Ralph J. Bernacki

Glycosidases in cancer and invasion

SummaryGlycosidases have been demonstrated to be elevated in the interstitial fluid of tumors, sera of animals and patients with tumors, and in some tumor tissue as compared to normal adjacent tissue. Elevations of serum β-N-acetylglucosaminidase and β-glucuronidase most commonly have been found to occur and these enzymes have been shown to be secreted into the extracellular medium by many different tumor cell types in vitro. The mechanism of cellular release of these hydrolytic enzymes probably involves tumor lysosomal exocytosis. Increased tumor glycosidase levels may promote increased tumor cell shedding from primary tumors, local invasion and perhaps be responsible directly, or indirectly for structural changes in tumor cell surface glycoconjugates. These cell surface changes could facilitate tumor cell thrombus formation, secondary site implantation and attachment in the microcirculation to endothelial cells and/or subendothelial basement membrane components.Other studies have demonstrated a correlation between metastatic cell potential and increased endoglycosidase and polysaccharide lyase activity. Generally, metastatic tumor cell variants have been found to be more invasive and capable of degrading proteoglycan basement membrane components, in part due to these increased levels of degradative enzymes. Hence, it is of considerable interest to develop inhibitors against these enzymes. Initial studies with glucuronidase inhibitors in the therapy of bladder tumors have been promising and with the advent of better agents and the use of appropriate in vitro metastatic models it may be possible to design and develop agents which interfere in various metastatic events and limit tumor progression.

Activity of paclitaxel liposome formulations against human ovarian tumor xenografts.

Although the current clinical formulation of paclitaxel (Taxol®) is an important new anti‐cancer agent, it has significant side effects, some of which are related to its formulation in Cremophor/ethanol, Paclitaxel is difficult to formulate for i.v. administration because of its poor aqueous solubility. Here, we report the therapeutic effects of 2 liposome formulations of paclitaxel against human ovarian A121 tumor growing as an s.c. xenograft in athymic nude mice. The liposome formulations used were ETL and TTL, which have 1 or 3 lipid components, respectively. TTL was used as a reconstituted lyophilate or as a stable aqueous suspension. ETL was used as a reconstituted lyophilate only. Both paclitaxel‐liposome formulations were much better tolerated than Taxol® after i.v. or i.p. administration. The acute reactions seen after Taxol® administration did not occur when paclitaxel‐liposome formulations were administered. All ETL and TTL preparations significantly delayed A121 tumor growth similarly to Taxol at equivalent doses and schedules. Based on pharmacokinetic data, it is possible that paclitaxel rapidly dissociates from ETL or TTL after i.v. administration and distributes in a manner similarly to Taxol. ETL and TTL formulations may be useful clinically not only for eliminating toxic effects of the Cremophor/ethanol vehicle but also for allowing alterations in route and schedule of drug administration. Int. J. Cancer, 71:103–107, 1997.

Anti-Angiogenic Activity of Selected Receptor Tyrosine Kinase Inhibitors, PD166285 and PD173074: Implications for Combination Treatment with Photodynamic Therapy

Angiogenesis, the formation of new blood vessels from an existing vasculature, is requisite for tumor growth. It entails intercellular coordination of endothelial and tumor cells through angiogenic growth factor signaling. Interruption of these events has implications in the suppression of tumor growth. PD166285, a broad-spectrum receptor tyrosine kinase (RTK) inhibitor, and PD173074, a selective FGFR1TK inhibitor, were evaluated for their anti-angiogenic activity and anti-tumor efficacy in combination with photodynamic therapy (PDT). To evaluate the anti-angiogenic and anti-tumor activities of these compounds, RTK assays, in vitro tumor cell growth and microcapillary formation assays, in vivo murine angiogenesis and anti-tumor efficacy studies utilizing RTK inhibitors in combination with photodynamic therapy were performed. PD166285 inhibited PDGFR-β-, EGFR-, and FGFR1TKs and c-src TK by 50% (IC50) at concentrations between 7−85nM. PD173074 displayed selective inhibitory activity towards FGFR1TK at 26nM. PD173074 demonstrated (>100 fold) selective growth inhibitory action towards human umbilical vein endothelial cells compared with a panel of tumor cell lines. Both PD166285 and PD173074 (at 10nM) inhibited the formation of microcapillaries on Matrigel-coated plastic. In vivo anti-angiogenesis studies in mice revealed that oral administration (p.o.) of either PD166285 (1−25 mg/kg) or PD173074 (25−100 mg/kg) generated dose dependent inhibition of angiogenesis. Against a murine mammary 16c tumor, significantly prolonged tumor regressions were achieved with daily p.o. doses of PD166285 (5−10 mg/kg) or PD173074 (30−60 mg/kg) following PDT compared with PDT alone (p<0.001). Many long-term survivors were also noted in combination treatment groups. PD166285 and PD173074 displayed potent anti-angiogenic and anti-tumor activity and prolonged the duration of anti-tumor response to PDT. Interference in membrane signal transduction by inhibitors of specific RTKs (e.g. FGFR1TK) should result in new chemotherapeutic agents having the ability to limit tumor angiogenesis and regrowth following cytoreductive treatments such as PDT.

Effect of Amphotericin B and Micafungin Combination on Survival, Histopathology, and Fungal Burden in Experimental Aspergillosis in the p47phox−/− Mouse Model of Chronic Granulomatous Disease

ABSTRACT Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by recurrent life-threatening bacterial and fungal infections. We characterized the effects of single and combination antifungal therapy on survival, histopathology, and laboratory markers of fungal burden in experimental aspergillosis in the p47phox−/− knockout mouse model of CGD. CGD mice were highly susceptible to intratracheal Aspergillus fumigatus challenge, whereas wild-type mice were resistant. CGD mice were challenged intratracheally with a lethal inoculum (1.25 × 104 CFU/mouse) of A. fumigatus and received one of the following regimens daily from day 0 to 4 after challenge (n = 19 to 20 per treatment group): (i) vehicle, (ii) amphotericin B (intraperitoneal; 1 mg/kg of body weight), (iii) micafungin (intravenous; 10 mg/kg), or (iv) amphotericin B plus micafungin. The rank order of therapeutic efficacy based on prolonged survival, from highest to lowest, was as follows: amphotericin B plus micafungin, amphotericin B alone, micafungin alone, and the vehicle. Lung histology showed pyogranulomatous lesions and invasive hyphae, but without hyphal angioinvasion or coagulative necrosis. Treatment with micafungin alone or combined with amphotericin B produced swelling of invasive hyphae that was not present in mice treated with the vehicle or amphotericin B alone. Assessment of lung fungal burden by quantitative PCR showed no significant difference between treatment groups. Serum galactomannan levels were at background despite documentation of invasive aspergillosis by histology. Our findings showed the superior efficacy of the amphotericin B and micafungin combination compared to either agent alone after A. fumigatus challenge and also demonstrated unique features of CGD mice as a model for experimental aspergillosis.

Synthesis and biological activity of novel 3′-trifluoromethyl taxoids

Second generation taxoids possessing a trifluoromethyl moiety in place of the 3′-phenyl group are synthesized by means of the β-Lactam Synthon Method. The in vitro cytotoxicities of these new taxoids are evaluated against several different human cancer cell lines and found to exhibit greatly enhanced activities as compared to those of paclitaxel and docetaxel. The activity enhancement is most remarkable against a drug-resistant breast cancer cell line, MCF7-R, expressing MDR phenotype.

High Correlation of Whole-Body Red Fluorescent Protein Imaging and Magnetic Resonance Imaging on an Orthotopic Model of Pancreatic Cancer

We have developed genetically fluorescent orthotopic models of human pancreatic cancer. In these models, noninvasive fluorescent protein imaging (FPI) of internal primary tumors and metastatic deposits has been carried out. Whole-body tumor images are easily and inexpensively obtained using FPI, permitting both detection and quantification of tumor load. In this study, we simultaneously compared single mice with a highly fluorescent, red fluorescent protein-expressing orthotopic pancreatic cancer xenografts with both FPI and high-resolution magnetic resonance imaging (MRI). Images were acquired at multiple time points after tumor implantation in the pancreas. Indwelling pancreatic primary tumors and metastatic foci were detected by both FPI and MRI. Moreover, a strong correlation existed between images taken with these two technologies. FPI permitted rapid, high-throughput imaging without the need for either anesthesia or contrast agents. Both FPI and MRI enabled accurate imaging of tumor growth and metastasis, although MRI enabled tissue structure to be visualized as well. FPI has high resolution and is exceedingly rapid with instant image capture. We suggest a complimentary role for these two imaging modalities.

New taxanes as highly efficient reversal agents for multi-drug resistance in cancer cells

New non-cytotoxic taxanes synthesized from 10-deacetylbaccatin III and special hydrophobic acylating agents show remarkable MDR reversal activity (< or = 99.8%) against drug-resistant human breast cancer cells when co-administered with paclitaxel or doxorubicin. This activity is ascribed to the highly efficient blocking of P-glycoprotein efflux by these new taxanes.

SYNTHESIS AND BIOLOGICAL ACTIVITY OF 3'-ALKYL- AND 3'-ALKENYL-3'-DEPHENYLDOCETAXELS

Abstract 3-Alkyl- and 3′-alkenyl-3′-dephenyldocetaxels are synthesized from 10-deacetylbaccatin III based on the β-lactam synthon method in good yields. The cytotoxicity of the new taxoids are evaluated against different human tumor cell lines and their ability to inhibit the microtubules disassembly examined. The 3′-isobutenyl, 3′-crotyl, and 3′-isobutyl analogs possess very strong cytotoxicity as well as antitumor activity in vivo . 3′-Isobutenyl- as well as 3′-crotyl-3′-dephenyl-10-acetyldocetaxel shows ca. 20 times stronger activity against an adriamycin-resistant human breast cancer cell line (MCF7-R) than those o docetaxel and paclitaxel.

Inhibition of human HT-29 colon carcinoma cell adhesion by a 4-fluoro-glucosamine analogue

Cell surface glycoconjugates play an important role in cellular recognition and adhesion. Modification of these structures in tumour cells could affect tumour cell growth and behaviour, including metastasis. 2-Acetamido-1,3,6-tri-O-acetyl-4-deoxy-4-fluoro-α-D-glycopyranose (4-F-GlcNAc) was synthesized as a potential inhibitor and/or modifier of tumour cell glycoconjugates. The effect of this sugar analogue on the adhesive properties of human colon carcinoma HT-29 cells was evaluated. Treatment of HT-29 cells with 4-F-GlcNAc led to reduced cell surface expression of terminal lactosamine, sialyl-Lex and sialyl-Lea, as determined by Western blotting and flow cytometry. The aberrant expression of these oligosaccharide structures on the HT-29 cell surface resulted in: (1) decreased E-selectin mediated adhesion of human colon cells to human umbilical cord endothelial cells (HUVEC); (2) impaired adhesion of HT-29 cells to β-galactoside binding lectin, galectin-1; and (3) reduced ability to form homotypic aggregates. After exposure to 4-F-GlcNAc, lysosomal associated membrane proteins (lamp) 1 and 2, and carcinoembryonic antigen (CEA) detected in HT-29 cells were of lower molecular weight, probably due to impaired glycosylation. These results strongly suggest that modification of tumour cell surface molecules can alter tumour cell adhesion and that tumour cell surface oligosaccharides may be suitable targets for therapeutic exploitation.

Broad-spectrum modulation of ATP-binding cassette transport proteins by the taxane derivatives ortataxel (IDN-5109, BAY 59-8862) and tRA96023

PurposeThe taxanes paclitaxel and docetaxel are substrates for P-glycoprotein (Pgp), an ATP-binding cassette (ABC) transport protein associated with multidrug resistance (MDR). In contrast, the synthetic taxane ortataxel (BAY 59-8862, IDN-5109) is effective against Pgp-expressing cells by virtue of modulation of Pgp-mediated transport. The synthetic taxane tRA96023 also modulates Pgp and is noncytotoxic due to removal of the tubulin-binding side chain at the C-13 position of the taxane backbone. We studied the effects of ortataxel and tRA96023 on the other MDR-associated ABC transport proteins, multidrug resistance protein (MRP-1) and breast cancer resistance protein (BCRP, MXR, ABCG2).MethodsModulation of mitoxantrone, daunorubicin and doxorubicin retention and cytotoxicity by ortataxel and tRA96023 was studied in established cell lines overexpressing Pgp, MRP-1 and wild type (BCRPR482) and mutant (BCRPR482T) BCRP, and was compared with modulation by the established Pgp-, MRP-1- and BCRP-specific modulators PSC-833, probenecid and fumitremorgin C, respectively.ResultsOrtataxel effectively modulated drug retention and cytotoxicity in cell lines overexpressing MRP-1 and BCRPR482, in addition to Pgp. tRA96023 modulated drug retention and cytotoxicity in cell lines overexpressing BCRPR482 and Pgp, but not those overexpressing MRP-1. Neither ortataxel nor tRA96023 modulated BCRPR482T.ConclusionsThe synthetic taxane derivatives ortataxel and tRA96023 are broad-spectrum ABC protein modulators. Further studies will seek to identify a noncytotoxic synthetic taxane that modulates Pgp, MRP-1 and BCRP.