Biocompatible Chemotherapy for Leukemia by Acid-Cleavable, PEGylated FTY720.

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■ INTRODUCTION
While modern medicine has made great advances in the fight against highly challenging diseases such as cancer, a need for specificity remains.Chemotherapeutics target cellular processes which are ubiquitously required, albeit to different extents, in all cells, thereby killing a subset of healthy cells and causing adverse side effects. 1 Furthermore, these side effects severely impact patient quality of life, at times resulting in discontinuation of therapy. 2 Enhancing specificity through conjugation of targeting molecules, exploiting the tumor microenvironment, and immunotherapy show much promise in addressing systemic toxicity.−5 A majority of targeted therapies are designed to interfere with a specific component of an oncogenic pathway; however, targets are not always specific to cancer, 6 and resistance mechanisms may activate parallel signaling pathways to allow for survival. 7,8Cancer metabolism is an attractive therapeutic target since oncogene-driven anabolism, dysregulation of growth, and defects in autophagy sensitize cancer cells to nutrient deprivation. 9−14 Cancer cells continue biosynthesis despite nutrient deprivation, which eventually limits ATP production and induces bioenergetic stress, ultimately resulting in cell death. 15−20 Importantly, FTY720 has shown minimal toxicity against normal cells while effectively killing cancer cells. 20,21Under the nutrient-limiting conditions induced by FTY720, normal cells can adapt by undergoing cell cycle arrest to become quiescent and catabolic, thus maintaining survival.Although various therapies currently exist that limit access to nutrients such as angiogenesis inhibitors and L-asparaginase, many do not possess the broad applicability of FTY720. 22,23−29 Furthermore, phosphorylated FTY720 is also known to induce immunosuppression by inhibiting lymphocyte egress from the thymus and secondary lymphoid organs, resulting in lymphopenia, a reduction of peripheral lymphocytes. 30FTY720 analogues designed to prevent phosphorylation through conformational restraint were shown to eliminate these adverse effects. 20,29,31The analogues down-regulate nutrient transport proteins and demonstrate potent anticancer efficacy in leukemia and prostate cancer models while maintaining normal heart rate.The promise of these compounds as anticancer therapies is counterbalanced by high monetary and temporal investments for new drug development.FDA approval, including clinical trials, may take up more than a decade with costs averaging over $1 billion and approval rate nearing only 10%. 32Most importantly, this approach is not generalizable to other chemotherapeutic agents.Prodrug formulation presents a less costly alternative through the 505(b)(2) development process, which could take as few as 30 months for FDA-approval. 33,34herefore, a generalized approach to the development of prodrug to specifically eliminate side effects of an already approved drug is highly meritorious from a scientific and translational standpoint.
Phosphorylation of FTY720 occurs during circulation, 35−37 and a prodrug form that would be stable in the bloodstream was developed by conjugating it with a biocompatible polymer, polyethylene glycol (PEG), via an acid-labile linkage.The prodrug should avoid bradycardia and immunosuppression by limiting phosphorylation (Figure 1).Once taken up by a cancer cell via endocytosis, the prodrug transforms to its active drug in the mildly acidic endosome and induces cell death by starvation.Both in vitro and in vivo studies demonstrated that the novel molecular approach in this study addresses a key clinical demand in developing effective and safe cancer chemotherapeutics.

■ RESULTS AND DISCUSSION
Design and Synthesis of FTY-k-PEG.Acetals (ketals) are commonly used hydroxyl-protecting groups and ideal for prodrug formulation because of their structural versatility, facile synthesis, and tunable cleavability under mildly acid conditions. 38The endosomal pH of ∼5.0 is an excellent Figure 1.Hypothetical illustration of FTY-k-PEG mechanism of action.FTY720 is rapidly phosphorylated to activate S1PRs and subsequently induces bradycardia and lymphopenia at the antineoplastic dose.An acid-transforming prodrug formulation shields FTY720 from phosphorylation, while maintaining its efficacy to downregulate nutrient transport proteins once endocytosed and reduced to the antineoplastic form.intracellular trigger for drug release from acid-labile drug conjugates. 39Initial synthetic methodology utilized 2-methoxypropene to generate methyl ketal-functionalized FTY720 (mkFTY) (Scheme S1), but the addition of hydrophobic methyl groups and loss of −OH hydrogen bonding resulted in loss of water solubility.To retain the ketal moiety while increasing solubility of the prodrug, N-Fmoc piperidone was used to generate a cyclic ketal in kFTY (7) (Scheme 1), eliminating the hydrophobic methyl groups and incorporating a secondary amine as a functional handle for further conjugation.The ketal formation was achieved using azeotropic distillation by which constant removal of water efficiently drives the reaction to completion. 40,41Although kFTY had increased water solubility compared to mkFTY, it was not readily soluble over concentrations of 10 mM.To further increase water solubility, kFTY was conjugated to succinimidyl carboxyl methyl ester polyethylene glycol (SCM-PEG 5k) prior to trifluoroacetamide (TFA)-deprotection to produce FTY-k-PEG (8).FTY-k-PEG was analyzed by MALDI-TOF to confirm PEGylation and TFA-deprotection (Figure S1).Average molecular weight of SCM-PEG 5K shifted by 479 Da after conjugation with kFTY-TFA (6) (MW 484.59 g/mol), and analysis of FTY-k-PEG (8) demonstrated a decrease in average molecular weight correlating to the loss of TFA (MW 97.02 g/mol).The synthetic methodology used in this synthesis proved to be straightforward and highyielding, and it uses low-cost, commercially available materials to further increase translational value.Other macromolecules may be used for conjugation, and a plethora of stimuliresponsive linkers may be explored to further enhance the specificity and safety of an FTY prodrug.−56 The versatility of the secondary amine on kFTY would allow development of novel combination therapy by conjugating another agent (e.g., drug and contrast agent) to further enhance its anticancer properties and combined imaging and therapy.
Triggered Release of FTY720 from FTY-k-PEG under Mildly Acidic Conditions.To assess the rate of drug release and demonstrate selective release under mildly acidic conditions, FTY-k-PEG was incubated at 37 °C in 10 mM deuterated acetate and tris buffers at pH 5.0 and 7.4, respectively, over the course of 2 days.The samples were analyzed by 1 H NMR taken at 0, 6, 28, and 48 h (Figure 2), and rate was determined by the peak at 2.23 ppm shifting to 1.91 ppm, correlating to the α-carbon protons as piperidone re-emerged.The spectra of FTY-k-PEG indicated that FTY720 was readily released at pH 5.0 (>80% after 28 h at a rate of 51 mM/h), while no detectable hydrolysis was observed at a physiological pH of 7.4 for a few days.This suggests that FTYk-PEG would be shielded from rapid phosphorylation during circulation and remain inactive toward S1PRs on off-target cells such as cardiomyocytes and lymphocytes, thus incapable of inducing onset bradycardia and lymphopenia.The drug release ratio at pH 5.0 to 7.4 was significantly higher than those using similar ketal chemistry. 57This might be attributed to the hydrophobic molecular environment of cyclic ketal at a neutral pH that dramatically changes to hydrophilic upon the protonation of the proximate amine at an acidic pH, greatly facilitating acid hydrolysis. 58,59The very slow hydrolysis of FTY-k-PEG at a neutral pH also implies reasonably long-term stability in saline, particularly under low-temperature storage conditions, in contrast to free FTY720 which requires daily fresh preparation.
Further studies in solid tumor cancer models are warranted since the tumor extracellular environment exhibits mildly acidic conditions with pH found to be as low as 5.7, a distinguishing phenotype often exploited for enhanced targeting. 60,61Considering the dramatically triggered release of FTY720 from FTY-k-PEG at pH 5.0, in contrast to no measurable release at pH 7.4, the drug release in the tumor environment is speculated to be elevated compared with other tissues/organs.
Eradication of Cancer Cells by FTY-k-PEG via Down-Regulated Nutrient Transporter Proteins.FTY720 is known to effectively inhibit cancer progression via downregulation of key nutrient transporters, selectively starving cancer cells to death. 20,21mkFTY and FTY-k-PEG were analyzed for affecting the viability and down-regulation of nutrient transporter associated protein 4F2hc in BCR-Abl p190-expressing FL5.12 murine hemopoietic cells, known to have a high expression of nutrient transporters. 62mkFTY demonstrated nearly identical efficacy to FTY720 in nutrient transporter down-regulation, confirming no loss of function after blocking hydroxyl groups by methyl ketal groups (Figure S2).FTY-k-PEG decreased expression of the amino acid transporter associated protein 4f2hc albeit at a reduced efficacy than that of FTY720 and had reduced efficacy for 24 h cell viability compared with FTY720 (Figure 3), possibly attributed to relatively inefficient cellular uptake of the PEGylated prodrug 63 than the free drug and slower acid-hydrolysis of cyclic ketal than methyl ketal. 57To confirm active drug was released selectively upon acid-hydrolysis, drugs were incubated in buffers of pH 7.4 and pH 5.0 prior to incubation with the cells (Figure 4).FTY720 was preincubated in the same buffers to rule out the possibility that the buffers would affect the drug's activity or contribute to cell apoptosis.A nonacidcleavable, PEGylated prodrug of FTY720, FTY-PEG, was also synthesized by conjugating SCM-PEG 5k to the free amine of FTY720 via amide bond (Figure S3), known to be extremely stable and hydrolyze only under harsh conditions or enzymatic cleavage. 64s expected, FTY-PEG had no effect on cell viability, indicating no release of FTY720.FTY-k-PEG preincubated at pH 7.4 yielded a significant decrease in efficacy compared with FTY720, while near identical efficacy to FTY720 is observed for FTY-k-PEG preincubated at pH 5.0.This demonstrates the acid-transforming nature of FTY-k-PEG and confirms its capability of releasing active drug under mildly acidic conditions.The cells were cultured in standard media where glucose concentration is not a limiting factor.The hypoglycemic microenvironment of tumors may further enhance the metabolic stress induced by released FTY720. 65,66The apoptotic effect of FTY720 is affected by nutrient transporter levels of cancer cells, and further studies would provide new insights in developing drugs for metabolicdependent targeted therapy for each cancer type.
Avoided Bradycardia and Lymphopenia in Vivo by FTY-k-PEG.To test whether FTY-k-PEG avoids bradycardia, C57BL/6 mice were surgically implanted with an electrocardiographic telemetry device, and heart rate was calculated from ECG data taken in the freely moving, conscious mice after intraperitoneal (i.p.) injection of saline, FTY720, or acidcleavable FTY-k-PEG (Figure 5A).Unmodified FTY720 clearly induces bradycardia, reducing heart rate by 50%, while FTY-k-PEG maintains normal heart rate, demonstrating that FTY-k-PEG fails to trigger bradycardia.To assess the effect of FTY-k-PEG on induction of lymphopenia, the numbers of circulating B and T lymphocytes were evaluated 12 h after i.p. injection of FTY720, FTY-k-PEG, and saline in C57BL/6 mice (Figure 5B).FTY720-induced lymphopenia was evident with nearly 60% reduction of lymphocytes, compared with that in saline or FTY-k-PEG-injected mice.These results suggest the safe administration of FTY-k-PEG at an antineoplastic dose of 155 mg/kg (10 mg/kg equivalent FTY720) without inducing the adverse effects associated with free FTY720.
Limited dosing of a chemotherapeutic agent due to adverse side effects is a significant barrier to accomplishing efficient therapy for cancer, often resulting in relapse. 67,68The failure of FTY-k-PEG to trigger bradycardia and lymphopenia in vivo indicates a possibility of high-dose chemotherapy.In addition, because the current work focuses on elimination of side effects, further studies can include extensive evaluation of FTY-k-PEG pharmacokinetic properties and interactions of S1PRs with FTY720 vs FTY-k-PEG to elucidate biochemical under-standing at a molecular level.To the best of our knowledge, this work presents a novel approach to prodrug-engineering by selectively preventing specific adverse effects through chemical modification, in contrast to many prodrugs aiming to avoid systemic side effects using a global approach.This concept could be applied to a multitude of cancer therapeutics to help decrease toxicity by evaluating detrimental side effects on a molecular level and designing prodrug moieties to prevent these unwanted effects.

−D).
There was a marked bioluminescence increase in mice treated with PBS and FTY-PEG indicating fast proliferation of BCR-Abl p190+ cells (Figure 6B,C).However, average bioluminescence signals in mice treated with FTY720 and FTYk-PEG were 64% lower than that of PBS on day 6 (p < 0.01).No significant difference was observed between FTY720 and FTY-k-PEG, confirming that FTY-k-PEG maintained the antileukemic properties of FTY720 without inducing bradycardia or lymphopenia (Figure 5).These results imply that the    acid-transforming prodrug formulation is a viable option to utilize the therapeutic benefits of FTY720 against leukemia and other cancers with avoided adverse side effects.Since FTY720 is an FDA-approved drug for multiple sclerosis and is selectively toxic to cancerous cells, a viable prodrug formulation with a preserved anticancer activity with lowered toxicity is highly beneficial for fast clinical translation, not only for therapeutic efficacy but also improved quality of patient life post-treatment. 2,33,34inimized Epigenetic Age by FTY-k-PEG.DNA methylation-derived biomarkers are currently gaining significant interest as changes in the epigenetic age are known to be associated with several pathological conditions including cancer 69 as well as treatment with certain chemotherapeutic drugs. 70In attempts to explore epigenetic age as a possible means to quantitatively evaluate systemic toxicity of a drug, healthy female Balb/c mice were daily treated with i.p. injections of FTY720, FTY-k-PEG, or PBS for 2 weeks, and blood samples were drawn prior to, during, and after treatment.Epigenetic age analysis was performed using Zymo Research Corporation's DNAge Epigenetic Aging Clock service, which assesses proprietary DNA methylation patterns known to specifically correlate epigenetic age to biological age.The methylated regions used for comparative analysis with corresponding methylation value (Figure 7A) showed clear differences in epigenetic biomarkers between FTY and FTY-k-PEG, and a significant increase in epigenetic age was observed in mice treated with FTY720 but not with FTY-k-PEG, 2 days post-treatment (Figure 7B).It was previously reported that bradycardia is most prevalent in the first 2 days after FTY720 administration, 25 and it was within this time frame when the greatest difference in epigenetic age was observed with FTY-k-PEG, possibly associated with avoided bradycardia and lymphopenia (Figure 5).−73

■ CONCLUSIONS
The limited clinical use of FTY720 for cancer therapy due to bradycardia and lymphopenia was addressed by an FTY720 prodrug that remains inactive during circulation but converts into its active form upon exposure to mildly acidic intracellular conditions.The prodrug engineering utilized an acid-labile cyclic ketal group to conjugated PEG, resulting in FTY-k-PEG, in an attempt to specifically inhibit phosphorylation of FTY720 that initiate the side effects.The synthetic methodology developed in this study is straightforward, scalable, and a less costly alternative to the development of an entirely new drug.FTY-k-PEG demonstrated nearly identical efficacy to free drug in BCR-Abl p190-driven leukemia model, both in vitro and in vivo, but without inducing bradycardia or lymphopenia.DNA methylation patterns known to be correlated with inflammation and disease implicated lower epigenetic damage by FTYk-PEG.This study demonstrated that prodrug engineering to disrupt specific off-target molecular mechanisms can effectively improve the safety profile of a drug.Future studies will be done on solid tumors to exploit the acid-cleavable prodrug for targeted anticancer effects, to elucidate the interactions of the prodrug at the molecular level, and to develop a combination therapy for synergistic cancer therapy.
■ EXPERIMENTAL SECTION Materials.All chemicals purchased from various vendors were used as received without further purification.FTY720• HCl was purchased from LC Laboratories (Woburn, MA).Succinimidyl PEG NHS 5K (mPEG SCM 5K) was purchased from Nanocs (Boston, MA).Anhydrous tetrahydrofuran (THF), anhydrous ethyl acetate (EtOAc), dichloromethane (DCM), anhydrous toluene, 3 Å molecular sieves, triethylamine (TEA), and pyridinium p-toluenesulfonate (PPTS) were purchased from Acros Organics (Morris Plains, NJ).para-Toluenesulfonic acid monohydrate (pTSA), anhydrous dimethylformamide (DMF), and dimethylsulfoxide (DMSO) were purchased from Sigma-Aldrich (Milwaukee, WI).Fmocn-piperidone, anhydrous magnesium sulfate, and all cell culture materials were purchased from Fisher Scientific (Hampton, NH). 1 H NMR spectra were obtained using a Bruker Advance 500 MHz NMR spectrometer (Billerica, MA).Electrospray mass spectra were obtained by using a Micromass LCT mass spectrometer (Milford, MA).For the determination of average molecular weight of mPEG SCM 5K and PEGylated prodrugs, an AB SCIEX MALDI TOF-TOF 5800 instrument (Framingham, MA) was used, and samples were prepared using 2,5dihydroxybenzoic acid (Acros Organics (Morris Plains, NJ) as matrix material.The samples were irradiated with 349 nm of diode-pumped solid state Nd:YAG laser and detected on linear high mass positive mode.For the molecular weight analysis, Mn calculated using Data Explorer software (Applied Biosystems, Foster City, CA).
Cell Viability.FL5.12 and FL5.12/p190 cells were seeded in a 48-well plate at a density of 4.0 × 10 4 cells/well with or without IL-3 (5 ng/mL) (Thermo Fisher, Waltham, MA).After incubation in varying concentrations of FTY720 and FTY-prodrugs for 24 h and rinsed with PBS, the cells were counted by flow cytometry (Guava Technologies, Hayward, CA) using Guava ViaCount Reagent (Guava Technologies, Hayward, CA) according to the manufacturer's recommended protocols.
Nutrient Transporter Expression.Surface 4F2hc expression was measured using phycoerythrin (PE)-conjugated rat antimouse 4F2hc antibody (BD Pharmingen, San Diego, CA); analysis by flow cytomery was restricted to viable cells.PE-conjugated mouse IgG1, κ (BD Pharmingen) was used as an isotype control.Cells were analyzed on a BD LSR II flow cytometer (BD Biosciences, San Jose, CA) and with FlowJo software (Treestar, Ashland, OR).
Heart Rate.Male C57BL/6 mice age 4−6 months were surgically implanted with DSI TA-ETAF20 electrocardiographic telemetry devices (Data Sciences International, St. Paul, MN) in the mouse's abdominal cavity with biopotential leads sutured in place in the chest wall.The electrocardiographic data was collected and recorded using the PhysioTel telemetry system and Dataquest A.R.T. 4.0 software (Data Sciences International).Mice were permitted a two week-long recovery after surgery before initiation of baseline telemetry recordings.Heart rate was calculated from ECG data taken in the freely moving, conscious mice.Mice (n = 3) were intraperitoneally treated with a single dose of vehicle (0.9% saline or 20% acidified DMSO in 0.9% saline), 10 mg/kg FTY720, or 155 mg/kg FTY-k-PEG, and heart rate was continuously monitored for 10 h.Mice were rested for 2 weeks at which point the experiment was repeated with the mice assigned to the alternate group.These experiments were performed as previously reported 29 in accordance with all national or local guidelines and regulations and were approved by the UCI Institutional Animal Care and Use Committee (IACUC).
Lymphocyte Sequestration.Female 8−24 week old C57BL6 mice were intraperitoneally injected with vehicle (0.9% saline or 20% acidified DMSO in 0.9% saline), 10 mg/kg (32 μmol/kg) FTY720, or 155 mg/kg FTY-k-PEG (32 μmol/ kg).After 12 h, blood was collected from the retro-orbital sinus under ketamine/xylazine anesthesia.Whole blood (10 μL) was added to 190 μL of ACK red blood cell lysis buffer.The mixture was incubated at RT for 3−5 min at 37 °C, and the white blood cells were recovered by centrifugation.Nucleated cells (Hoechst33342-positive) were counted using a hemocytometer to obtain the white blood cell count.Separately, 50 μL of whole blood was added to 1 mL of ACK red blood cell lysis buffer and incubated for 3−5 min at RT. Cells were washed with 2% fetal calf serum (FCS, Atlanta Biologicals (Flowery Branch, GA) in PBS with 0.05% NaN3 (Sigma-Aldrich (Milwaukee, WI)), and red blood cell lysis was repeated.Tubes were decanted and resuspended in 100 μL of 10% FCS in PBS with 0.05% NaN3 and directly conjugated antibodies against B220, CD4, or CD8 (all from Biolegend, San Diego, CA) for 30 min on ice.Cells were analyzed on a BD LSR II flow cytometer; the analysis was restricted to live cells (DAPInegative).These experiments were performed in accordance with all national or local guidelines and regulations and were approved by the UCI IACUC.
In Vivo Bioluminescence Imaging.In an immune competent syngeneic model, 6−7 weeks old Balb/c female mice were intravenously injected with FL5.12/p190/Luc cells (1.0 × 10 5 cells per mouse) in the tail vein.After 5 days, the mice received daily i.p. injections of FTY720, FTY-k-PEG, FTY-PEG (all at a dose of 32 μmol/kg) or PBS for 5 days (n = 5).Mice were placed under anesthesia using isoflurane (Patterson Veterinary, Greeley, CO) and interperitoneally injected with 120 mg/kg of synthetic firefly luciferin (Promega, Madison, WI) in PBS at a concentration of 20 mg/mL immediately prior to bioluminescence imaging with an IVIS Lumina system (Caliper Life Sciences, Hopkinton, MA) for 4 min.Mice were under anesthesia for the entire duration of imaging and bioluminescence signal intensities in mice were quantified using Living Image 3.2 software associated with the imaging system.These experiments were performed as reviewed and approved by the UCI IACUC.
DNAge (Epigenetic Aging Clock) Analysis.Blood samples (100 μL) were collected in triplicate from healthy female BALB/c mice treated as described above.Upon immediate preservation in DNA/RNA Shield (Zymo Research, Irvine, CA), genomic DNA was purified using Quick-DNA Plus Kit (Zymo Research) as instructed by the manufacturer.Sample library preparation and data analysis for mouse DNAge(epigenetics aging clock) were performed by service provider (Zymo Research).Briefly, genomic DNA (200 ng) was bisulfite-converted using EZ DNA Methylation-Lightning Kit (Zymo Research).Bisulfite-converted DNA libraries for targeted bisulfite sequencing platform, called SWARM (Simplified Whole-panel Amplification Reaction Method), were sequenced on a HiSeq 1500 sequencer for >1000× coverage.Sequence reads were identified using Illumina base calling software (San Diego, CA) and aligned to the reference genome using Bismark, an aligner optimized for bisulfite sequence data and methylation calling.The methylation level of each sampled cytosine was estimated as the number of reads reporting a C, divided by the total number of reads reporting a

Figure 7 .
Figure 7. Healthy BALB/c mice were treated with FTY720 (10 mg/kg [32 μmol/kg]) or FTY-k-PEG (155 mg/kg [32 μmol/kg]) or PBS by daily i.p. injections for 14 days, and blood samples were taken 2 days post-first treatment, 5 days after treatment ended, and 1 month after treatment.(A) Heatmap representation of differentially methylated regions used to calculate epigenetic age at various time points.(B) Epigenetic age generated from samples analyzed using proprietary markers for DNA methylation status (n = 3).