Zhisong Cao

Dependence of Anticancer Activity of Camptothecins on Maintaining Their Lactone Function

Abstract: Camptothecins contain a lactone ring that exists in the closed form below ph 7. Above 7, the open (CPT+) and the closed (CPT) form coexist in a 50–50 ratio in mouse plasma and in a 90–10 ratio in human plasma due to the high affinity of human serum albumin (HSA) for CPT+. CPT+ is much less toxic than CPT and it is excreted much faster. In complete RPMI 1640 culture medium, the equilibrium CPT+‐CPT is 50–50. If 4% HSA is added, it moves to 90–10 modeling for the human physiological situation.

Development and validation of a reverse-phase HPLC with fluorescence detector method for simultaneous determination of CZ48 and its active metabolite camptothecin in mouse plasma

A simple and sensitive high-performance liquid chromatography (HPLC) assay for the analysis of CZ48, a potent anticancer candidate, and its active metabolite camptothecin (CPT) in mouse plasma was developed and validated. CZ44 was used as an internal standard (IS). The samples were injected onto a C18 Synergi Polar-RP column (4 microm, 150 mm x 4.60 mm) maintained at 30 degrees C. The identification of peaks showed high specificity. Shimadzu RF-10AXL fluorescence detector was used at the excitation and emission of 380 and 418 nm, respectively. The mean recoveries were 81.41+/-0.035%, 86.00+/-0.053% and 82.21+/-0.020% for CZ48 and 76.01+/-0.028%, 77.04+/-0.042% and 85.93+/-0.023% for CPT at three concentrations of 10, 100 and 900 ng/ml, respectively. The calibration curve was linear (r(2)=0.9999) over CZ48 and CPT concentrations ranging from 5 to 1000 ng/ml and 10-1000 ng/ml (n=6), respectively. The method had an accuracy of >95% and intra- and inter-day precision (RE%) of <1.2% and <2.2% for CZ48 and CPT, respectively, at three different concentrations (10, 100 and 900 ng/ml). The lower limit of quantification (LLOQ) using 0.1 ml mouse plasma was 10 ng/ml for CZ48 and 5 ng/ml for CPT. Stability studies showed that CZ48 and CPT were stable in mouse plasma after 4h incubation at room temperature or after 1 month storage at -80 degrees C with three freeze/thaw cycles. The method reported is simple, reliable, precise and accurate and confirmed by the determination of plasma samples in the mice after oral administration of CZ48.

Structure-activity relationship of alkyl camptothecin esters.

Abstract: The cytotoxicity of camptothecin (CPT) esters 1–6 was measured. Like parental camptothecin, esters 2 and 3, but not 1, 4, 5, and 6, inhibited proliferation of human leukemia cells in culture and induced programmed cell death as assessed by flow cytometry studies. Exhibition of similar levels of antiproliferative activities of CPT 2 and 3 required different incubation time periods in cell cultures, with CPT and 3 requiring the shortest and longest periods, respectively. Both 2 and 3 were inactive against cells resistant to the semisynthetic CPT derivative 9‐nitrocamptothecin and unable to stabilize DNA‐topoisomerase I (Topo I) “cleavable complexes” in a cell‐free system, suggesting that Topo I activity was required but insufficient for the mechanism of action of 2 and 3. Mouse liver homogenate converted esters to parental CPT, but the conversion rates were different with different esters. Of four tested esters in this experiment, ester 2 had the fastest conversion rate. In vivo studies showed that ester 2 had an exceptional lack of toxicity in nude mice, even at enormous doses, and demonstrated extensive activity against human breast and colon tumors grown as xenografts in immunodeficient nude mice, whereas no antitumor activity was observed for the other esters. In conclusion, ester 2 is a prodrug of the antitumor compound CPT, and it can be administered at very high doses in mice with no appearance of toxicity. This study provides a basis for further evaluation of CPT ester 2 as an investigational anticancer agent.

Crystalline Camptothecin-20(S)-O-Propionate Hydrate:A Novel Anticancer Agent with Strong Activity against 19 Human Tumor Xenografts

To find a more effective and less toxic chemotherapeutic agent, we have successfully prepared crystalline camptothecin-20(S)-O-propionate hydrate (CZ48) by reacting camptothecin with propionic anhydride using concentrated sulfuric acid as catalyst. The biological effectiveness of this new anticancer agent was evaluated by using xenografts of human cancers in nude mice as the testing models. The extensive treatment of 21 human tumors with various dose levels of CZ48 has shown that this agent is highly effective against many different human tumors tested with a striking lack of toxicity. Of the 21 human tumor lines tested, 9 regressed, 5 were <10% of the control, 3 were <20%, and 2 were <40%. Two tumors did not respond. The total response rate was 90% (19 of 21). No toxicity was observed in mice. The effective doses required to achieve the positive response varied from 100 to 1,000 mg/kg/d depending on the tumors. The maximum tolerated dose was not reached because of the nontoxic nature of the drug in mice. Thus, this compound has a much wider therapeutic index compared with that of the existing anticancer drugs currently in use.

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.

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.

An Efficient Preparation of 7-Methoxychromanone

Abstract The reaction of m-methoxyphenol 7 with 3-chloropropionaldehyde diethyl acetal 8 and the subsquent treatments gave the corresponding 7-methoxychromanone 6 in overall 54% yield. This method represents an efficient preparation of chromanone.

Enhanced Lactone Stability of CZ48 in Blood Correlates to its Lack of Toxicity in Mice

PURPOSE The aim of this study was to correlate the relationship between the pharmacokinetic behaviors and the toxicity of a new investigational anticancer agent CZ48, a C20-propionate ester of camptothecin (CPT) in mice. METHODS In this study, the safety and pharmacokinetics of oral doses of CZ48 were compared with the oral doses of CPT. Mice were administered orally one of three single doses of CZ48 (50, 200 and 1000 mg/kg) and two single doses of CPT (1.5 mg/kg and 6.0 mg/kg). Blood samples were collected from all mice at the defined time points after drug administration for assessment of plasma CZ48 and CPT concentrations. RESULTS The study showed that CZ48 was very stable in mouse blood and the majority of this agent stayed intact as the lactone form when in circulation, with only a small fraction of the CZ48 molecules metabolized into CPT. The concentration of the metabolite CPT measured in the mouse blood was only 3% of the concentration found for the maximum tolerated dose (6.0 mg/kg) of plain CPT. The stability difference between CZ48 and CPT in blood was structurally explained by the geometry of these two molecules. CONCLUSION The lack of toxicity of CZ48 at effective doses in mice is attributed to its enhanced stability in their blood.

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