Albert DeJesus

Partial characterization of human leukemia U-937 cell sublines resistant to 9-nitrocamptothecin P

Abstract: Human leukemia U‐937 cell sublines exhibiting various levels of resistance to 9‐nitrocamptothecin (9NC) were developed after exposure to progressively increased 9NC concentrations. Increases in 9NC resistance of the cells were accompanied by decreases in proliferation rate; appearance of morphological and functional features that correlate with granulocytic maturation; decreased synthesis of topoisomerase I; increased synthesis of topoisomerase II; and inability or decreased ability to induce tumors when xenografted in nude mice. 9NC‐resistant cells, transferred and propagated in 9NC‐free media for 6 months, continue to exhibit resistance and other features similar to cells propagated in continual presence of 9NC. Finally, 9NC‐resistant U‐937 cells respond to physiological and non‐physiological agents of cell differentiation, indicating that alternative treatments can be successfully used to inhibit growth of 9NC‐resistant U‐937 cells and tumors.

Sensitivity of camptothecin-resistant human leukemia cells and tumors to anticancer drugs with diverse mechanisms of action.

Human leukemia U-937 cell clones resistant to 9-nitrocamptothecin (9NC) appear after exposure to increase 9NC-concentrations. Drug resistance is irreversible, regardless of whether the 9NC-resistant (U-937/CR150) cells grow in media with or without 9NC. U-937/CR150 cells are more sensitive than wild type U-937 (U-937/wt) cells to topoisomerase II-directed drugs, amsacrine, daunorubicin, and etoposide. The mitotic inhibitor, vincristine, induces hyperdiploidy in U-937/wt, but not in U-937/CR150 cells, whereas the antimetabolites, cytarabine and methotrexate, and the nitrosourea, carmustine, elicit similar responses in both U-937/wt and U-937/CR150 cells. U-937/CR150-generated tumors in nude mice are sensitive to etoposide. The clinical implications of increased sensitivity of 9NC-resistant tumors to some anticancer drugs are discussed.

Anticancer Activity of New Haloalkyl Camptothecin Esters against Human Cancer Cell Lines and Human Tumor Xenografts Grown in Nude Mice

All chemotherapeutic agents currently in use have a narrow window of therapeutic index of 1 to 1.2. Camptothecin ester compounds are reported to have a wider therapeutic index when being used to treat human xenografts in nude mice. As a continuous effort in searching for better chemotherapeutic agents for treating cancers, new haloalkyl camptothecin and 9-nitrocamptothecin ester derivatives 2a-b and 3a-d were prepared by respective acylation of camptothecin 1a and 9-nitrocamptothecin 1b with the corresponding acylating agents. These new derivatives were tested in vitro against 8 human cancer cell lines using 7 different concentrations ranging from 5 to 300 nM and also in vivo against various types of human tumor xenografts grown in nude mice. Most of these new compounds started showing inhibitory effects on the growth of 8 cancer cell lines at concentration of 80 nM and achieved greater than 70% inhibitions against these cell lines when the concentration increased to 300 nM. Compound 2a and 3a showed good activity against human tumor xenografts in nude mice. Compared to mother compound camptothecin, 3a was much less toxic in mice with a better therapeutic index, having the potential to be further developed as a safer treatment for cancers.

Metabolic Difference of CZ48 in Human and Mouse Liver Microsomes

CZ48, chemically camptothecin-20-O-propionate hydrate, is currently under clinical investigation. The kinetics of the metabolite camptothecin (CPT) formation and of CZ48 depletion in mouse and human liver microsomes in the presence or absence of NADPH was examined. The formation rate of camptothecin in human liver microsomes was significantly higher than that in mouse with mean Kms of 1.9 and 0.5 nM and Vmaxs of 9.3 and 2.2 pmol/min/mg, respectively. However, the apparent intrinsic clearance (Vmax/Km) ratios for camptothecin in human and mouse liver microsomes were not significantly different from each other (4.9 versus 4.4) in the presence of NADPH. The depletion of CZ48 in human microsomes was four times faster with 4.55% of CZ48 remaining intact while in mouse 19.11% of the drug remained unchanged after 60 min. These results suggest that there is a remarkable species difference of CZ48 biotransformation between human and mouse. The depletion rate of CZ48 in human liver microsomes is considerably higher than that in the mouse.

Monocytic differentiation and synthesis of proteins associated with apoptosis in human leukemia U‐937 cells acquiring resistance to Vincristine

Abstract: Human leukemia U‐937/WT cells were exposed to stepwise increased concentrations of Vincristine so that Vincristine‐resistant cell sublines (termed U‐937/RV) were developed. Established U‐937/RV cell sublines have continuously propagated over a year, both in absence and presence of VCR, and have demonstrated similar features. In contrast to U‐937/WT cells, U‐937/RV cells have longer doubling time, and are more differentiated as determined by appearance of distinct morphological features and synthesis of mRNA that codes for the monocyte colonystimulating factor‐1 receptor (c‐fms). Both apoptosis‐suppressing Bcl‐2 and Bcl‐XL proteins were undectable in U‐937/WT cells, whereas Bcl‐2 was nearly detectable and Bcl‐XL readily detectable in U‐937/RV cells. The apoptosis‐promoting Bax protein was also absent in U‐937/WT cells and readily detected in U‐937/RV cells. Vincristine‐resistant cells with different levels of resistance synthesize similar levels of c‐fms mRNA and Bax protein. Finally, unlike U‐937/WT cells, U‐937/RV cells have no ability to induce tumors when xenografted in immunodeficient mice. The findings collectively suggest that development of resistance to Vincristine in U‐937/WT cells may correlate with cell differentiation and synthesis of proteins that regulate apoptosis.

Synthesis and anti-tumor activity of alkenyl camptothecin esters

AbstractAim:To study the degrees of influence of changing side ester chains at position C20 of camptothecin on the anti-tumor activity of the molecules.Methods:The esterification reaction of camptothecin 1 and 9-nitrocamptothecin 2 with crotonic anhydride in pyridine gave the corresponding esters 3 and 4, respectively. The acylation of 1 and 2 with cinnamoyl chloride gave products 7 and 8. Epoxidation reaction of 3 and 4 with m-chloroperoxybenzoic acid in benzene solvent gave the products 5 and 6. Esters 3, 4, and 5 were tested for anti-tumor activity against 14 human cancer cell lines.Results:Both in vitro and in vivo anti-tumor activity studies for these esters were conducted and the data demonstrated positive results, that is, these esters were active against the tested tumor lines.Conclusion:Alkenyl esters 3 and 4 showed strong anti-tumor activity in vitro against 14 different cancer cell lines. Ester 3 was active against human breast carcinoma in mice and the toxicity of the agent was not observed in mice during the treatment, implying that this agent is effective for treatment with low toxicity.

Abstract 4337: In vitro biotransformation of and inhibitory effects of CZ48 in human liver microsomes

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA BACKGROUND AND PURPOSE CZ48 is a novel potent anticancer agent currently in a phase I clinical trial. The absorption of CZ48 in humans is approximately less than 10% based on the excretion ratio of the unchanged form in the feces following oral administration. It was reported that CZ48 acts as a pro-drug and exerts its anticancer activity through the active metabolite CPT in vivo. The specific enzymes involved in its biotransformation have not been identified. To date, it has been very difficult to predict drug-drug interactions with CZ48 in the clinical setting due to a lack of information. Cytochrome P450 enzymes are primarily found in liver cells but are also located in other organ cells throughout the body. Therefore, in vitro studies were conducted to identify and characterize the cytochrome P450 enzymes involved in the formation of the major metabolite CPT. In this study, we conducted in vitro experiments by using in vitro human liver models which have been developed in the past few decades, including isoenzymes, microsomes, cytosol, S9 fraction, statins and inhibitors/inducers to allow us to evaluate the potential for drug-drug interactions. METHODS Using an integrated approach CZ48 biotransformation was studied for CYP-mediated metabolic reaction with human liver microsomes (HLM), cytosol and S9 fraction. Seven major isoenzymes were used to investigate, through relative activity factor approach, the contribution of selective statins or of several inhibitors/inducers in the phase-I metabolism of CZ48. RESULTS Experiments revealed that 97.9% of CZ48 was metabolized in HLM in 2 hours, and 64.8% of CZ48 was biotransformed in the S9 fraction, while cytosol was less responsible (13.4%) for CZ48 biotransformation. The majority (68.5%) of CZ48 was metabolized into CPT when co-incubated with CYP3A4. The statins, mevastatin (lovastatin), atorvastatin and simvastatin, are specific CYP3A4 substrates and inhibited CZ48 biotransformation. In contrast, rosuvastatin showed no inhibitory effect. At CZ48 concentrations of 0.25, 0.5 and 1 uM, verapamil, erythromycin, clarithromycin and CYP3A4 showed the strong inhibitory effects on CZ48 biotransformation. However, based on results with human liver microsomes in the presence of the CYP3A4 inducers phenytoin, rifampicin and carbamazepine, it was suggested that cz48 was partly metabolized by other enzymes in the microsomes. CONCLUSIONS Our results establish the metabolic pathways of CZ48, revealing the significant role of CYPs in its metabolism. Of the major human CYPs, CYP3A4 is responsible for catalyzing the biotransformation of CZ48. Indeed, CYP3A4 has extremely broad substrate specificity and is responsible for the metabolism of 50% of drugs in current use. Our results provide insight into the underlying biochemical mechanisms of CZ48, which would help to predict clinical outcomes. Note: This abstract was not presented at the meeting. Citation Format: Xing Liu, Albert DeJesus, Dana Vardeman, Zhisong Cao, Beppino Giovanella. In vitro biotransformation of and inhibitory effects of CZ48 in human liver microsomes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4337. doi:10.1158/1538-7445.AM2014-4337

Abstract 670: Bis-nitrophenylphosphate (BNPP) but not phenylmethylsulfonyl fluoride (PMSF) inhibits the activation of the novel anti-cancer pro-drug CZ48

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL CZ48, the 20-O-propionate ester of Camptothecin (CPT), is a non-toxic prodrug of CPT first described by Cao et al. (1998). The propionate side-chain is enzymatically cleaved in target tissues. This gives rise to CPT, an inhibitor of Topoisomerase I, which then blocks cell division causing selective tumor toxicity. We have previously established the preferential activation of CZ48 in tumor tissues compared to normal tissues and an ultra sensitive assay to detect it (2009). The present studies explore the specifics of that activation in vitro using both cells in culture and surgically removed xenografted tumors from nude mice. DOY lung adenocarcinoma was grown subcutaneously in nude mice and then surgically excised, homogenized and serially centrifuged to produce specific subcellular fractions. The bulk of esterase activity was found to be concentrated in the cytosol, while the microsomal fraction showed minimal activity. All other fractions tested were below the limit of detection. HT-29 colon carcinoma cells were grown in culture in the presence and absence of varying concentrations of bis-nitrophenylphosphate (BNPP), a potent inhibitor of carboxylesterase and butyrylcholinesterase (BChE), in an attempt to find the highest possible non-toxic concentration to the cells. This concentration was then included during a 7-day IC50 determination for CZ48. In the presence of BNPP, the IC50 of CZ48 increased 4-fold from 181.4 nM to 710.8 nM. In vitro conversion of CZ48 to CPT by cytosolic fraction of a HT-29 tumor xenograft grown subcutaneously in nude mice was decreased by BNPP in a dose dependent manner. In contrast, the serine protease and carboxylesterase inhibitor phenylmethylsulfonyl fluoride (PMSF) and other esterase inhibitors did not affect the activation of CZ48. Purified butyrylcholinesterase and acetylcholinesterase (SigmaAldrich, St. Louis MO) did not show any capacity to activate CZ48. In conclusion, the enzymatic activation of CZ48 is not mediated by butyrylcholinesterase but by another cytosolic enzyme inhibitable by BNPP. These data constitute a significant step forward in unraveling the exact mechanism for the activation of CZ48. This will help design a pre-treatment assay to establish tumor sensitivity, as well as give us direction for designing new, more potent prodrugs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 670. doi:10.1158/1538-7445.AM2011-670

Abstract 3547: The differences in intestinal absorptions between CZ48 and its parental Camptothecin

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC The determination of oral bioavailability is a critical step in early phase drug development. In part, the ability to predict whether a drug will be effectively transported across the gastrointestinal mucosa can be estimated from the physicochemical properties of the compound. Currently, the Caco-2 human colon carcinoma cell line is often used by the pharmaceutical industry to evaluate intestinal absorption of drugs. The purpose of the present study was to investigate the membrane transport mechanism of CZ48, a hydrated C20-propionate ester of Camptothecin (CPT), in the Caco-2 cell culture model, in order to understand the possible role of membrane transporters on its oral bioavailability and disposition. Caco-2 cells were grown onto Millicell™ to form monolayers. Directional transport of CZ48 and CPT was determined. The results indicated that apical (AP) to basolateral (BL) transport of CZ48 and CPT was distinctively different from BL to AP transport, with AP to BL transport surpassing the BL to AP transport. The ratio of BL-AP/AP-BL transport rate was 0.36 and 3.0 for CZ48 and CPT at the concentration of 5µM, respectively. The results also showed that AP to BL permeability of CZ48 was ten times lower than CPT, but the accumulation of CZ48 in intact cells was higher than CPT (0.67 and 0.49 nmol, respectively). In conclusion, the p-glycoprotein may be involved in the transepithelial transport of CPT. An additional carrier mechanism may be involved in the AP uptake of CZ48, since Apical transport of CZ48 in the Caco-2 cells is mediated, at least in part, by uptake of a nucleic bases into cells via an nucleobase transporter. 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 3547.