Chuan Shih

Multiple folate enzyme inhibition: mechanism of a novel pyrrolopyrimidine-based antifolate LY231514 (MTA)

Extensive biochemical and pharmacological evidence indicates that LY231514 is a novel antifolate antimetabolite. LY231514 is transported into cells mainly through the reduced folate carrier system and extensively metabolized to polyglutamated forms. The polyglutamates of LY231514 inhibit at least three key folate enzymes: TS, DHFR, and GARFT, and to a lesser extent AICARFT and C1-tetrahydrofolate synthase. The combined effects of the inhibition exerted by LY231514 at each target give rise to an unusual end-product reversal pattern at the cellular level that is distinct from those of other inhibitors such as methotrexate and the quinazoline antifolates. The metabolic effects exerted by LY231514 on the folate and nucleotide pools are also quite distinct from those of MTX and LY309887. The efficient polyglutamation, longer cellular retention and the multiple folate enzyme inhibition mechanism may all have contributed directly to the exciting antitumor responses now observed in Phase I and II studies. The multitargeted inhibition mechanism of LY231514 is particularly intriguing. This new level of mechanistic insight, which has evolved from the study of LY231514, challenges the traditional concept and paradigm of antifolate drug discovery and development which focused on developing very potent and selective inhibitors of single folate enzyme targets, such as DHFR, TS or one of the enzymes along the de novo purine biosynthetic pathway. Given the complex nature of folate metabolism and the critical role of folates in maintaining the physiological functions of living systems, it is completely reasonable to suspect that agents which can interfere at multiple sites in the folate pathway may trigger and cause more biochemical imbalance in the cellular DNA and RNA synthesis of malignant cells than agents that act on a single point (Fig. 5). In conclusion, LY231514 (MTA) is a new generation antifolate antimetabolite demonstrating inhibitory activity against multiple folate enzymes including TS, DHFR and GARFT. In current phase II studies, MTA is broadly active as a single agent and is showing very encouraging antitumor activity in multiple solid tumors including colorectal, breast and non-small cell lung cancers (38-43). The every three week dosing schedule has proven to be convenient and easy to administer and the clinical toxicities of LY231514 seem to be well tolerated. More advanced and extensive clinical trials of LY231514 are currently in progress.

Synthesis of quinolinyl/isoquinolinyl[a]pyrrolo [3,4-c] carbazoles as cyclin D1/CDK4 inhibitors.

A novel series of pyrrolo[3,4-c] carbazoles fused with a quinolinyl/isoquinolinyl moiety were synthesized and their D1/CDK4 inhibitory and antiproliferative activity were evaluated. Compound 8H, 14H-isoquinolinyl[6,5-a]-pyrrolo[3,4-c]carbazole-7,9-dione (1d) was found to be a highly potent D1/CDK4 inhibitor with an IC(50) of 69 nM. Compound 1d also inhibited tumor cell growth, arrested tumor cells in G1 phase and inhibited pRb phosphorylation.

Biochemistry and pharmacology of glycinamide ribonucleotide formyltransferase inhibitors : LY309887 and lometrexol

SummaryLometrexol, a tight-binding antifolate inhibitor of the purine de novo enzyme glycinamide ribonucleotide formyltransferase (GARFT), was the first GARFT inhibitor to be investigated clinically. Unexpected observations of delayed cumulative toxicity prompted a search for a second generation antimetabolite with a more favorable biochemical, pharmacological and toxicological profile. LY309887, 6R-2′,5′-thienyl-5,10-dideazatetrahydrofolic acid, had 9-fold greater potency to inhibit GARFT (Ki = 6.5 nM) compared to lometrexol. Like lometrexol, LY309887 was activated by folypolyglutamate synthetase, however, it had a lower first order rate constant. In vitro and in vivo data were consistent with these observations: polyglutamation of LY309887 was less extensive compared to lometrexol and livers of mice accumulated fewer polyglutamates of LY309887 than polyglutamates of lometrexol. The affinities of these two compounds for isoforms of human folate receptors (FR) were compared. Lometrexol had a 6-fold higher affinity for FRα than LY309887 and both compounds had higher affinity for the α isoform compared to the β isoform. The selectivity of LY309887 for FRα (β(Ki)/α(Ki) = 10.5) was twice that of lometrexols (β/α = 5.0). Lometrexol and LY309887 were potent cytotoxic compounds against the human leukemia cell line CCRF-CEM with IC50s of 2.9 nM and 9.9 nM, respectively. In vivo, LY309887 was more potent than lometrexol at inhibiting tumor growth in the C3H mammary murine tumor model and several tumor xenografts. Excellent efficacy was achieved by both compounds in several colon xenografts. In two pancreatic human xenografts, LY309887 achieved greater efficacy than lometrexol. In summary, the biochemical and pharmacological properties of lometrexol and LY309887 support the hypothesis that these antifolates will have clinical activity against human solid tumors. LY309887 is a second generation GARFT inhibitor with biochemical and pharmacological properties which distinguish it from lometrexol and suggest that it will have broad antitumor activity, a different pharmacokinetic profile and produce less toxicity than lometrexol in cancer patients.

Synthesis and biological evaluation of cryptophycin analogs with substitution at C-6 (fragment C region).

Analogs of the antitumor agents cryptophycins 1 and 8 with dialkyl substitution at C-6 (fragment C) were synthesized and evaluated for in vitro cytotoxicity against human leukemia cells (CCRF-CEM). The activity of these analogs decreased as the size of the substituents at C-6 increased. The C-6 spirocylopropyl compound (2g) was highly potent in vitro and showed excellent antitumor activity in animal models.

Discovery and preclinical antitumor efficacy evaluations of LY32262 and LY33169

The discoveries of a new antitumor agent (LY32262) (N-[2,4-dichlorobenzoyl]phenylsulfonamide) and a close analog (LY33169) are described. For this discovery, a disk-diffusion-soft-agar-colony-formation-assay was used to screen a portion of the Eli Lilly inventory, with the evaluation of each agent against normal cells, leukemic cells and several solid tumors, including a multidrug-resistant solid tumor (with marked selective cytotoxicity for Colon-38 and Human-Colon-15/MDR compared to normal fibroblasts and L1210 leukemic cells characterizing the discovery). In mice, LY32262 and/or LY33169 had curative activity against Colon Adenocarcinoma-38, Human Colon-116, Human Prostate LNCaP, and Human Breast WSU-Br-1. In addition, many other tumors were highly sensitive: Panc-03=2.4 log kill (LK); Panc-02=2.9–4.1 LK; Squamous Lung LC-12=2.1 LK; Colon-26=2.2 LK; AML1498=2.7 LK; Human Sm Cell Lung DMS-273=6.3 LK; Human Squamous Lung 165=3.7 LK; Human Ovarian BG-1=3.7 LK; Human Colon CX-1 (H29)=1.6 LK; Human Colon-15/MDR (a p-glycoprotein positive multidrug resistant tumor)=2.3 LK; Human CNS-gliosarcoma-SF295=3.8 LK. Several tumors were only marginally responsive or totally unresponsive: Mammary Adenocarcinoma-16/C=0.6 LK; Mammary Adenocarcinoma-17=no kill; Colon Adenocarcinoma-11=no kill; L1210 leukemia=1.3 LK; Human Prostate PC-3=0.5 LK; Human Adenosquamous Lung H125=no kill; and Human Breast Adenocarcinoma MX-1=0.9 LK. There was no absolute tissue of origin correlation with antitumor efficacy, although colon tumors were most responsive and mammary tumors least responsive. The cause of the “hit and miss” efficacy has not been determined.

Dietary folate and folylpolyglutamate synthetase activity in normal and neoplastic murine tissues and human tumor xenografts.

The importance of polyglutamation for the activation of natural folates and classical antifolates and recent evidence for the role of dietary folate as a biochemical modulator of antifolate efficacy led us to investigate the influence of changes in dietary folate on folylpolyglutamate synthetase (FPGS) activity. Activities were measured using lometrexol (6R-5,10-dideazatetrahydrofolic acid) as a substrate for FPGS with extracts of murine tissues, murine tumors, and human tumor xenografts from mice on standard diet or low folate diet. Tissues and tumors from mice on standard diet exhibited a 6-fold range of FPGS activity. Kidney had the lowest activity (36 pmol/hr.mg protein), followed by the human xenograft PANC-1 pancreatic carcinoma (46 pmol/hr.mg protein), liver (109 pmol/hr.mg protein), murine C3H mammary tumor (112 pmol/hr.mg protein), and the human xenograft MX-1 mammary carcinoma (224 pmol/hr.mg protein). In response to restricted dietary folate, four out of five tissues had significantly increased (25-50%) FPGS activity. Only the tumor with highest FPGS activity under standard diet conditions (MX-1 mammary) did not respond to low folate diet. The results indicate that changes in dietary folate intake can modulate FPGS activity significantly in vivo and suggest that the tissue distribution and toxicities of classical antifolates requiring polyglutamation for activation and cellular retention will be influenced significantly by folate status of the host.

Expression of pRB, cyclin/cyclin-dependent kinases and E2F1/DP-1 in human tumor lines in cell culture and in xenograft tissues and response to cell cycle agents.

Purpose: Cell cycle regulatory components are interesting targets for cancer therapy. Expression of pRb, cyclin D1, cdk4, cyclin E, cdk2, E2F1 and DP-1 was determined in MCF-7 and MDA-MB-468 breast carcinoma cells, H460 and Calu-6 non-small cell lung carcinoma cells, H82 and SW2 small cell lung carcinoma cells, HCT116 and HT29 colon carcinoma cells and LNCaP and DU-145 prostate carcinoma cells. Methods: For Western blotting, the ratio with actin expression was used to normalize the data; all lines were run on the same gels. Results: In cell culture, pRb was not detected in MB-468 and H82 was low in SW2 and DU-145 and highest in HCT116; in tumors, pRb was not detected in MB-468, H82, SW2, and DU-145 and was highest in LNCaP and Calu-6. Cyclin D1 was not detected in SW2 cells in culture, was low in MB-468 and H82, and was highest in LNCaP and H460; in tumors, cyclin D1 was low in MB-468, H460, SW2 and DU145, and was highest in LNCaP. In cell culture, cdk4 was lowest in Calu-6, HCT116, HT29 and DU-145 and highest in H82 and SW2; in tumors, cdk4 was low in MCF-7, MB-468, H460, Calu-6 and HCT116 and was very high in the SW2. Expression of cyclin E was very low in MCF-7 and HT29 and high in H460 in culture and was very low in MCF-7, H460, Calu-6, H82, HT29 and DU-145 in tumors and high in HCT116 and LNCaP. In cell culture, E2F1 was lowest in MB-468, Calu-6, HT29 and DU-145 cells and highest in LNCaP cells; in tumors, E2F1 was lowest in MCF-7, MB-468 and Calu-6 and highest in LNCaP. In cell culture, DP-1 was lowest in MB-468, HCT116 and HT29 and highest in SW2. The MCF-7 and MB-468 lines were most resistant to flavopiridol and olmoucine and the H460 and Calu-6 lines were most resistant to genistein. The SW2 tumor was most responsive to flavopiridol and olomoucine. Conclusions: There is a high degree of variability in the expression of cell cycle components in human tumor cell lines, resulting in complexity in predicting response to cell cycle directed agents.

Synthesis and biological activity of nor- and homo-5,10-dideazatetrahydrofolic acid

Abstract A palladium(0)-catalyzed Heck coupling reaction was used in the synthesis of nor- and homo-DDATHF which are close analogs of the potent glycinamide ribonucleotide formyltransferase inhibitor, 5,10-dideazatetrahydrofolic acid (DDATHF).

Abstract 3611: LY2801653, an orally available small molecule inhibitor of c-Met, demonstrated broad antitumor efficacy in patient derived xenograft models

The receptor tyrosine kinase c-Met and its ligand hepatocyte growth factor (HGF) play important roles in cell growth, migration, and survival. Dysregulation of the c-Met/HGF signaling pathway has been found in many human malignancies. LY2801653 was identified and developed as a novel, potent, and orally active small molecule inhibitor of human c-Met. It demonstrated dose dependent inhibition of c-Met phosphorylation in xenograft tumors with a long lasting PD effect. In the current study, the anti-tumor activity of LY2801653 was evaluated in a panel of human patient derived xenograft models. LY2801653 displayed potent anti-tumor efficacy in a number of non small cell lung, renal, pancreatic, and breast tumor models. Examination of c-Met expression in these tumors by immunohistochemistry (IHC) revealed a good correlation between response and c-Met expression in the tumor tissue. Interestingly, the two non small cell lung adenocarcinoma models LXFA526 and LXFA1647 that were resistant to EGFR inhibitors Cetuximab and Erlotinib, displayed high sensitivity towards LY2801653. High level of genomic amplification of c-Met locus was found in these models, consistent with the hypothesis that c-Met activation represents one of the resistant mechanisms to EGFR inhibition. LY2801653 treatment led to increase in functional vessel areas, and decrease in tumor hypoxia. Enhanced anti-tumor efficacy was achieved when Erlotinib was combined with LY2801653. In summary, these results demonstrated the promising therapeutic potential of LY2801653 in mono and combination therapy of cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3611.

Abstract 4511: LY2801653 treatment increases survival of mice bearing H441 orthotopic lung tumors

Lung cancer is the leading cause of cancer-related death for both men and women in the world. c-Met receptor tyrosine kinase (RTK) signaling pathway has been implicated in tumor growth, angiogenesis, and metastasis. Sustained activation, over-expression, or mutation of c-Met is associated with poor prognosis in several tumors, including NSCLC. Therefore, c-Met is emerging as a novel target of therapeutic potential for lung cancer. We have discovered and developed a novel, ATP competitive, and orally active small molecule inhibitor of c-Met, LY 2801653. It is a potent inhibitor of c-Met phosphorylation in vivo, and demonstrated anti-tumor activity in several xenograft tumor models. In the current study, we evaluated the efficacy of LY2801653 in both c-Met dependant and c-Met independent NSCLC cells in vitro and in vivo orthotopic lung models that recapitulated the local and regional growth pattern seen in lung cancer patients. In c-Met dependant H441 model, LY2801653 demonstrated the significant inhibition of both primary tumor (90% inhibition) and metastasis (65% inhibition of lymph node and 70% inhibition of chest wall); whereas in c-Met independent H1299 model, LY281653 only inhibited primary tumor growth (70% inhibition). The benefit of LY2801653 treatment was further demonstrated in the survival of mice bearing H441 lung tumors. Treatment with LY2801653 significantly increased the survival of mice to 192% compared to that in the vehicle treated mice. The anti-tumor and/or anti-metastasis mechanism of LY2801653 was also investigated. These results demonstrated therapeutic potential of LY2801653 in the treatment of lung cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4511.