Letter pubs.acs.org/macroletters
Versatile Synthesis of Polymer-Temozolomide Conjugates Matthew Skinner, Sarah M. Ward, and Todd Emrick* Polymer Science and Engineering Department, University of Massachusetts, 120 Governors Drive, Amherst, Massachusetts 01003, United States S Supporting Information *
ABSTRACT: A new and versatile synthesis was developed to produce polymer prodrugs composed of poly(2-methacryloyloxyethyl phosphorylcholine) (polyMPC) conjugated covalently to the glioblastoma drug Temozolomide (TMZ). Through a controlled free radical copolymerization of a TMZ-substituted methacrylate and MPC, water-soluble polyMPC-TMZ conjugates were realized with TMZ loadings of >50 mol %. After polymerization, the structural fidelity of the pendent TMZ moieties was confirmed by NMR and UV− vis spectroscopy, while molecular weight estimation by gel permeation chromatography (GPC) confirmed the desired control over the polymerization, with isolated polymer-TMZ conjugates exhibiting narrow molecular weight distributions. This synthetic design opens new possibilities for formulating numerous types of polymeric TMZ-based structures that are of interest for treating glioblastoma.
P
olymer−drug conjugation is effective for augmenting the antitumor efficacy of many small molecule chemotherapeutics, affording polymer prodrugs with enhanced aqueous solubility and improved pharmacokinetic characteristics over the drug alone.1,2 As a result of the leaky vasculature and poor lymphatic drainage of solid tumors, accumulation of polymer-bound drugs in malignant tissue is enhanced, leading to greater antitumor activity and reduced off-target toxicity.3 Temozolomide (TMZ) is a first-line chemotherapeutic for treating glioblastoma, the most commonly diagnosed malignant central nervous system tumor in the United States. 4 Administered orally, TMZ is a lipophilic, acid-stable DNA methylating agent that crosses the blood−brain barrier and presents relatively mild side effects.5,6 Serving as a small molecule prodrug, TMZ hydrolyzes spontaneously at physiological pH, as shown in Figure 1, liberating methyldiazonium cations that elicit antitumor activity by methylation of guanine and adenine nucleobases.7,8 Owing to the slightly alkaline microenvironment of brain tumors,9 this pH-induced mechanism confers selective cytotoxicity for malignant versus healthy brain tissue, resulting in limited off-target toxicity.7 Unfortunately, the rapid in vivo decomposition and clearance of TMZ requires frequent dosing to maintain antitumor activity.7,8,10 Several strategies have been studied for sustained TMZ delivery, including encapsulation in nanoparticles and liposomes for systemic administration,11−13 as well as entrapment in hydrogels,14 degradable matrices,15 and microspheres for localized treatment.16 We found only one example of a polymer-TMZ conjugate, prepared from poly(β-L-malic acid).17 While this conjugate demonstrated in vitro activity against glioblastoma tumor cells, the chemical and physical characterization of the polymer prodrug was limited, and the © XXXX American Chemical Society
Figure 1. Chemical decomposition of TMZ: hydrolysis/decarboxylation at physiological pH affords the labile 5-(3-methyltriazen-1yl)imidazole-4-carboxamide (MTIC); further acid-catalyzed degradation yields 5-aminoimidazole-4-carboxamide (AIC) and finally the cytotoxic methyldiazonium cation.
synthetic strategy is unsuitable for preparing well-defined structures with tunable drug loading. The current prognosis for glioblastoma is poor: with combined surgery, radiation, and TMZ chemotherapy, median survival is 12−15 months.5,6,18 In addition to a short in vivo half-life, TMZ efficacy is hampered by chemoresistance induced by O6-methylguanine-DNA methyl transferase (MGMT), a native enzyme that repairs damaged DNA following methylation.7,8 MGMT activity in glioblastoma tumors can be depleted by sustained TMZ exposure,19 and recurrent glioblastoma treated with protracted dosing schedules improves Received: January 5, 2017 Accepted: February 3, 2017
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DOI: 10.1021/acsmacrolett.7b00007 ACS Macro Lett. 2017, 6, 215−218
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yield as a flaky white solid, with the expected molar mass confirmed by fast-atom bombardment HRMS (HRMS-FAB) ([M + H]+: 308.0989 g/mol). In the 1H NMR spectrum of 2, resonances corresponding to imidazole (δ = 8.85 ppm) and methyltriazene (δ = 3.88 ppm) protons were observed (Figure S2); the UV−vis absorption spectra of TMZ (λmax = 325 nm) and TMZ-methacrylate 2 (λmax = 323 nm) were nearly identical (Figure S3). The RAFT-mediated copolymerization of MPC and 2 was attempted initially in a MeOH/DMSO solvent mixture to ensure homogeneity. Although monomer conversion was high (87%), UV−vis characterization of the isolated copolymer showed a significant absorption at (λmax = 287 nm), indicative of TMZ decomposition to the AIC byproduct.27 This observation prompted an investigation of the stability of TMZ in organic solvents, findings not reported previously to our knowledge. Solutions of TMZ in 1:1 MeOH/DMSO, DMSO, acetonitrile, and 2,2,2-trifluoroethanol (TFE) were incubated at room temperature, 50 °C, and 70 °C. UV−vis spectroscopy showed the absorption spectrum of TMZ to remain unchanged after 24 h incubation in DMSO and acetonitrile at all temperatures, as shown in Figures 2 and S4. In
tumor response.20−23 As these alternative regimens can increase the occurrence of dose-limiting hematoxicity, new synthetic designs are needed that extend TMZ circulation and mask offtarget toxicity. We are examining the potential for polymer prodrugs to improve TMZ efficacy through either systemic or local treatment. We previously studied polymer zwitterions, specifically poly(2-methacryloyloxyethyl phosphorylcholine) (polyMPC), in chemotherapy.24,25 For example, water-soluble polyMPC-Doxorubicin (Dox) prodrugs exhibited extended circulation in breast and ovarian tumor animal models and led to greater drug accumulation in tumor tissue versus healthy organs. Mice treated with polyMPC-Dox prodrugs exhibited enhanced survival, retarded tumor growth, and lower incidence of dose-limiting toxicity relative to animals treated with Dox alone. Here we seek to extend the polyMPC prodrug platform to TMZ and, as a first step, aim to develop an efficient synthesis of water-soluble polyMPC-TMZ conjugates. Through a novel TMZ-methacrylate derivative, the drugs were incorporated as pendent moieties in polyMPC-based copolymers using reversible addition−fragmentation chain-transfer (RAFT) polymerization. The polymerization experiments afforded polyMPC-TMZ conjugates with tunable drug incorporation and narrow molecular weight distribution. Polymerization conditions were optimized to ensure fidelity of the TMZ structure such that the conjugates would retain the efficacy of TMZ. TMZ-methacrylate 2, the key monomer in this study, was prepared as shown in Scheme 1. TMZ-carboxylic acid 1 was Scheme 1
Figure 2. (a) UV−vis absorption spectra of TMZ solutions incubated in 1:1 MeOH/DMSO at room temperature (left), 50 °C (middle), and 70 °C (right); (b) UV−vis absorption spectra of TMZ solutions incubated at 70 °C in DMSO (left), acetonitrile (middle), and TFE (right).
contrast, TMZ heated to 50 or 70 °C in 1:1 MeOH/DMSO degraded completely in 1 h; significant degradation was observed even at room temperature. Importantly, TMZ proved stable in TFE, a generally good solvent for polymer zwitterions, at temperatures up to 70 °C for >24 h. The marked difference in TMZ stability in MeOH versus TFE is attributed to the weaker nucleophilicity of the latter, which slows or precludes imidazotetrazine solvolysis.28 The insight derived from this TMZ stability analysis led us to conduct RAFT copolymerizations of MPC and 2 at 70 °C in TFE, as shown in Figure 3, utilizing 4,4′-azobis(4-cyanovaleric acid) (ACVA) and 4-cyano-4-(phenylcarbonothioylthio)pentanoic acid as initiator and chain-transfer agent, respectively. Reagent stoichiometries were adjusted to target copolymers with approximate number-average molecular weight (Mn) values of 20000 g/mol, and monomer feed ratios were intended to afford 11, 21, 35, and 50 mol % incorporation of 2 into polymers 3A−3D, respectively. Monomer conversions of 88−94% were achieved in polymerization times of 6−9 h. Polymerizations were terminated by exposure to air, and the
synthesized by diazotization and hydrolysis of the TMZ amide precursor in nitrous acid using a procedure similar to that described by Stevens and co-workers.26 Precipitation over ice afforded 1 as a fine white solid in yields of up to 75%. The absence of TMZ amide resonances in the 1H NMR spectrum of 1 (Figure S1) indicated complete carbamoyl hydrolysis, and electron ionization high-resolution mass spectrometry (HRMSEI) confirmed the expected structure of 1 ([M]+: 195.0395 g/ mol). Esterification of 1 with 2-hydroxyethyl methacrylate (HEMA) was initially attempted via the corresponding acid chloride by reaction of 1 with thionyl chloride,27 resulting in imidazotetrazine degradation. The comparatively mild carbodiimide-mediated esterification proved more effective. As shown in Scheme 1, methacrylate 2 was synthesized from 1 and HEMA in the presence of 1-(3-(dimethylamino)propyl)-3ethylcarbodiimide hydrochloride (EDC) and catalytic 4(dimethylamino)pyridine (DMAP). Methacrylate 2 was purified simply by aqueous extraction and isolated in 71% 216
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in TFE. Molecular weight distributions were decidedly narrow, as shown by the representative chromatogram in Figure 4.
Figure 3. (a) Synthesis of polyMPC-TMZ 3A−3D by RAFT copolymerization of MPC and 2 in TFE; (b) Photograph of a sample of a lyophilized polyMPC-TMZ conjugate; (c) UV−vis absorption spectra of 2 and polyMPC-TMZ copolymers 3A−3D in TFE, and an absorption spectrum of degraded TMZ.
polymer products were purified by repeated precipitation into THF, followed by centrifugal dialysis against aqueous 0.1 M HCl. The conjugates were then lyophilized, giving 3A−3D in yields of 60−74% as pink solids that readily dispersed in water at concentrations >20 mg/mL. NMR and UV−vis spectroscopy confirmed the presence of TMZ pendent groups in copolymers 3A−3D. In the 1H NMR spectrum of 3D (Figure S5), resonances corresponding to intact imidazole (δ = 8.46 ppm) and methyltriazene (δ = 3.88 ppm) groups were observed. Resonances at 140.9, 131.4, and 38.0 ppm in the 13C NMR spectrum of 3D (Figure S5) correspond to the urea, imidazole, and methyltriazene moieties, respectively. Figure 3 shows the UV−vis absorption spectra of solutions of 2 and conjugates 3A−3D in TFE, and the spectrum of an aqueous solution of TMZ that was allowed to decompose fully. Each copolymer possessed an absorption maximum at 323 nm, with a notable absence of any spectral features to suggest TMZ degradation. Using 1H NMR spectroscopy, copolymer compositions were estimated by comparing the relative signal intensities of the imidazole and trimethylammonium groups (δ = 2.80−3.30 ppm), with estimated incorporations of 2 summarized in Table 1. In general, the copolymer compositions were in reasonable agreement with the theoretical values. Molecular weight estimation of polymers 3A−3D was performed by gel permeation chromatography (GPC) eluting
Figure 4. (a) Representative GPC chromatogram of polyMPC-TMZ (sample 3D) eluting in TFE; (b) Number-average molecular weights (Mn) and PDI values of 3A−3D estimated by GPC eluting in TFE (calibrated against PMMA standards).
Copolymers 3A−3D were obtained with low PDI values, estimated relative to poly(methyl methacrylate) (PMMA) calibration standards; Mn values were in reasonable agreement with those targeted. Additionally, GPC chromatograms of polyMPC-TMZ copolymers (UV detection) showed a notable absence of residual methacrylate 2 or other small molecule impurities (Figure S7). This molecular weight characterization confirmed that RAFT methodology is amenable to preparing well-defined polymer-TMZ conjugates using monomer 2. The effect of polyMPC conjugation on the hydrolytic stability of TMZ was investigated by UV−vis spectroscopy. For example, TMZ and copolymer 3D were incubated in pH 7.4 PBS at 37 °C, and the decrease in intensity of the absorption maximum corresponding to intact imidazotetrazine moieties (328 or 326 nm for TMZ or 3D, respectively) was monitored to gauge TMZ decomposition.27 Plotting relative absorbance (A/A o) as a function of incubation time, degradation profiles were constructed (Figure S8) and halflives (t1/2) measured. Under these conditions, TMZ degraded rapidly (t1/2 = 1.0 h), while polyMPC conjugation enhanced stability significantly, affording a greater than 2-fold extension of TMZ half-life. This analysis suggests that polymer conjugation is a facile method for improving the physiological stability of TMZ while additionally confirming the occurrence of TMZ degradation required for its antitumor activity. In summary, TMZ, a first-line glioblastoma chemotherapeutic, was incorporated successfully into a series of polyMPC conjugates by RAFT copolymerization of the novel TMZmethacrylate 2. TMZ was integrated into the copolymers at tunable drug loadings (up to 50 mol % or greater), and conjugates were obtained with narrow molecular weight distributions. This method for preparing well-defined polymer-TMZ prodrugs uses simple and effective polymerizations
Table 1. Incorporation of TMZ-Methacrylate Estimated by 1 H NMR Spectroscopy polymer
target incorporation of 2 (mol %)
measured incorporationa of 2 (mol %)
3A 3B 3C 3D
11 21 35 50
16 25 37 52
a
Molar incorporation estimated by 1H NMR (500 MHz) spectroscopy in TFE-d3. 217
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that are metal-free, avoid the need for post-polymerization drug conjugation, and preserve the potent chemical structure of TMZ. Owing to the versatile reactivity of the TMZmethacrylate derivative, opportunities for introducing TMZ into a variety of biocompatible polymer compositions and networks are now available, providing access to a polymerTMZ therapeutic platform suitable for systemic and local chemotherapy. Going forward, effective use of such polymers by systemic administration will require retention of the blood− brain barrier crossing properties inherent to TMZ itself, which may require biorecognition with small molecules,29,30 peptides,31−33 and antibodies to facilitate transcytosis.34,35 Our ongoing studies seek to incorporate such functionality in polymer-TMZ prodrugs and move toward establishing the efficacy of this novel polyMPC-TMZ platform in vivo.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsmacrolett.7b00007. Materials, methods, HRMS, and additional NMR, UV− vis, and GPC data (PDF).
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Todd Emrick: 0000-0003-0460-1797 Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors acknowledge the financial support provided by the National Institutes of Health under Award Number R21 CA167674, the National Science Foundation Graduate Research Fellowship under Grant Number 1451512 (S.M.W.), and facilities provided by the National Science Foundation Materials Research Science and Engineering Center (MRSEC) on Polymers (DMR-0820506). Mass spectral data were obtained at the University of Massachusetts Mass Spectrometry Center.
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REFERENCES
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