Tyrosine kinase inhibitors. 14. Structure-activity relationships for methylamino-substituted derivatives of 4-[(3-bromophenyl)amino]-6-(methylamino)-pyrido[3,4-d]pyrimidine (PD 158780), a potent and specific inhibitor of the tyrosine kinase activity of receptors for the EGF family of growth factors.

The 4-[(3-bromophenyl)amino]pyrido[3,4-d]pyrimidine PD 158780 is a very potent in vitro inhibitor of the tyrosine kinase activity of the epidermal growth factor receptor (EGFR) (IC50 0.08 nM), and other members of the erbB family, by competitive binding at the ATP site of these signal transduction enzymes. A series of analogues of PD 158780 bearing solubilizing functions off the 6-methylamino substituent were prepared by reaction of the 6-fluoro derivatives with appropriate amine nucleophiles. These were evaluated for their ability to inhibit the tyrosine phosphorylating action of EGF-stimulated full-length EGFR enzyme and for inhibition of autophosphorylation of the EGFR in A431 human epidermoid carcinoma cells in culture. The most effective analogues were those bearing weakly basic substituents through a secondary amine linkage, which proved water-soluble (> 10 mM) and potent (IC50S generally < 1 nM). No clear SAR could be discerned for these compounds with respect to amine base strength or the distance of the cationic center from the chromophore, suggesting that 6-substituents are in a favorable area of bulk tolerance in the enzyme binding site. More distinct SAR emerged for the ability of the compounds to inhibit EGFR autophosphorylation in A431 cells, where analogues bearing lipophilic weak bases were preferred. Representative analogues were evaluated for antitumor effectiveness against four in vivo tumor models. Significant in vivo activity was observed in estrogen-dependent MCF-7 breast and A431 epidermoid tumors. Marginal activity was seen in an EGFR-transfected tumor model, suggesting that while this cell line requires EGF for clone formation in soft agar, other growth factors may be able to replace EGF in vivo. Also, no activity was seen against the SK-OV-3 ovarian cancer model, which is known to express other EGF receptor family members (although it is not clear whether these are absolutely required for growth in vivo). While substantial growth delays were seen in A431 and MCF-7 tumor models, the treated tumors remained approximately the same size throughout therapy, suggesting that the compounds are cytostatic rather than cytotoxic under these test conditions. It remains to be determined if more prolonged therapy has cytotoxic effects in vivo, resulting in net tumor cell kill.

Tyrosine kinase inhibitors. 6. Structure-activity relationships among N- and 3-substituted 2,2'-diselenobis(1H-indoles) for inhibition of protein tyrosine kinases and comparative in vitro and in vivo studies against selected sulfur congeners.

A small series of 2,2'-diselenobis(1H-indoles) was synthesized as redox-modified congeners of our earlier reported 2,2'-dithiobis(1H-indole) series. Utilizing chemistry similar to that developed earlier for the disulfur series, compounds were made from 2-halogeno-3-indolecarboxylic acid precursors bearing various polar functionality at the C-3 position and small alkyl substituents at the N-1 position of the indole nucleus. Additional compounds were derived from (R)- or (S)-tryptophan via a novel application of diselenium dichloride as an electrophilic source of diselenium, and a much improved process to a 2,2'-dithiobis(1H-indole) congener was developed utilizing disulfur dichloride as a source of disulfur. Against isolated epidermal growth factor receptor (EGFr), platelet-derived growth factor receptor (PDGFr), and v-src tyrosine kinases, compounds in this series displayed broad inhibitory activity with IC50 = 0.9 to > 100 microM vs EGFr, 3.4 to > 50 microM vs PDGFr, and 0.4-6.7 microM vs v-src. In general, compounds derived from tryptophan displayed the greatest potency against EGFr and those from 2-halogeno-3-indolecarboxylic acids greater potency against PDGFr and v-src. Enzyme kinetics studies showed that both classes of compounds display primarily noncompetitive inhibition with respect to either ATP or peptide substrate. The sulfhydryl reducing agent dithiothreitol (DTT) caused a general decrease in inhibition of the EGFr and v-src tyrosine kinases by both the diselenium and disulfur series with the reversal of enzyme inhibition occurring less readily within the diselenium series. In whole cell studies, compounds of this class were growth inhibitory against Swiss 3T3 mouse fibroblasts with IC50 values from 0.5 to 19.5 microM, and the observed SAR was different from that of the 2,2'-dithiobis(1H-indoles). A comparative study in the same cell line on the effects of the 2,2'-diselenobis(1H-indole) derived from (R)-tryptophan vs its disulfur congener on growth factor mediated tyrosine phosphorylation showed that this compound significantly inhibited EGFr and PDGFr (in response to its ligand) autophosphorylation with complete suppression at 25 and 5 microM, respectively. Tyrosine phosphorylation of an 85 kDa protein typically phosphorylated in response to bFGF was also exquisitely sensitive to this compound, and it displayed inhibitory effects on DNA, RNA, and protein synthesis at submicromolar concentrations. The disulfur congener exhibited a qualitatively similar pattern; however, its potency was 10-fold less. This same diselenium/disulfur pair was evaluated in vivo against the B16 melanoma, colon carcinoma 26, and M5076 sarcoma murine tumors, and the A431 epidermoid, and C6 glioma human tumor xenografts. At maximum tolerated doses (1.8 and 5.0 mg/kg/injection, respectively), neither the diselenium nor disulfur congener was effective against the C6 glioma when administered intraperitoneally on a d1-9 schedule. Studies were also carried out against the A431 epidermoid xenograft to evaluate the same pair of compounds via continuous subcutaneous infusion from Alzet miniosmotic pumps. The maximum dose that could be administered daily was limited by compound solubility. Neither compound produced an antitumor effect in a 7-day continuous infusion study. In the 27-day study, the disulfur compound was inactive whereas the diselenium compound produced a 10.8-day growth delay without appreciable treatment related weight loss. The in vitro and in vivo findings offer a mechanistic rationale as to why the 2,2'-diselenobis(1H-indoles) are more potent inhibitors than their disulfur congeners.

Inhibition of the epidermal growth factor receptor tyrosine kinase by PD153035 in human A431 tumors in athymic nude mice.

PD153035 is a potent (Ki = 6 pm) and specific inhibitor of the epidermal growth factor (EGF) receptor tyrosine kinase that suppresses tyrosine phosphorylation of the EGF receptor in A431 cells at nanomolar concentrations in cell culture. We have examined the pharmacokinetics of this compound and its ability to rapidly suppress phosphorylation of the EGF receptor in A431 human epidermoid tumors grown as xenografts in immunodeficient nude mice. Following a single i.p. dose of 80 mg/kg, the drug levels in the plasma and tumor rose to 50 and 22 microM within 15 minutes. While the plasma levels of PD153035 fell below 1 microM by 3 hours, in the tumors it remained at micromolar concentrations for at least 12 hours. The tyrosine phosphorylation of the EGF receptor was rapidly suppressed by 80-90% in the tumors. However receptor phosphorylation returned to control levels after 3 hours despite the continued presence of the drug at concentrations which, based on previous in vitro results, were predicted to maintain inhibition. EGF-stimulated tyrosine kinase activity in tumor extracts was decreased and recovered in parallel with the effects of PD153035 on receptor phosphorylation though the activity had reached only about half of the control activity after three hours. These results demonstrate the potential for using small molecule inhibitors to inhibit the EGF receptor tyrosine kinase in vivo, though a fair evaluation of their potential anti-cancer activity will have to wait for solutions to problems with sustained delivery which may allow us to maintain suppression of EGF receptor phosphorylation.

Chemotherapy with [SP-4-3-(R)]-[1,1-cyclobutanedicarboxylato(2-)](2- methyl-1,4-butanediamine-N,N')platinum (CI-973, NK121) in combination with standard agents against murine tumors in vivo.

[SP-4-3-(R)]-[1,1-cyclobutanedicarboxylato(2-)](2-methyl-1,4- butane-diamine-N,N')platinum (CI-973) is a cisplatin analogue that is currently in clinical trial. Preclinically, CI-973 retained activity against L1210, P388, K562, and human ovarian carcinoma sublines resistant to cisplatin in vitro. CI-973 also retained substantial activity [ratio of median life span of treated to control groups x 100% (%T/C) > 190] against cisplatin resistant L1210 and P388 sublines in vivo. Good activity (stasis or tumor burden reduction) was also obtained against five murine solid tumors including breast, colon, and sarcoma. Binary combination therapy with CI-973 and seven clinically utilized anticancer agents was evaluated against murine tumors in vivo for the ability of each combination to produce a superior therapeutic response compared to optimal single agent therapy alone at tolerated doses. The seven agents combined with CI-973 were mitomycin C, cyclophosphamide, doxorubicin, vinblastine, etoposide, ifosfamide, and methotrexate. Of the combination regimens evaluated, only the combination of CI-973 and methotrexate was therapeutically superior to single agent therapy. Against i.v. inoculated P388 leukemia, combination therapy with CI-973 at 32 mg/kg/injection and methotrexate at 43 mg/kg/injection produced 5.3 logs greater cell kill (%T/C = 284, with one cell surviving therapy) than either methotrexate therapy (%T/C = 208, with 2.5 x 10(5) cells surviving therapy) or CI-973 therapy (%T/C = 193 with 2.5 x 10(6) cells surviving therapy) alone. The combination toxicity index of 1.0 indicated additive normal host tissue toxicity with the same target organs for dose limiting toxicity. To better understand the enhanced cell kill in vivo, the combination of CI-973 and methotrexate was evaluated against P388 leukemia in vitro. Clonogenic survival studies showed that the combination of CI-973 and methotrexate was additive rather than synergistic with respect to cell killing in vitro. The lack of greater than additive cell kill in vitro suggested that the mechanism responsible for synergistic kill in vivo was not operative in vitro. The in vivo results suggest that the combination of CI-973 and methotrexate may be useful in the clinic.

Sequence and schedule-dependent synergy of trimetrexate in combination with 5-fluorouracil in vitro and in mice.

The purpose of this study was to determine the conditions for optimum synergistic efficacy of the two-drug combination of trimetrexate and 5-fluorouracil. Synergistic cell killing of Chinese hamster ovary cells in these clonogenic survival assays was observed only when the cells had been exposed to trimetrexate (25 microM) for 2 to 4 h prior to 5-fluorouracil exposure (either 125 or 250 microM). The schedule dependence of the observed synergy in vitro was closely linked to trimetrexate-induced changes in cellular 5-phosphoribosyl 1-pyrophosphate (PRPP) pools. Exposure to 25 microM trimetrexate induced increases in PRPP pools to 398% and 761% of control values at 2 and 4 h, respectively. Methotrexate (20 microM) also increased Chinese hamster ovary cell PRPP content in a time-dependent fashion to values of 280 and 511% of control after 2 and 4 h of drug exposure. Previous in vivo studies demonstrated a modest degree of therapeutic synergy between trimetrexate and 5-fluorouracil against P388 leukemia. Our in vitro results suggested that the degree of synergy seen in vivo could be increased with appropriate schedule changes. Mice were implanted i.p. with 10(6) P388 leukemia cells on Day 0 and were treated with trimetrexate (every 3 h for eight injections; Days 1, 5, and 9) and 5-fluorouracil (Days 1, 5, and 9) as single agents or in combination on one of two schedules; 5-fluorouracil was administered with either the first or the last of the eight trimetrexate doses on Days 1, 5, and 9. Both treatment regimens demonstrated therapeutic synergy but, as predicted from the in vitro data, the "5-fluorouracil last" was superior to the "5-fluorouracil first" sequence. Treatment with the optimal doses on the "5-fluorouracil last" sequence (trimetrexate, 31; 5-fluorouracil, 33 mg/kg/injection) produced an increased life span of 183% and a net reduction in tumor cell burden of 6.7 logs compared with a 111% increased life span (net reduction in tumor burden of 2.6 logs) produced by the most active of the single agents, 5-fluorouracil. Thus the efficacy of the combination of trimetrexate with 5-fluorouracil was sequence and time dependent both in vitro and in vivo. The synergy, observed in vitro and probably in vivo, was linked to a trimetrexate-induced elevation of intracellular PRPP, thus facilitating the production of 5-fluoropyrimidine nucleotides. These data are similar to the sequence and schedule dependency of the methotrexate/5-fluorouracil combination with important differences.(ABSTRACT TRUNCATED AT 400 WORDS)

Assay of plant proteins with bicinchoninic acid for high resolution two-dimensional polyacrylamide gel electrophoresis.

A method is described for estimating proteins in the same plant tissue sample that is solubilized for separation by two-dimensional polyacrylamide gel electrophoresis. The method uses a modified bicinchoninic acid (BCA) protein assay procedure and a modified standard urea solubilization buffer to estimate microgram values of unknown protein concentration, in the presence of 9 M urea and 4% Nonidet P-40, from a linear standard curve. A method for a quantitative determination of protein concentration by BCA in a sample containing 9 M urea and 4% Nonidet P-40 is also described. This method is effective for the determination of proteins in minute non-green and green plant tissue, and is especially designed for vegetative and floral shoot apices, and the primordia of inflorescences.