Tyos
`
`nnual meeting of the
`r’can Associatio
`Cancer Researc
`
`May 15-18, 1991
`Houston Texas
`
`-~"
`
`Sandoz Inc.
`Exhibit 1011-0001
`
`JOINT 1011-0001
`
`

`
`PRECLINICAL PHARMACOLOGY/EXPERIMENTAL THERAPEUTICS
`
`1920
`Folate Analogues: Nonpolyglutamylatable inhibitors of thymidylate
`synthase (TS) and GAR-formyltransferase (GARFTase). Nair, M.G,
`Abraham, A., Kisliuk, R.L., McGuire, J3;, Galivan J. Univ. of. South
`Alabama, Mobile, AL 36688, Tufts University, Boston, MA 02111, Roswell
`Park Cancer Institute, Buffalo, NY 14263, and Wadsworth Center for
`Laboratories and Research, Albany, NY 122011
`We have previously shown that replacement of the L-glutamate moiety
`of classical 4-amino antifolates such as methotrexate and 10-deazaamino-
`pterins with a 4-methylene glutamate results in a) potent inhibition of
`dihydrofolate reductase, b) high level of antifolate activity during continuous
`exposure to tumor ceils, c) enhanced ability to inhibit folinic acid transport
`to tumor cells relative to methotrexate and d) complete loss of substrate
`activity to folylpolyglutamate synthetase. These studies have now been
`extended to other antifolates, by r~placing the glutamate moiety of TS
`inhibitors PDDF and DMPDDF and the GARFTase inhibitor DDATHF, with
`4-methylene glutamate. The new compounds are non-polyglutamylatable and.
`effective inhibitors of folinic acid transport to H35 hepatoma cells. How-
`ever, the 4-methyleneglotamate derivatives are much weaker antifolates than
`their predecessors as judged by their ability to inhibit the growth of H35
`hepatoma and CCRF-CEM human leukemia cell growth. The results clearly
`indicate that polyglutamylation is a major determinant of non-DHFR type
`antifolate cytotoxicity.
`
`1921 ~
`Reversal of the toxicity but not the autitumor activity of
`Lometrexol by folic acid:
`Grindey, G.B., Alati, T,, and Shih, C. Lilly Research Labs.
`Indianapolis, Ind. 46285.
`Lometrexol, 5,10-dideazatetrahydrot’olic acid (DDATHF) has
`broad spectrum activity and good therapeutic index in routine and
`human xenograft carcinomas. However, severe toxicity was
`observed during initial Phase I trials (R.L. Nelson, pers. comm.).
`DDATHF (12.5-50 mg/kg/day X 5) is non-toxic and achieves >95%
`inhibition at all dose levels using C3H mammary carcinoma. In
`C3H mice placed on a relate-free diet for two weeks, DDATIIF
`toxicity is increased 100-fold. Antitumor activity is markedly
`reduced with only 21% and 88% inhibition o[ tumor growth
`observed at maximally tolerated doses of 0.25 and 0.5 mg/kg,
`respectively. High doses or, relic acid in the drinking water
`completely reverse both toxicity and activity. In contrast, low
`doses of relic acid (0~003%) prevent this dietary-induced toxicity
`and >90% inhibition of tumor growth is achieved from 6.25-50
`mg/kg of DDATHF. The tight-binding of DDATHF to the relic acid
`transport protein may be involved in these observations. The
`activity and toxicity of related analogs under these conditions is
`under evaluation. These results support the use of low doses of
`oral relic acid to reduce the toxicity of DDATHF in clinical trials.
`
`1923
`An altered Kt for the reduced folate transport
`system confers reslsta~ce to Lometrexol
`( (6R) DDATHF). O.Russello, B.A.Moroson,
`A.R.Cashmore, A.D.Cross, G.P.Beardsley. Dept.
`Pediatrics, Yale Univ. ,New Haven, CT 06510.
`A highly DDATHF resistant human lymphoblastic
`leukemia cell line (CCRF-CEM/CR 14) was obtained
`by further exposure of a previously developed
`resistant cell line (CRI5), to 1 mM (6R) DDATHF.
`The CRI5 line has defective polyglutamylation,
`but normal levels of GAR TFase and normal
`reduced folate transport. The derived CRI4 line
`can be mantained in 0.i mM (6R) DDATHF, 5 orders
`of magnitude above the ED 50 for the parent cell
`line. The CRI4 line showed the same
`polyglutamylation defect seen in CRI5. Kinetic
`parameters for GAR TFase from CRI4 were not
`different from those from the parent cell line.
`Study of the transport system revealed an
`increased Kt, but no differences in V ax or
`number of blndlng sltes. The same alteratlons in
`affinity were found for (6S)DDATHF, methotrexate
`and leucovorin. (CA 50721,IST and AIRC, Italy.)
`
`1924
`High-level methotrexate resistance in a human breast
`cancer cell line secondary to a novel membrane
`translocation defect.
`Pinard M-F and Jol~vet J. Institut du Cancer de
`Montr6al, 1560 Sherbrook~ east, Mont~4ai H2L 4MI.
`A 1,000-fold methotrexate (MTX) resistant ZR-75-1
`human breast cancer cell line w~th deficient
`polyglutamylation (J B~ol Chem 259: 10793, 1984) was
`found to be unable to accumulate MTX during short
`exposures to high drug concentrations. MTX initial
`uptake was only mildly abnormal w~th a 2.3-fold
`increased Kt in the resistant compared to the wild type
`cells and no Vmax alterations. At MTX concentrations
`~IO#M, initial uptake kinetics became identical and the
`cytotoxicity curves superimposable in both sensitive
`and resistant cells. The latter still could not
`accumulate or transstimulate MTX however, suggesting
`abnormal translocation of the reduced folate/MTX
`membrane carrier across the cell membrane and likely
`explaining the almost absent polyglutamate formation.
`Impaired MTX translocation can thus be responsible for
`high levels of drug resistance.
`
`1922
`Trimetrexate resistance in human colon cancer cells is
`associated with acute induction of dihydrofelatc rcductasc.
`JL Grem, DM Boatman, P Daychild, CJ Allegra. Medicine
`Branch, NCI, Bethesda, MD.
`The IDs~ for TMTX (24 h exposure (exp.)} in a cloning
`assay was 0.44 pM in SNU-C4 (C4) cells, while IICT-I!6 (116)
`and NCI-H630 (630) cells were 37- and 200-fold less sensi-
`tive. We studied several 8otentia[ resistance mechanisms.
`Uptake and retention o; |’~C]TMTX were similar i,, all 3
`lines. Differences in dTTP, ATP and GTP deplel, ion with TMTX
`did not account for the variable sensitivity~’ nor did dif-
`feret~ees in baseline DIIFR properties including catalytic
`act., binding capacity and binding affinity. ’rhymidylate
`synthase act. was 45% and 73% lower in 116 and 630 cells.
`Exp. to I ~M TMTX for 24 h resulted in a signif, increase
`(1.8- to 2.2-fold) in total DIIFR content in the 2 resistant
`lines (in binding assays and Western blot analysis). The
`increase in DilFR Content during TMTX was evident by 6-12 h
`and was prevented 5y concomitant exp. to tile protein
`synthesis inh~bitor cyclobeximide. The relevance of this
`phenomemon was suggested by rapl~ recovery of t,ree,
`bio]oglcally active DHFR ~u~d [6-’ll]dtJrd incorporation
`after TMTX removal in the resistant cells.
`
`1925
`In vlvo antitumor activity and metabolism of a series of
`open chain folate based GAR transformylase inhlbitors.
`Mullin, R.J., Bigham, E.C., Duch, D.S., Ferone, R., Keith,
`B.R., Smith, G.K., and Waters, K.A. Wellcome Research
`Laboratories 3038 Cornwallis Rd., R.T.P~, NC 27789
`The activity of 5-deazaacylotetrahydrofolate (5-DACTHF)
`an inhibitor of purlne de novo biosynthesis and potential
`antitumor agent has been reported. This study compares the
`properties of 5-DACTHF and a series of analogs, 2’-F,
`3’-F, 18-S and IO-CH=. All analogs have similar ICs~
`values for inhibition of MCF-7 cell growth, GAR trans-
`formylase, and methotrexate uptake by Molt-4 cells, a
`measure of cellular uptake potential. Only 5-DACTHF, 2’-F,
`and 3’-F demonstrate significant inhibition of colon 38
`adenocarcinoma growth in vivo. This correlates with the Km
`of these compounds for folylpolyglutamate synthetase. In
`support of this correlation, 24 hours after dosing, only
`those compounds with antitumor activity were detectable in
`tumor tissue, and they were present nearly exclusively as
`the polyglutamated species despite similar blood levels
`for all compounds. These results indicate that, following
`uptake, polyglutamation represents a critical step in the
`in vivo anti tumor activity of these compounds.
`
`PROCEEDINGS OF THE AMERICAN ASSOCIATION FOR CANCER RESEARCH
`VOLUME 32 m MARCH 1991
`324
`
`Sandoz Inc.
`Exhibit 1011-0002
`
`JOINT 1011-0002