is almost insoluble at acid and neutral pH, and is rather heterogeneous on alkaline gels with or without sodium dodecyl sulphate. Group II shows strong similarity to beta-casein from other species, as concluded from its amino acid composition and its N- and C-terminal sequences. This group consists of five electrophoretically distinguishable forms, all containing ester phosphate groups but no carbohydrate. Group III is composed of C-terminal fragments of the beta-like (group II) fraction and probably arises from the action of a plasmin-like enzyme present in horse milk. It does not contain phosphate or carbohydrate. Homology of this group with bovine gamma-caseins is demonstrated. Both beta- and gamma-like caseins are more soluble at 4 degrees C than at room temperature.