Background
Tamoxifen remains a safe and effective agent for women diagnosed with ER (+) breast cancer. It is a first-line agent for pre-menopausal breast cancer patients and for women requiring secondary chemoprevention after a DCIS or LCIS diagnosis. It is an option for other ER+ breast cancer patients who do not tolerate the side effects of aromatase inhibitors. Results of the ATLAS trial show that 10-years treatment with tamoxifen further improves long-term survival compared to 5-years treatment [
1]. However, the response to tamoxifen shows well-known individual variability [
2‐
8]. Tamoxifen is a pro-drug, which needs to be converted into active metabolites for optimal clinical activity. Cytochrome P450 enzyme CYP2D6 is required to convert tamoxifen into 4-hydroxytamoxifen (4-OHT) and endoxifen [
9], both of which are about 100 times more potent than tamoxifen [
10,
11]. Genetic polymorphism in CYP2D6 affects the rate of metabolic activation of tamoxifen. This may account for poor initial response to tamoxifen and worse disease outcome after standard therapy. Multiple clinical studies have shown that poor metabolizer (PM) patients tend to have shorter overall survival rate than those who are extensive metabolizers (EM) [
4‐
8]. Existing clinical and laboratory data support the hypothesis that bioavailable 4-OHT or endoxifen could offer improved therapeutic efficacy and potentially lower dose requirements, with reduced adverse effects [
12‐
14]. Indeed, 4-OHT is being developed as a topically applied gel currently in Phase II clinical trials [
15‐
18]. The use of orally available 4-OHT is hampered by its rapid first-pass clearance due to O-glucuronidation [
19] and the resulting poor bioavailability compared to oral tamoxifen. Clinical trials utilizing high-dose tamoxifen have been conducted in PM patients in order to increase blood levels of active metabolites. However, this also increases the risk with adverse effects including hot flashes and thrombosis [
20].
We have recently developed several boron-derived prodrugs of 4-OHT that demonstrated potent antiestrogenic activities
in vitro at significantly lower concentrations than tamoxifen [
21]. We propose a novel endocrine therapy regimen using ZB497, an orally bioavailable prodrug form of 4-OHT that can be administered at lower doses than standard tamoxifen treatment, thereby not only circumventing the need for CYP2D6 enzyme to catalyze the hydroxylation of tamoxifen or N-desmethyltamoxifen, but also potentially reducing or eliminating side effects by virtue of significantly reduced dosage. In order to further evaluate the prodrug as a potential new option in breast cancer treatment and/or prevention, we conducted in vivo efficacy studies using a well-characterized mouse xenograft model based on the ERα positive MCF-7 breast cancer cells. We determined whether the boron-based 4-OHT prodrug can achieve acceptable in vivo efficacy in an ERα + breast cancer xenograft model as compared to tamoxifen in a dose dependent manner. Pharmacokinetic studies were performed in mice to investigate the metabolism, distribution, and concentration change over time after a single dose of ZB497, in comparison with tamoxifen and 4-OHT. Moreover, tumor tissues from mice were analyzed for drug accumulation after 21 days of treatment of ZB497 or tamoxifen.
Methods
Reagents and materials
All reagents, solvents, and analytical standards were purchased from Sigma Aldrich (St. Louis, MO) and Fisher Scientific (Fairfield, NJ). ZB497 were synthesized following the synthetic route described in detail in a previous report [
21].
In vivo efficacy study in mice
Four- to six-week old female ovariectomized Nu/Nu mice were purchased from Charles River Laboratories (Wilmington, MA). The mice were given a period of adaptation in a sterile and pathogen-free environment with phytoestrogen-free food and water ad libitum. MCF-7 cell line was purchased from ATCC (ATCC #HTB-22, Manassas, VA), and routinely cultured in phenol red-free DMEM medium supplemented with 5 % FBS, 4 mM glutamine, 1 mM sodium pyruvate, 100 IU/mL penicillin, 100 μg/mL streptomycin and 0.25 μg/mL amphotericin. The cells were harvested in the exponential growth phase using a PBS/EDTA solution. The animals were injected bilaterally in the mammary fat pad (MFP) with 5 × 106 viable cells suspended in 50 μL sterile PBS mixed with 100 μL Matrigel (reduced growth factor; BD Biosciences, Bedford, MA). 17β- estradiol pellets (0.72 mg, 60-day release; Innovative Research of America, Sarasota, FL) were implanted subcutaneously in the lateral area of the neck using a precision trochar (10 gauge) at the time of cell injection. All procedures in animals were carried out under anesthesia using a mixture of isofluorane and oxygen delivered by mask. Tumors were allowed to form and at day 15 post cell injection mice were randomized into groups of 5 mice each. Mice were treated daily with intraperitoneal (i.p.) injection or oral gavage of either vehicle (1:20 DMSO/PBS for i.p. or 1:10 ethanol/PBS for oral gavage) or a drug for 21 days. For the dose-dependent efficacy study, five groups (5 mice/group) of tumor-bearing nude mice (one control and one group each per dose per drug). Tumor size was measured 3 times weekly using digital calipers. Tumors were surgically removed from sacrificed mice treated with a daily dose of either 1 mg/kg tamoxifen or 1 mg/kg ZB497 by oral gavage for 7 days, weighed, and stored at −80 °C until sample preparation and analysis.
Pharmacokinetic studies
Female C57BL/6 mice were used for the pharmacokinetic study of ZB497. For intraperitoneal administration of drugs, mice were injected with PBS containing ZB497, tamoxifen, or 4-OHT by adding appropriate amounts of individual stock solutions of the drugs dissolved in DMSO. For oral administration, mice were given oral gavage containing PBS and ethanol-dissolved ZB497, 4-OHT, or tamoxifen at a single dose of 1 mg/kg/mouse. After i.p or oral administration, blood samples were collected from the orbital sinus of the mice at various time points with each group of mice subjected to only one sampling. Mice blood was collected with a capillary into 1.5 mL microcentrifuge tubes containing 0.1 mL of 10 % EDTA anticoagulant. Plasma was then separated from red cells by centrifugation in a refrigerated centrifuge at 4 °C and transferred to a separate tube. The plasma samples were frozen at −80 °C until analysis.
Analysis of drug concentrations in plasma and tumor tissues
Plasma samples were extracted with chloroform:methanol (2:1) using traditional Folch method for lipid extraction. Methanol (1 mL) and chloroform (2 mL) were added to each plasma sample followed by addition of 5 ng trans-Tamoxifen-13C2, 15 N to each sample as the internal standard. The mixtures were stored at −20 °C overnight. Next, the samples were sonicated for 5 min and centrifuged with a Thermo Scientific Heraeus Megafuge16 Centrifuge. The top layer was transferred to another test tube. The bottom layer was washed with 1 mL chloroform:methanol (2:1), centrifuged, and the top layer was transferred and combined with the previous top layer. Eight tenth of a milliliter HPLC grade water was added to the extracts. After vortexing, the mixture was centrifuged. The bottom layer was dried out with nitrogen and re-suspended in 100 μL HPLC grade acetonitrile. An aliquot of 10 μL sample was injected onto a Hypersil Gold column (50 × 2.1 mm; particle size 1.9 μm, Thermo Scientific) on a Dionex Ultimate 3000 UPLC system equipped with a TSQ Vantage triple quadrupole mass spectrometer for analysis. A binary mobile phase (A: water with 0.05 % formic acid; B: acetonitrile with 0.05 % formic acid) was used to achieve a gradient of initial 30 % B for 1 min and then to 80 % B at 8 min, to 100 % B at 9 min, and returned to 30 % B for 4 min. The flow rate was controlled at 0.6 mL/min. The settings of HESI source were as follows: spray voltage (3200 volt); vaporizer temperature (365 °C); sheath gas pressure (45 psi); auxiliary gas pressure (10 psi); capillary temperature (330 °C). Nitrogen was used as the sheath gas and auxillary gas. Argon was used as the collision gas.
For determination of drug concentrations in tumor tissues, tumors were initially homogenized in 3 mL chloroform:methanol (2:1 v:v) with a PYREX™ Tenbroeck tissue grinder. The same solvent (1 mL) was used to wash the tissue grinder three times and the washings were combined with the initial homogenized tumor suspension. After adding 5 ng trans-tamoxifen-13C2, 15 N to each sample as an internal standard, the mixtures were stored at −20 °C overnight. The mixtures were then sonicated for 5 min and centrifuged. The top layer was transferred to another test tube. The bottom layer was washed with 1 mL chloroform:methanol (2:1), centrifuged, and the top layer from this wash was transferred and combined with the previous top layer. After adding water (1.4 mL) to the extract, vortexing and centrifuging, and the bottom layer was dried out with nitrogen and re-suspended in 100 μL HPLC grade acetonitrile for analysis on the HPLC-TSQ instrument under the same conditions as those used for the analysis of the plasma samples.
Ethical considerations and statistical analysis
All procedures involving the animals were conducted in compliance with State and Federal laws, standards of the U.S. Department of Health and Human Services, and guidelines established by the Institutional Animal Care and Use Committee at Xavier University. All animal experiments were approved by Xavier’s Institutional Animal Care and Use Committee. The facilities and laboratory animals program of Xavier University are accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care. Statistical analyses were performed using Microsoft excel software. Pharmacokinetic data analyses were performed using the PKsoftware [
22].
Discussion
Tamoxifen is a prodrug and its full therapeutic effect depends on the biotransformation of tamoxifen into its active metabolites, 4-OHT and endoxifen. Thus the use of 4-OHT as an agent for endocrine therapy is attractive in that not only it is 100-fold more potent as an antiestrogen than tamoxifen but also 4-OHT has well-known pharmacological and toxicological profiles. It can circumvent the problem associated with deficient CYP2D6 metabolism and potentially lower the dosage requirement owing to its inherent high potency. The problem lies in 4-OHT’s rapid first-pass metabolism, and the poor bioavailability of 4-OHT when used directly via oral administration.
By introducing a cleavable boron-aryl carbon bond to replace the hydroxyl group in 4-OHT, we have designed and tested a boronic prodrug, ZB497 for its ability to overcome the poor bioavailability of 4-OHT. Our study has demonstrated that ZB497 can not only effectively deliver the desired form of the drug, 4-OHT in an animal model, but also at a much higher plasma concentration. The facile oxidative deboronation of the prodrug under physiological conditions is likely facilitated by the presence of P450 enzymes and/or reactive oxygen species [
24]. In mouse blood after oral or intraperitoneal administration of ZB497, the predominant drug form is 4-OHT, constituting about 75-85 % of total drug concentration in blood. In addition, compared to tamoxifen or 4-OHT treatment, ZB497 also afforded higher levels of endoxifen in plasma, independent of CYP2D6 status. Such guaranteed delivery of 4-OHT can effectively overcome differences in therapeutic efficacy due to variations in patient metabolism.
But more importantly, the boronic prodrug yielded an astonishingly higher concentration of 4-OHT in systemic circulation than was expected, lending itself to an equally enhanced bioavailability that would have favorable clinical implications. The 30- to 40-fold increase in plasma concentration of 4-OHT upon i.p. or oral administration of ZB497, compared to 4-OHT administration at equal dosage, strongly suggests that the boronic structure does more than just delivering the desired 4-OHT form while preventing its rapid clearance from the body via glucuronidation. Through a mechanism not yet fully accounted for, the boronic structure must be responsible for enabling the remarkable enrichment in plasma concentration of 4-OHT as measured in our pharmacokinetic studies. From the metabolism data both
in vitro [
21] and in vivo in the current study, we know that the predominant form of ZB497 is B415, the boronic acid form of the prodrug. It is well known that boronic acids can form reversible complexes with 1,2 and 1,3 diol groups common in sugar molecules and glycoproteins [
25,
26], which upon hydration would release the boronic acids. We hypothesize that such reversible, covalent complexes between boronic acid and a diol group may facilitate the enrichment in plasma due to the abundance of molecules containing the diol groups. Moreover, such complexes may serve as a reservoir to release the boronic acid as the prodrug undergoes deboronation to be converted to 4-OHT. Indeed, this unique property of boronic acid has recently been exploited for an enhanced delivery of insulin for treatment of diabetes [
27]. Consequently, in addition to markedly greater plasma concentration, the clearance time of 4-OHT from ZB497 (t
1/2 = 39.5 hrs) is significantly prolonged compared to direct administration of 4-OHT (t
1/2 = 31.7 h) or tamoxifen (t
1/2 for 4-OHT = 22.6 h).
As an immediate result of increased plasma 4-OHT concentration, we found that xenograft tumor tissues also have significantly higher drug accumulation. We also noted that in tumor tissues, the percentage of boron-containing drugs was significantly higher than in plasma after repeated doses of ZB497, suggesting a preferential uptake of the boronic acid form of the prodrug by cancer cells. Nevertheless, 4-OHT concentration in tumor tissues from ZB497-treated mice was still 4 times higher than found in mice treated with tamoxifen.
We believe that enhanced overall bioavailability of 4-OHT conferred by ZB497, as evidenced in the over 30 fold increase in peak plasma concentration and the 8-fold increase in total drug accumulation in tumor tissues culminated in ZB497’s superior in vivo efficacy. At the dosage lowered to 1/10 of a milligram per kilogram, ZB497 remained therapeutically effective in inhibiting xenograft tumor growth, whereas tamoxifen largely lost its efficacy for tumor inhibition at this dose. On the other hand, at the higher dosage of 1 mg/kg, the efficacy difference between ZB497 and tamoxifen was not as prominent. In addition, the degree of tumor growth inhibition by ZB497 at two doses was also similar, and not proportional to the 10-fold difference in dosage. We speculate that the maximum therapeutically effective drug concentration in systemic circulation may have been reached by ZB497 at 0.1 mg/kg and by tamoxifen at 1 mg/kg. Thus, further increasing the dosage of ZB497 to 1 mg/kg may not lead to proportional increase in efficacy in mice.
As demonstrated in the efficient metabolic conversion of ZB497 to 4-hydroxytamoxifen in mice, the prodrug aims to deliver a therapeutically effective dose of the active drug form, 4-OHT. Therefore, it is conceivable that the main possible side effects of the prodrug would be associated with 4-OHT, which is a SERM that behaves similarly as tamoxifen with up to 100-fold greater potency than tamoxifen. It is unknown if 4-OHT may exert 100-fold greater side effects if given at the same dose, but at a fraction of the dose that may be required to reach a therapeutic level, the side effects may not exceed those of tamoxifen. Moreover, because use of ZB497 will eliminate tamoxifen and other related metabolites in systemic circulation, side effects due to these substances may also be eliminated.
Each dose of one mg of ZB497 will generate 0.23 mg of pinacol and 0.12 mg of boric acid. At these low levels, both pinacol and boric acid are not known to be toxic to humans. The LD50 of pinacol in mouse is 3,380 mg/kg oral dose [
28] and the LD50 of boric acid in rat is 2660 mg/kg [
29]. Notably, the mean daily intakes of boron from food and beverages alone for male and female adults are 1.28 and 1.00 mg respectively [
30]. These data suggest that side effects due to the boronic structure of ZB497 may be minimal to nonexistent.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
QZhong, CZ, QZhang carried out experiments and participated in manuscript writing. SZ and GW conceived the study and wrote the manuscript. LM participated in design of study and writing, review, and revision of the manuscript. All authors read and approved the final manuscript.