Paula Pera

Polyamine biosynthetic activity in normal and neoplastic human colorectal tissues

Polyamine biosynthetic activity was assessed in various colorectal tissue samples consisting of noninvolved mucosa, benign adenomatous polyps and adenocarcinomas taken at surgery from a total of 40 patients. Ornithine decarboxylase (ODC) displayed a gradient of enzyme activity (i.e., adenocarcinoma > polyps > mucosa) which seemed to correlate positively with the neoplastic status of the tissue. In 10 of the patients, samples were obtained for all three tissue types. Five of these exhibited a clear repetition of the trends in enzyme activity seen with the mixed patient tissue sampling whereas the remainder differed by having the highest ODC activity in the polyps. In nine of the ten cases, ODC activity was substantially lower in the mucosa than in either of the neoplastic lesions. Trends in enzyme activity were the same for tissues obtained from either the colon or rectum. The ODC activity in adenocarcinomas could not be correlated with histologic differentiation, stage or site of the disease, however, in samples from female patients (all postmenopausal) the activity was elevated over normal mucosa to a greater extent (ten‐fold) than in male patients (seven‐fold). S‐adenosylmethionine decarboxylase activity was assessed in 27 of the 40 patients and found to follow the same distribution as ODC; however, the mean value differences ± SEM between tissues were less distinct. In general, tissue polyamine pool analysis of these same specimens reflected the levels of ornithine and S‐adenosylmethionine decarboxylase activities. Overall, the data reveal an increase in polyamine biosynthetic activity in colorectal neoplasms, relative to surrounding mucosa, which may correlate with (1) progression of the neoplastic process, (2) the proportion of proliferating cells, (3) the rate of cell proliferation, or (4) a combination of two or all of these possibilities.

The Tyrosine Kinase Inhibitor Imatinib Mesylate Enhances the Efficacy of Photodynamic Therapy by Inhibiting ABCG2

Purpose: The ATP-binding cassette protein ABCG2 (breast cancer resistance protein) effluxes some of the photosensitizers used in photodynamic therapy (PDT) and, thus, may confer resistance to this treatment modality. Tyrosine kinase inhibitors (TKI) can block the function of ABCG2. Therefore, we tested the effects of the TKI imatinib mesylate (Gleevec) on photosensitizer accumulation and in vitro and in vivo PDT efficacy. Experimental Design: Energy-dependent photosensitizer efflux and imatinib mesylates effects on intracellular accumulation of clinically used second- and first-generation photosensitizers were studied by flow cytometry in murine and human cells with and without ABCG2 expression. Effects of ABCG2 inhibition on PDT were examined in vitro using cell viability assays and in vivo measuring photosensitizer accumulation and time to regrowth in a RIF-1 tumor model. Results: Energy-dependent efflux of 2-(1-hexyloxethyl)-2-devinyl pyropheophorbide-a (HPPH, Photochlor), endogenous protoporphyrin IX (PpIX) synthesized from 5-aminolevulenic acid, and the benzoporphyrin derivative monoacid ring A (BPD-MA, Verteporfin) was shown in ABCG2+ cell lines, but the first-generation multimeric photosensitizer porfimer sodium (Photofrin) and a novel derivative of HPPH conjugated to galactose were minimally transported. Imatinib mesylate increased accumulation of HPPH, PpIX, and BPD-MA from 1.3- to 6-fold in ABCG2+ cells, but not in ABCG2− cells, and enhanced PDT efficacy both in vitro and in vivo. Conclusions: Second-generation clinical photosensitizers are transported out of cells by ABCG2, and this effect can be abrogated by coadministration of imatinib mesylate. By increasing intracellular photosensitizer levels in ABCG2+ tumors, imatinib mesylate or other ABCG2 transport inhibitors may enhance efficacy and selectivity of clinical PDT.

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.

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.

Novel Methods to Incorporate Photosensitizers Into Nanocarriers for Cancer Treatment by Photodynamic Therapy

A hydrophobic photosensitizer, 2‐[1‐hexyloxyethyl]‐2‐devinyl pyropheophorbide‐a (HPPH), was loaded into nontoxic biodegradable amine functionalized polyacrylamide (AFPAA) nanoparticles using three different methods (encapsulation, conjugation, and post‐loading), forming a stable aqueous dispersion. Each formulation was characterized for physicochemical properties as well as for photodynamic performance so as to determine the most effective nanocarrier formulation containing HPPH for photodynamic therapy (PDT).

Synthesis, photophysical, electrochemical, tumor-imaging, and phototherapeutic properties of purpurinimide-N-substituted cyanine dyes joined with variable lengths of linkers.

Purpurinimide methyl esters, bearing variable lengths of N-substitutions, were conjugated individually to a cyanine dye with a carboxylic acid functionality. The results obtained from in vitro and in vivo studies showed a significant impact of the linkers joining the phototherapeutic and fluorescence imaging moieties. The photosensitizer-fluorophore conjugate with a PEG linker showed the highest uptake in the liver, whereas the conjugate linked with two carbon units showed excellent tumor-imaging and PDT efficacy at 24 h postinjection. Whole body imaging and biodistribution studies at variable time points portrayed enhanced fluorescent uptake of the conjugates in the tumor compared to that in the skin. Interestingly, the conjugate with the shortest linker and the one joining with two carbon units showed faster clearance from normal organs, e.g., the liver, kidney, spleen, and lung, compared to that in tumors. Both imaging and PDT efficacy of the conjugates were performed in BALB/c mice bearing Colon26 tumors. Compared to the others, the short linker conjugate showed poor tumor fluorescent properties and as a corollary does not exhibit the dual functionality of the photosensitizer-fluorophore conjugate. For this reason, it was not evaluated for in vivo PDT efficacy. However, in Colon26 tumor cells (in vitro), the short linker was highly effective. Among the conjugates with variable linkers, the rate of energy transfer from the purpurinimide moiety to the cyanine moiety increased with deceasing linker length, as examined by femtosecond laser flash photolysis measurements. No electron transfer from the purpurinimide moiety to the singlet excited state of the cyanine moiety or from the singlet excited state of the cyanine moiety to the purpurinimide moiety occurred as indicated by a comparison of transient absorption spectra with spectra of the one-electron oxidized and one-electron reduced species of the conjugate obtained by spectroelectrochemical measurements.

Synthesis and biological activity of 14-hydroxydocetaxel

Abstract New taxoids, 14β-hydroxydicetaxek abd uts 1m14-acetonide, are synthesized from 14β-hydroxy-10-deacetylbaccatin III 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. 14β-hydroxydocetaxel shows very strong cytotoxicity, especially against A549 human non-small cell lung cancer cell line (IC 50 = 0.8 nM).

Modeling the Combination of Amphotericin B, Micafungin, and Nikkomycin Z against Aspergillus fumigatus In Vitro Using a Novel Response Surface Paradigm

ABSTRACT Response surface methods for the study of multiple-agent interaction allow one to model all of the information present in full concentration-effect data sets and to visualize and quantify local regions of synergy, additivity, and antagonism. In randomized wells of 96-well plates, Aspergillus fumigatus was exposed to various combinations of amphotericin B, micafungin, and nikkomycin Z. The experimental design was comprised of 91 different fixed-ratio mixtures, all performed in quintuplicate. After 24 h of drug exposure, drug effect on fungal viability was assessed using the tetrazolium salt 2,3-bis {2-methoxy-4-nitro-5-[(sulfenylamino) carbonyl]-2H-tetrazolium-hydroxide} (XTT) assay. First, we modeled each fixed-ratio combination alone using the four-parameter Hill concentration-effect model. Then, we modeled each parameter, including the 50% inhibitory concentration (IC50) effect, versus the proportion of each agent using constrained polynomials. Finally, we modeled the three-agent response surface overall. The overall four-dimensional response surface was complex, but it can be explained in detail both analytically and graphically. The grand model that fit the best included complex polynomial equations for the slope parameter m and the combination index (equivalent to the IC50 for a fixed-ratio concentration, but with concentrations normalized by the respective IC50s of the drugs alone). There was a large region of synergy, mostly at the nikkomycin Z/micafungin edge of the ternary plots for equal normalized proportions of each drug and extending into the center of the plots. Applying this response surface method to a huge data set for a three-antifungal-agent combination is novel. This new paradigm has the potential to significantly advance the field of combination antifungal pharmacology.

Design and synthesis of de novo cytotoxic alkaloids by mimicking the bioactive conformation of paclitaxel.

Novel paclitaxel-mimicking alkaloids were designed and synthesized based on a bioactive conformation of paclitaxel, that is, REDOR-Taxol. The alkaloid 2 bearing a 5-7-6 tricyclic scaffold mimics REDOR-Taxol best among the compounds designed and was found to be the most potent compound against several drug-sensitive and drug-resistant human cancer cell lines. MD simulation study on the paclitaxel mimics 1 and 2 as well as REDOR-Taxol bound to the 1JFF tubulin structure was quite informative to evaluate the level of mimicking. The MD simulation study clearly distinguishes the 5-6-6 and 5-7-6 tricyclic scaffolds, and also shows substantial difference in the conformational stability of the tubulin-bound structures between 2 and REDOR-Taxol. The latter may account for the large difference in potency, and provides critical information for possible improvement in the future design of paclitaxel mimics.

In vitro antitumor activity of 9-nitro-camptothecin as a single agent and in combination with other antitumor drugs.

Abstract: Preclinical studies at Roswell Park Cancer Institute by Minderman, Cao, and Rustum (unpublished results) showed that a combination of SN‐38 and 5‐FU against HCT‐8 human colon carcinoma cells in vitro was synergistic, with the best interaction occurring when the drugs were added sequentially, SN‐38 first. Their in vivo studies using HCT‐8 tumor xenografts implanted s.c. in nude athymic mice demonstrated superior efficacy for a sequential i.v. administration of CPT‐11, 24 hr before 5‐FU. On the basis of these studies, our group has begun to evaluate effects of RFS2000 (9‐nitro‐20(S)‐camptothecin) (9‐NC) in combination with a series of other antitumor agents. Using a panel of human tumor cell lines including A121 ovarian cancer, HCT‐8 colon cancer, H‐460 NSCLC, HT‐1080 fibrosarcoma, and MCF7 mammary cancer, we found that a 2‐hr exposure to 9‐NC resulted in ID50 values of <1.0 μM, whereas continuous exposure to drug resulted in ID50 values of <1.0 nM. Tumor growth inhibitory activities of 5‐FU, gemcitabine, and paclitaxel were determined for comparison. Combinations of these agents were evaluated with 9‐NC using the human HCT‐8 colon tumor cell line. Concurrent and sequential combinations of 9‐NC with 5‐FU had some regions of the concentration‐effect surface with local synergy and some with local antagonism. However, sequential combination of 9NC or SN‐38 followed by 5‐FU, 24 hr later appeared to be highly synergistic at high dose‐effect levels (i.e., ID90), suggesting that sequential drug administration may be more efficacious at high effect level and that the order of drug addition is very important. Overall, our results were similar to that found earlier by Rustums group with CPT11 (or SN‐38) and 5‐FU, suggesting that sequential combination of 9‐NC (or other camptothecin analogues) followed by 5‐FU has potential for the treatment of cancer in man.