Department of Pharmacology and Therapeutics
Roswell Park Cancer Institute
Elm and Carlton Streets
Buffalo NY USA 14263
Tel: 716 - 845 - 3058
Fax: 716 - 845 - 8857
E-mail: ralph.bernacki@roswellpark.org

Program

Anticancer Drug Development; Drug Resistance; Metastasis and Angiogenesis

The major objective of Dr. Bernacki’s laboratory research program is the development of new chemotherapeutic agents and treatments based on the identification of novel tumor cell determinants. Major emphasis is being placed on studying tumor drug resistance mechanisms, metastasis, and angiogenesis as a means of identifying specific targets for therapeutic manipulation. This is leading to the development and selection of experimental drugs and treatments with the potential to overcome intrinsic and/or acquired drug resistance, decrease tumor angiogenesis and limit metastatic spread. Recent studies, with Dr. Mazurchuk using whole animal MRI imaging, have identified 1-[2-C-cyano-2-deoxy-beta-D-arabino-pentafuranosyl]-N4-palmitoyl cytosine, supplied by Dr. Kobayashi (Sankyo Co.), to be more efficacious than either gemcitabine or 5-FU in inhibiting the development and growth of liver metastases and increasing the life span of nude athymic mice transplanted orthotopically by Dr. Hoffman (AntiCancer Inc.) with a human colon tumor xenograft. These studies suggest that CS-682 may have an impact in the treatment of colon cancer in man.

Progress
Overcoming Multidrug Drug Resistance in Cancer: Paclitaxel is a clinically active agent useful against a wide spectrum of malignancies. It acts in a cytostatic fashion by facilitating a G2/M arrest, and as a cytotoxic agent through induction of cellular apoptosis. Paclitaxel efficacy is limited due to several resistance mechanisms. Our studies have involved overcoming resistance mediated through the cell membrane efflux pump P-glycoprotein (Pgp), which is a mdr1 gene product that confers multi-drug resistance to cells. Numerous chemotherapeutic agents, including paclitaxel have demonstrated high affinity to Pgp, thus greatly decreasing their intracellular accumulation and limiting anti-tumor activity.

Orataxel (formerly known as IDN-5109 and BAY 59-8862) is a semisynthetic, orally bioavailable, taxane which has shown superior efficacy against various Pgp expressing tumor cell lines in comparison to paclitaxel and docetaxel. Orataxel facilitates a G2/M arrest in the Pgp transfected MDA435/LCC6mdr1 breast cell line at much lower drug concentrations than paclitaxel and docetaxel. In P-glycoprotein expressing tumor cell lines we have observed that orataxel increases the intracellular levels and decreased efflux of Pgp substrates Rh-123 and Doxorubicin, inferring Pgp modulation. Using [3H]-labeled orataxel, we demonstrated that it accumulates to significantly higher levels than paclitaxel in Pgp expressing tumor cell lines in-vitro. The non-toxic, taxane based, multi-drug resistance reversal agent, tRA96023, also was able to markedly increase intracellular levels of [3H]-paclitaxel and [3H]-orataxel in Pgp expressing tumor cells. This finding likely explains the increase in efficacy observed both in-vivo and in-vitro when tRA96023 is combined with paclitaxel or orataxel. Studies using [3H]-orataxel and [3H]-paclitaxel in mice have demonstrated differences in plasma half-life and biodistribution in mice implanted with Pgp positive tumors. Orataxel is retained significantly longer in the plasma, brain and Pgp-positive tumors in comparison to paclitaxel and may have increased efficacy for the treatment of brain tumors.

Recent studies with Dr. Baer and Minderman, designed to evaluate the modulatory ability and structure activity relationships of orataxel and taxane reversal agents against not only P-glycoprotein (Pgp)-, but also multidrug resistance protein (MRP-1)-, and breast cancer resistance protein (BCRP)- mediated mitoxantrone transport. Twenty tRA’s were selected to study their modulatory activity against non-Pgp-mediated drug efflux. Human myeloma cell lines 8226/Dox6 and 8226/MR20, expressing Pgp and BCRP, respectively, and the human myeloid leukemia cell line HL60/Adr, expressing MRP-1 served as human MDR cell line models. Orataxel effectively modulated mitoxantrone retention and cytotoxicity in cell lines expressing MRP-1 and BCRP, in addition to Pgp. Orataxel, however due to its cytotoxic potency, is not optimal for development as a clinical MDR modulator so we sought to select a non-toxic taxane drug resistance reversal agent (tRA) capable of inhibiting not only Pgp but MRP and BCRP. Four taxane-based reversal agents (tRAs) strongly modulated doxorubicin and mitoxantrone efflux and enhanced their cytotoxicity in cell lines expressing the MDR proteins, decreasing IC50s by as much as 97%. These tRAs, especially tRA98006, have promise for development as clinical broad-spectrum MDR modulators and show great promise as new agents for clinical combination chemotherapy in the treatment of multidrug resistant leukemias.

Preclinical Drug Development Studies: An integral function of this laboratory is the preclinical therapeutic evaluation (or screening) of diverse chemotherapeutic agents or treatments as part of a Pharmacology & Therapeutic programmatic effort in antitumor drug development.
Several chiral analogs of tamoxifen, a potent anti-estrogen, synthesized by Dr. Huw Davies, SUNYAB, have been found to have superior growth inhibitory activity against an ER positive, human breast tumor cell line, in comparison to the parent compound. These agents are being evaluated with a series of tamoxifen resistant breast tumor cell lines,to develop second generation analogs with broad therapeutic potential for the treatment of breast cancer.

Metastasis Research: In order to facilitate preclinical evaluation of potential antimetastatic agents, we examined the metastatic spread of human colorectal tumors implanted into nude athymic mice. To date, our studies have indicated that most human tumor xenografts implanted sc in nude athymic mice metastasize poorly. Implantation of human colon tumor intraspenically in mice facilitated metastasis to the liver, but in low incidence. However, AC3488UM human colon tumor cell line, provided by Dr. Hoffman, AntiCancer Inc., was implanted orthotopically into the cecum of mice, resulting in multiple tumor foci quickly appearing in liver. MRI images, obtained by Dr. Mazurchuk in the CCSG Animal MRI Resource, clearly demonstrated increasing primary tumor growth and evidence of frank tumor metastases and growth of multiple tumor foci in liver. This human tumor xenograft model was used to assess the antimetastatic efficacy of several clinical candidate agents including CS-682, a lipophilic deoxycytosine analog. CS-682 was administered po to mice implanted orthotopically with AC3488UM human colon tumor xenograft. Relative to control, the overall occurrence of metastases was decreased by 62% with CS-682 treatment. ILS (increased lifespan) above the 29-day mean survival without treatment, was 28 days with CS-682. Overall, CS-682 administration significantly reduces primary and secondary tumor growth in mice and has potential for the treatment of colon cancer in man.

Angiogenesis Research: Tumor angiogenesis is a prerequisite for tumor growth and progression; it also offers the tumor a route for facilitating metastasis. The development of the vasculature in the tumor from surrounding, normal endothelial cells offers yet another site for interfering in primary tumor growth and metastasis. With the discovery of various growth factors (e.g. bFGF), released by tumor cells, capable of inducing endothelial cell migration, growth and formation of capillary vessels, specific approaches at interfering with these processes is emerging. This laboratory has recently developed the capability for screening inhibitors of endothelial cell growth, migration, micromotion (with Dr. Sawhney) and the formation of capillary vessels in vitro. Certain signal transduction inhibitors, more specifically, two bFGF-receptor tyrosine kinase inhibitors, and a semisynthetic taxane IDN5390 have demonstrated potent antiangiogenic activity in both the in vitro and in vivo assays. We are currently utilizing functional MRI (fMRI), performed by Dr. Mazurchuk, to measure drug-induced changes in tumor oxygenation levels as a correlate of altered tumor blood flow. It is anticipated that treatment with antiangiogenic agents will reduce tumor blood flow, and that fMRI will allow pharmacodynamic measurement in prediction of antitumor efficacy.

Constitutive E-selectin expression on dermal microvascular endothelial cells plays a critical role in mediating adhesive interactions of human skin-homing T-cells and in pathologic accumulation of lymphocytes in skin and in T-cell lymphoma metastases to skin. The major E-selectin ligand on human skin-homing T-cells is cutaneous lymphocyte-associated antigen (CLA), a specialized glycoform of P-selectin glycoprotein ligand-1 (PSGL-1). A novel fluorinated analog of N-acetylglucosamine (4-F-GlcNAc), synthesized by Drs. M. Sharma, Matta and Paul at RPCI was shown by Drs. Dimitroff and Sakstein (Harvard) to inhibit the expression of poly-lactosaminyl-containing HECA-452 on human CLA+ T-cells, preventing tethering and rolling on selectins under shear stress. 4-F-GlcNAc was incorporated directly into native CLA expressed on T-cells, indicating direct inhibition on poly-N-acectyllactosamine elongation and selectin-binding determinants on PSGL-1 O-glycans. These observations establish a potential treatment approach for targeting pathologic lymphocyte trafficking to skin and indicate that 4-F-GlcNAc may be a promising agent for treatment of dermal tropism associated with malignancies and inflammatory disorders.

Select Publications

• Leontieva OV, Preobrazhenskaya MN, Bernacki RJ. Partial circumvention of P-glycoprotein-mediated multidrug resistance by doxorubicin 14-O-hemiadipate. Investigational New Drugs, 20: 35-48, 2002
• Marasco CJ Jr, Kramer DL, Miller J, Porter CW, Bacchi CJ, Rattendi D, Kucera L, Lyer N, Bernacki R, Pera P, Sufrin JR. Synthesis and evaluation of analogues of 5’-([(Z)-4-amino-2-butenyl]-methyl-amino)-5’-deoxyadenosine as inhibitors of tumor cell growth, trypanosomal growth, and HIV-1 infectivity. J. Med. Chem. 45: 5112-5122, 2002
• Ojima I, Geng X, Lin S, Pera P, Bernacki RJ. Design, synthesis and biological activity of novel C2-C3’ N-linked macrocyclic taxoids. Bioorg. Med. Chem Lett 12: 349-352, 2002
  Dimitroff CJ, Bernacki RJ, Sackstein R. Glycosylation-dependent inhibition of cutaneous lymphocyte-associated antigen expression: implications in modulating lymphocyte migration to skin. Blood 101: 602-610, 2003
• Wu M, Mazurchuk R, Chaudhary ND, Spernyak J, Veith J, Pera P, Greco W, Hoffman RM, Kobayashi T, Bernacki RJ. High resolution magnetic resonance imaging of efficacy of cytosine analog CS-682 in a liver-metastasis nude mouse model. Cancer Res. 63: 2477-2482, 2003
• Brooks T, Minderman H, O’Louglin K, Pera P, Ojima I, Baer M, Bernacki RJ. Taxane-based reversal agents modulate drug resistance mediated be P-glycoprotein, multidrug resistance protein and breast cancer resistance protein. Mol Cancer Thera. 2:1195-1205, 2003