Sami
Osman
a,
William R.
Waud
b,
Gregory S.
Gorman
c,
Billy W.
Day
ad and
Kazunori
Koide
*a
aDepartment of Chemistry, University of Pittsburgh, 219 Parkman Avenue, Pittsburgh, Pennsylvania 15260. E-mail: koide@pitt.edu; Fax: +1 412 624 8611; Tel: +1 412 624 8767
bDepartment of Cancer Therapeutics and Immunology, Southern Research Institute, Birmingham, Alabama
cDepartment of Toxicology and Bioanalytical Science, Southern Research Institute, Birmingham, Alabama
dDepartment of Pharmaceutical Sciences, University of Pittsburgh, 3501 Fifth Avenue, Pittsburgh, Pennsylvania 15213
First published on 2nd November 2010
This study was designed to determine the in vitro and in vivo antitumor behaviour of three analogues of the natural product FR901464. All analogues demonstrated effective inhibition of cell proliferation. Minimal in vivo antitumor activity was observed, warranting further PK and antitumor efficacy studies.
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Fig. 1 Structures of FR901464, meayamycins and pladienolides. |
The structural and biological activities of FR901464 inspired other groups to discover novel natural products with similar biological activity. One such notable class of natural products is the pladienolides.4–7 Recently, it was found that both FR9014648 and the pladienolides9 bind to a subunit of the human spliceosome, splicing factor 3b (SF3b). Structure–activity relationship (SAR) and pharmacokinetic studies of the pladienolides led to the development of E7107 for its anticancer activity.9 E7107 showed remarkable antitumor activity in mouse xenograft models.9 It was then examined in Phase I clinical trials for solid tumors.10 This was presumably the first pre-mRNA splicing inhibitor to be tested in humans. In that study, E7107 was shown to reversibly inhibit pre-mRNA processing in humans.10 This clinical study has now been suspended.11
Although FR901464 was discovered and tested in xenograft models prior to the pladienolides, its progress in terms of drug development lags behind. Studies of FR901464 and its analogues through total chemical syntheses and biological analyses have provided substantial insights into the SAR of FR901464. The structural similarities between FR901464 and the pladienolides enabled the design and synthesis of simplified analogues in large quantities for animal studies.12 A preliminary study shows somewhat promising antitumor activity with one of the simpler analogues.12
We too have been engaged in the chemical and biological studies of FR901464 and its analogues. After our total synthesis of FR901464,13,14 we reported the chemically more stable analogue, meayamycin,15 which exhibited GI50 values in the low to middle picomolar range.16 Meayamycin was also shown to inhibit pre-mRNA splicing in a cell-free system and in live cells.16 Meayamycin was more potent against human small lung cancer A549 cells than against nontumorigenic human lung fibroblasts IMR-90.16 Meayamycin was very effective against a multidrug-resistant cell line.16 More recently, meayamycin B was synthesized and found to be even more potent than meayamycin and more stable in serum.17 Here, we wish to report our preliminary studies of meayamycins in vivo.
Antitumor activity was assessed on the basis of delay in tumor growth (T-C). The delay in tumor growth is the difference in the median of times post-staging for tumors of the treated and control groups to double in mass two times. Animals whose tumor failed to attain the evaluation size were excluded. Tumors were measured in two dimensions (length and width) twice weekly, and the tumor weight was calculated using the formula (length × width2)/2 and assuming unit density. The mice were also weighed twice weekly. As a positive control, tumor-bearing mice treated with COMPOUND LINKS
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Download mol file of compoundpaclitaxel intravenously at 15 mg kg−1/inj on a Q1D × 5 schedule were also examined.
Animals were anesthetized with 2:
1 CO2/O2 and heparinized microhematocrit tubes were used to collect blood from the retro-orbital sinus. Samples were held on ice until processed. Plasma was separated by centrifugation at 2,400 rpm and stored at −84 °C until assayed.
Cell Line | Tissue | Meayamycin GI50/nM | Meayamycin C GI50/nM |
COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundDoxorubicin GI50/nM |
Velcade GI50/nM |
---|---|---|---|---|---|
HCT-116 | Colon | 0.07 | 1.4 | 70 | 5 |
MCF-7 | Breast | 0.02 ± 0.0116 | 0.4 | N/A | N/A |
MDA-MB231 | Breast | 0.07 ± 0.0616 | 1.1 | N/A | N/A |
NHDF | Skin | 0.44 | 39 | 462 | 334 |
NHLF | Lung | 0.91 | 12 | 223 | 1.7 |
HMEC | Mammary | 0.11 | 9 | 276 | 3.5 |
HUVEC | Vein | 0.06 | 4 | 109 | 1.1 |
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Fig. 2 Dose tolerance of meayamycin and meayamycin B in mice (n = 3). (A) Tolerance of meayamycin in male athymic nude mice Q4D × 3. (B) Tolerance of meayamycin B in male athymic nude mice Q4D × 3. (C) Tolerance of meayamycin B in male athymic nude mice Q1D × 5. Arrows denote times of injections. |
Following the dose tolerance studies, we proceeded to evaluate the antitumor efficacy of meayamycin. With HCT-116 human colon tumor xenografts, tumor size was between 100 and 245 mm3 (100–245 mg) in size at the initiation of treatment (Day 16) (Fig. 3A). Intravenous administration of meayamycin at doses of 3.0 and 1.5 mg kg−1/inj on a Q4D × 3 treatment schedule lacked antitumor activity. Minimal antitumor activity was observed with a dose of 6 mg kg−1/inj administered Q4D × 3, resulting in a maximum inhibition rate (IR = [1-(median tumor weight treated/median tumor weight control)] × 100%) of 24% on day 41, although this may not be statistically significant (p > 0.05).
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Fig. 3 Activity of meayamycin, meayamycin B and COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundpaclitaxel in human tumor models (n = 10). (A) Response of HCT-116 tumor implanted sc to treatment with meayamycin (Day 16) and COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundpaclitaxel (Day 16). (B) Response of HCT-116 tumor implanted sc to treatment with meayamycin B (Day 16) and COMPOUND LINKS Read more about this on ChemSpider Download mol file of compoundpaclitaxel (Day 16). (C) Response of PC-3 tumor implanted sc to treatment with meayamycin B (Day 11). Data in (A) and (B) represent the same control group (paclitaxel-treated mice). Statistical significance was evaluated by comparing the time to 2 tumor mass doubling values of vehicle-treated groups to drug-treated groups. p values of less than 0.05 were considered statistically significant. The * signifies 0.05 < p < 0.80 (statistically not significant), and ** signifies p < 0.05. |
Next, meayamycin B was evaluated against the same xenograft model (Fig. 3B). Intravenous administration of meayamycin B at doses of 3.0 and 1.5 mg kg−1/inj on a Q4D × 3 schedule was tolerated without deaths and lacked antitumor activity. Administration of meayamycin B at a dose of 6 mg kg−1/inj on a Q4D × 3 schedule led to a maximum IR of 41% on Day 41. It should be noted that this result may be statistically insignificant (p > 0.05) and that one of the ten meayamycin B-treated mice died on Day 20. A vehicle given on the same schedule was tolerated without deaths. Intravenous administration of COMPOUND LINKS
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Download mol file of compoundpaclitaxel (positive control) at a dose of 15 mg kg−1/inj on a Q1D × 5 schedule was also tolerated without deaths and showed a maximum IR of 81% on Days 41 and 44.
Meayamycin B's antitumor efficacy was also evaluated in sc-implanted PC-3 human prostate tumor in male mice (Fig. 3C). These mice had tumor sizes of 100–234 mm3 (100–234 mg) at the initiation of treatment. A Q1D × 5 intravenous administration of meayamycin B at a dose of 1 mg kg−1/inj lacked antitumor activity while a dose of 2 mg kg−1/inj led to a maximum IR of 26% on Day 22 and a maximum average bodyweight loss of 15%. Treatment with saline, administered intravenously on a Q1D × 5 treatment schedule, was tolerated without deaths or appreciable bodyweight loss.
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Fig. 4 Plasma mean concentration time profiles of meayamycin B and meayamycin C in the plasma of female non-tumor bearing athymic nude mice. n = 3 for each time point. |
In order to gain insights into the pharmacokinetics of meayamycin B, we initially attempted to measure meayamycin B and its metabolites in the plasma viaLC-MS. Due to the complexity of the plasma matrix, initial Q1 data were not definitive. Next, targeted mixed reaction monitoring (MRM) transitions were designed based on the predicted metabolites from LightSight (Applied Biosystems, Foster City, CA). The targeted MRM transitions were designed on the basis of the mass shifts of the predicted metabolites. Mass shifts of the parent compound and the fragment ions were monitored. The observed peaks indicated dioxidation, demethylation, and glucuronidation. These metabolites were not quantified because authentic standards were not available. Nonetheless, multiple peaks were observed for the glucuronide mass transition, implying that the glucuronidation of meayamycin B might be a predominant metabolic pathway.
We hypothesized that meayamycin C, which has no hydroxy group, might be resistant to glucuronidation, thereby prolonging its half-life in plasma. To test this hypothesis, meayamycin C was synthesized17 and intravenously administered to female non-tumor-bearing athymic nude mice at a single dose of 6 mg kg−1. As shown in Fig. 4, meayamycin C plasma concentration fell quickly within 1 h after the injection and then remained below 10 ng ml−1 for the next 4 h, suggesting that the glucuronidation of meayamycin B was not the major reason for its poor pharmacokinetics.
To ensure that the measured levels in plasma were accurate and not affected by poor extraction of meayamycin B caused by covalent binding to proteins, we evaluated the extraction efficiency after incubation. Samples were prepared in triplicate by spiking mouse plasma at a concentration of 10 μM of meayamycin B and incubating them in a shaking water bath for 15 min at 37 °C. This is the same sample generation procedure that would be used to determine the plasma protein binding properties of drugs.19,20 After incubation, the samples were extracted using COMPOUND LINKS
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Download mol file of compoundacetonitrile and analyzed using tandem mass spectrometry with the identical methodology as employed for the previous samples described above. The amount of meayamycin B extracted from the incubated plasma samples was compared to a 10 μM solvent standard prepared in a starting mobile phase. The analysis showed that ∼ 95% of meayamycin B spiked into the incubated plasma samples was recovered. This result suggested it was not likely that strong irreversible protein binding occurred.
This study shows an attempt to evaluate the anticancer activities of novel synthetic FR901464 analogues in in vitro and in vivo cancer models. The antiproliferative activity of meayamycin and derivatives was first determined by measuring their GI50 values with various cancer cell lines. Our results, together with previously reported data, showed that all of the FR901464 derivatives used in this study exhibited strong antiproliferative activity against these cell lines. The order of potency was meayamycin B > meayamycin > meayamycin C. Despite the potency of meayamycin and meayamycin C in cancer and non-cancerous cells, the therapeutic index increased from meayamycin to meayamycin C (if, e.g., we compare GI50 in the HCT-116 cell line and the non-cancerous cell lines in Table 1). The therapeutic index for meayamcyin is 6 (NHDF cell line), 2.7 (NHLF cell line), 1.6 (HMEC cell line), and 1 (HUVEC cell line). On the other hand, the therapeutic index for meayamycin C is 15 (NHDF cell line), 4.6 (NHLF cell line), 3.5 (HMEC cell line), and 1.5 (HUVEC cell line). This indicates that there is some degree of selectivity of meamycin C towards the HCT-116 cancer cell line. This quantitative analysis should be met with caution because the margin of error could be greater when the growth inhibition of nontumorigenic cells was measured.
Subsequently, we initiated an in vivo study to determine whether the compounds could inhibit tumor growth in HCT-116 and PC-3 xenograft models. First, we determined a maximum tolerated dose of meayamycin and meayamycin B. Doses for both compounds ranged from 0.03 to 3 mg kg−1/inj administered intravenously on a Q4D × 3 schedule. The treated animals were monitored for weight loss and mortality. No considerable weight loss or deaths were observed in any of the mice during or after treatment, except that a maximum average body weight loss of 11% was observed with meayamycin B at a dose of 3 mg kg−1/inj. Meayamycin B was also tolerated at a different dosing schedule, Q1D × 5, at doses of 0.5 and 1.0 mg kg−1/inj. A dose of 1.0 mg kg−1/inj led to only a maximum average body weight loss of 10%. Higher doses (1.5–6.0 mg kg−1/inj) were lethal for mice. Webb et al. designed structurally simpler analogues that were tolerated at much higher doses (≤50 mg kg−1 at a Q1D × 5 schedule), but that may be attributed to the lower antiproliferative activity (GI50 were at high nM to single-digit μM).23 These data show that doses of meayamcyin and meayamycin B can be administered safely by the intravenous route on Q4D × 3 and Q1D × 5 schedules.
The in vivo antitumor activity of meayamycin and meayamycin B was initially evaluated in the HCT-116 xenograft model in mice. Dosing schedule studies revealed that intravenously administered meayamycin and meayamycin B achieved IR values of 24% (Day 41) and 41% (Day 41), respectively, using a Q4D × 3 dosing schedule. With the low IR values of both meayamycin and meayamycin B, we decided to increase the frequency of injections. We chose meayamycin B for this experiment because meayamycin B was more stable than meayamycin in mouse serum (t1/2 = 13 h vs. 2 h).17 Due to the availability of xenograft models at the time, we switched from HCT-116 to PC-3 in the next study. Against the PC-3 tumor xenograft, meayamycin B elicited an IR of 26% on Day 22 using a Q1D × 5 dosing schedule. A higher dose was lethal to mice at this injection frequency, which limited our window of optimizations. Although a positive control was omitted in this experiment, the xenograft model might be considered valid because the tumor growth (200 mg to 400 mg) of the vehicle-treated group occurred over 3.1 days, a rate within the range we previously observed (1.5–3.9 days).24 In HCT-116 tumor xenografts, COMPOUND LINKS
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Download mol file of compoundpaclitaxel exhibited an IR of 81% on Days 41 and 44 using a Q1D × 5 dosing schedule. The interpretation of the available preclinical data is limited by the lack of information of plasma FR901464-like PK parameters that are associated with antitumor activity in animal models.
Our study illustrated that less than 1% of the injected meayamycin B was found in plasma within 30 min after administration. A glucuronidated meayamycin B metabolite exhibited the strongest signal in the mass spectrometric evaluations when compared to other detected metabolites. This result led us to hypothesize that meayamycin C might be resistant to glucuronidation. To test this hypothesis, meayamycin C was synthesized and its plasma concentrations were evaluated in female non-tumor-bearing mice after intravenous administration. The plasma concentrations of meayamycin C fell quickly within 1 h after the injection and remained below 10 ng ml−1 for the next 4 h, suggesting that the glucuronidation of meayamycin B was not the major reason for its poor pharmacokinetics. Although meayamycin B rapidly diminished in mouse plasma, the compound showed some antitumor activity, IR = 41%. It is possible that some of the metabolites of this compound, formed by the action of metabolizing enzymes, are the active form(s) of the agent.
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