Sustained delivery of a camptothecin prodrug - CZ48 by nanosuspensions with improved pharmacokinetics and enhanced anticancer activity.

Background and aim: We have synthesized a novel lactone-stabilized camptothecin (CPT) analog named CZ48 and demonstrated its potent anticancer effects via bioconversion to the active CPT in earlier studies. Herein, we aimed to develop, optimize and characterize CZ48 nanosuspensions, for a sustained delivery of this drug in humans with an intravenous (i.v.) administration. Methods and materials: A three-factor, five-level central composite design (CCD) was employed to establish the impacts of the critical influencing factors (concentrations (wt%) of CZ48, polysorbate 80 (Tween-80), and Pluronic® F-108 (F-108)) on the responses (particle size and zeta potential). Based on the quantitative influencing factor-response relationships, two optimized CZ48 nanosuspensions of 197.22 ± 7.12 nm (NS-S) and 589.35 ± 23.27 nm (NS-L) were developed with the zeta potential values of -26.5 mV and -27.9 mV, respectively. Results: CZ48 released from the nanosuspensions in a sustained manner in contrast to the rapid release from cosolvent in both PBS and human plasma. Moreover, NS-S exhibited more favored pharmacokinetic properties than NS-L, with a 31-fold prolonged elimination half-life of CPT, and a 2.4-fold enhanced CPT exposure over cosolvent. In efficacy study, NS-S exhibited significant tumor suppression and an improved survival rate with a higher tolerable dose, compared to CZ48 cosolvent. Conclusion: We have successfully developed CZ48 nanosuspensions with significantly favorable pharmacokinetics and improved efficacy using CCD approach. The formulation offers potential merits as a preferred candidate for clinical trials with the prolonged CPT exposure, which is known to correlate with the clinical efficacy.

Desmoplastic small round cell tumor (DSRCT) xenografts and tissue culture lines: Establishment and initial characterization.

Desmoplastic small round cell tumor (DSRCT) is an extremely rare and aggressive neoplasm, which mainly affects young males and generally presents as a widely disseminated tumor within the peritoneal cavity. Due to the rarity of the tumor, its younger and overall healthier patient population (compared with other tumor types) and the fact that it lacks definitive histological and immunohistological features, the diagnosis of DSRCT may be frequently delayed or the tumor may be entirely misdiagnosed as a different type of abdominal sarcoma. The present study aimed to rectify the lack of models that exist for this rare neoplasm, through the development of several DSRCT tissue cultures and xenograft lines. Samples were received from surgeries and biopsies from patients worldwide and were immediately processed for xenograft development in nude mice. Tumor tissues were minced and fragments were injected into the dorsal flanks of nude mice. Of the 14 samples received, nine were established into xenograft lines and five into tissue culture lines. Xenografts displayed the microscopic histology of their parent tumors and demonstrated two different growth rates among the established xenograft lines. Overall, the establishment of these xenograft and tissue culture lines provides researchers with tools to evaluate DSRCT responses to chemotherapy and to investigate DSRCT-specific signaling pathways or mechanisms.

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.

TERT promoter mutations occur frequently in gliomas and a subset of tumors derived from cells with low rates of self-renewal.

Malignant cells, like all actively growing cells, must maintain their telomeres, but genetic mechanisms responsible for telomere maintenance in tumors have only recently been discovered. In particular, mutations of the telomere binding proteins alpha thalassemia/mental retardation syndrome X-linked (ATRX) or death-domain associated protein (DAXX) have been shown to underlie a telomere maintenance mechanism not involving telomerase (alternative lengthening of telomeres), and point mutations in the promoter of the telomerase reverse transcriptase (TERT) gene increase telomerase expression and have been shown to occur in melanomas and a small number of other tumors. To further define the tumor types in which this latter mechanism plays a role, we surveyed 1,230 tumors of 60 different types. We found that tumors could be divided into types with low (<15%) and high (≥15%) frequencies of TERT promoter mutations. The nine TERT-high tumor types almost always originated in tissues with relatively low rates of self renewal, including melanomas, liposarcomas, hepatocellular carcinomas, urothelial carcinomas, squamous cell carcinomas of the tongue, medulloblastomas, and subtypes of gliomas (including 83% of primary glioblastoma, the most common brain tumor type). TERT and ATRX mutations were mutually exclusive, suggesting that these two genetic mechanisms confer equivalent selective growth advantages. In addition to their implications for understanding the relationship between telomeres and tumorigenesis, TERT mutations provide a biomarker that may be useful for the early detection of urinary tract and liver tumors and aid in the classification and prognostication of brain tumors.

Correlation between the sensitivity of tumors to treatment with CZ48 and local concentrations of the active metabolite CPT within the tumors.

Crystalline camptothecin-20-O-propionate hydrate (CZ48) is an esterification product from the reaction of natural camptothecin with propionic anhydride. CZ48 has been tested against 29 human tumor lines grown in nude mice as xenografts. Of the tested tumor lines, 28 were found to be responsive to CZ48, by regression or significant inhibition. The total response rate was 97%. However, the effective dose required to achieve the positive response varied from 100 to 2000 mg/kg/day depending on the tumor type. Thus, the sensitivity of tumors to CZ48 treatment varied from tumor to tumor. The most sensitive CLO-breast carcinoma achieved regression when treated with 100 mg/kg/day, while PC3-prostate carcinoma required as high as 1000 mg/kg/day to achieve a definitive response. To determine the reason for these differences in sensitivities among the tumors, we treated 9 human xenografts grown in nude mice with 1000 mg/kg/day CZ48 until saturation and measured the local concentrations of the parental CZ48 as well as the corresponding metabolite camptothecin (CPT) in the tumors with the established high-performance liquid chromatography procedure. Results showed that the sensitivities of these tumors to CZ48 treatment were not affected by local concentrations of the active metabolite CPT in the tumors, but instead by the types of tumors.

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 K(m)s of 1.9 and 0.5 nM and V(max)s of 9.3 and 2.2 pmol/min/mg, respectively. However, the apparent intrinsic clearance (V(max)/K(m)) 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.

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.

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.

ISG15 as a novel tumor biomarker for drug sensitivity.

Tumor cells are known to exhibit highly varied sensitivity to camptothecins (CPT; e.g., irinotecan and topotecan). However, the factors that determine CPT sensitivity/resistance are largely unknown. Recent studies have shown that the ubiquitin-like protein, IFN-stimulated gene 15 (ISG15), which is highly elevated in many human cancers and tumor cell lines, antagonizes the ubiquitin/proteasome pathway. In the present study, we show that ISG15 is a determinant for CPT sensitivity/resistance possibly through its effect on proteasome-mediated repair of topoisomerase I (TOP1)-DNA covalent complexes. First, short hairpin RNA-mediated knockdown of either ISG15 or UbcH8 (major E2 for ISG15) in breast cancer ZR-75-1 cells decreased CPT sensitivity, suggesting that ISG15 overexpression in tumors could be a factor affecting intrinsic CPT sensitivity in tumor cells. Second, the level of ISG15 was found to be significantly reduced in several tumor cells selected for resistance to CPT, suggesting that altered ISG15 regulation could be a significant determinant for acquired CPT resistance. Parallel to reduced CPT sensitivity, short hairpin RNA-mediated knockdown of either ISG15 or UbcH8 in ZR-75-1 cells resulted in increased proteasomal degradation of CPT-induced TOP1-DNA covalent complexes. Taken together, these results suggest that ISG15, which interferes with proteasome-mediated repair of TOP1-DNA covalent complexes, is a potential tumor biomarker for CPT sensitivity.

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.

Synthesis and antitumor activity of aromatic camptothecin esters.

Twenty-eight new aromatic esters of camptothecins 2-29 were prepared in yields of 5 to 96% by straight acylation of camptothecin (1a) and 9-nitrocamptothecin (1b) with various aromatic acids as acylating agents. All of these esters were tested against 14 different human cancer cell lines. The antitumor activity of these compounds was related to the nature of the substituting groups of their side aromatic chains. In general, esters with strong electron-withdrawing groups on their side aromatic chains were active; esters with halogen-substituted side aromatic chains were slightly active; and esters without any substituting groups on their side aromatic chains were practically inactive. The IC50 studies showed that the majority of these esters were not as potent as their parental compounds 1a and 1b; whereas, the potencies of esters 6 and 25 were exceptionally high, much higher than the commercial camptothecin analogues and comparable to (or slightly more potent than) their parental compounds.

Synthesis and anti-tumor activity of alkenyl camptothecin esters.

AIM: 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.

Establishment and characterization of cancer cell cultures and xenografts derived from primary or metastatic Mullerian cancers.

PURPOSE: The purpose of this study was to characterize cell cultures and xenografts derived from patients with ovarian cancer. EXPERIMENTAL DESIGN: Ninety specimens from 67 patients were plated in RPMI 1640 or inoculated in nude mice. Growth characteristics of cell cultures and xenografts were determined. Expression of receptors for estrogen, progesterone, androgen, epithelial growth factor, fibroblast growth factor, HER-2/erbB-2/c-neu proto-oncogene, and the P53 expression were characterized by immunocytochemistry in 28 cell cultures. RESULTS: Forty-nine percent of samples were cultured successfully in vitro. Ascitic and pleural effusion specimens were more likely to produce a cell culture or a xenograft than solid tissue specimens (P < 0.005). All of the cell cultures had an epithelial morphology, and 89% were aneuploid with a mean DNA index of 1.6 (range, 0.9-3.0). Of 54 and 61 specimens inoculated into nude mice i.p. and s.c., 15 (28%) and 18 (30%) produced a xenograft, respectively, with two-thirds of these xenografts being reproducibly tumorigenic. The median time to first passage was 21 weeks for cell cultures and 8-12 weeks for xenografts. Expression of epithelial growth factor receptor, HER-2/erbB-2/c-neu proto-oncogene, fibroblast growth factor receptor, estrogen, progesterone, and androgen was seen in 24, 21, 31, 17, 43, and 18%, respectively. P53 was overexpressed in 62% of cell cultures analyzed. CONCLUSIONS: Ovarian cancer cells collected from effusions are easier to grow in vitro than in vivo. The only characteristic that may be associated with tumorigenicity was abnormal P53 expression. This panel of ovarian cancer materials provides useful models for biological or therapeutical studies.

Structure-activity relationship of alkyl 9-nitrocamptothecin esters.

AIM: To study the structure-activity relationship of alkyl 9-nitrocamptothecin esters. METHODS: Two alkyl 9-nitrocamptothecin (9NC) esters 5g and 5h were prepared by esterification reactions of 9NC with valeric anhydride and heptanoic anhydride, respectively. Eight 9NC esters 5a-5h were tested for cytotoxicity against human leukemia cell lines HL-60 and U-937. Flow cytometry analysis was used to identify the cell cycle phase targeted by the esters and quantify the extent of ester-induced cell death (apoptosis). RESULTS: Esters 5b and 5c demonstrated great abilities to inhibit growth of the leukemia cells followed by induction of apoptosis; esters 5a, 5e, and 5g induced slight perturbations in the cell cycle at high concentrations; and esters 5d, 5f, and 5h were completely inactive against the cell lines tested. Thus these esters showed the cell anti-proliferative activity in an order of 5b approximately 5c>5a approximately 5e approximately 5g>5d approximately 5f approximately 5h. Esters 5b, 5c, and 5e were tested in vivo against various human carcinomas in nude mice grown as xenografts. Only 5b and 5c showed a significant antitumor activity. Particularly, ester 5b demonstrated an antitumor activity against a broad spectrum of human carcinomas including breast, lung, colon, pancreas, stomach, ovarian, and melanoma, etc. CONCLUSION: These esters act like prodrugs of their parental 9-nitrocamptothecin. High drug doses need to be administered to animals in order to inhibit growth, and induce regression, of human tumor xenografts in nude mice. These compounds may be developed into potent anticancer drugs due to their low toxicity.

Preclinical evaluation of the anticancer activity and toxicity of 9-nitro-20(S)-camptothecin (Rubitecan).

The purpose of this study is to establish the maximum tolerated dose of rubitecan in mice, dogs and men and to establish the anticancer activity of such dose against human tumors xenografted in nude mice. Nude mice received increasing doses of Rubitecan by intrastomach injection until the maximum tolerated dose (MTD) had been established for both the single dose and the multiple doses at the schedule of 5 days on, 2 days off. Extrapolating from the mouse data, MTD was determined for oral administration in dogs and man. Levels of the drug in plasma were determined by high pressure liquid chromatography (HPLC). Using maximum tolerated multiple doses, the sensitivity of human cancer xenografts in nude mice to Rubitecan was determined. MTD of Rubitecan in mice for multiple doses intrastomach at the schedule of 5+,2- was 1 mg/kg/day. MTD in dogs was also 1 mg/kg/day, administered orally but at the schedule of 4+,3-. In man, it was 1 mg/m2/day at the schedule of 5+,2-. Treatment of human cancer xenografts in nude mice with MTD of Rubitecan resulted in 100% growth inhibition of 30/30 tumors tested and in 24/30 in their total disappearance. These 30 tumors comprised all the most common human cancers: lung, colorectal, breast, pancreatic, ovarian, prostate, stomach, melanoma and a leukemia. From the data collected, it appears that rubitecan is a very promising anticancer drug with high potency against a wide spectrum of human cancers. These cancers growing as xenografts in nude mice are always growth inhibited (30/30) and frequently (24/30) totally destroyed by the administration of non-toxic doses of Rubitecan.