PEG-g-poly(GdDTPA-co-l-cystine): A Biodegradable Macromolecular

Biodegradable PEGylated Gd-DTPA l-cystine copolymers, PEG-g-poly(GdDTPA-co-l-cystine), were prepared and tested as a blood pool contrast agent in mice...
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Bioconjugate Chem. 2004, 15, 1424−1430

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PEG-g-poly(GdDTPA-co-L-cystine): A Biodegradable Macromolecular Blood Pool Contrast Agent for MR Imaging Aaron M. Mohs,† Xinghe Wang,† K. Craig Goodrich,‡ Yuda Zong,† Dennis L. Parker,§ and Zheng-Rong Lu*,† Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah, Salt Lake City, Utah 84112, Department of Radiology, LDS Hospital, Salt Lake City, Utah 84112, and Department of Radiology, University of Utah, Salt Lake City, Utah 84112. Received July 16, 2004

Biodegradable PEGylated Gd-DTPA L-cystine copolymers, PEG-g-poly(GdDTPA-co-L-cystine), were prepared and tested as a blood pool contrast agent in mice. The biodegradable macromolecular agent was designed to be broken down into smaller Gd complexes by endogenous thiols via the disulfidethiol exchange reaction to facilitate the clearance of Gd complexes after the contrast-enhanced MRI examination. Gd-DTPA L-cystine copolymers were synthesized by condensation polymerization of L-cystine and DTPA-dianhydride in water followed by chelating with Gd(OAc)3. MPEG-NH2 (MW ) 2000) was then conjugated to the polymeric backbone in different ratios. The macromolecular contrast agent was readily degraded with the incubation of L-cysteine. It also demonstrated superior contrast enhancement in the heart and blood vessels as compared to a low molecular weight control agent, Gd-(DTPA-BMA). At 1 h postcontrast, the PEGylated macromolecular agent still showed prominent enhancement, while little contrast enhancement was detectable in the blood pool by the control agent. PEG-g-poly(GdDTPA-co-L-cystine) shows promise as an MR blood pool imaging agent.

INTRODUCTION

Gadolinium complexes, Gd-DTPA, GD-DOTA, or their derivatives, are routinely used as extracellular MRI contrast agents in the detection and diagnosis of various cardiovascular pathological conditions by enhancing the morphology and functionality of the condition. Albeit these low molecular weight agents have low toxicity, their inherent pharmacokinetic properties, including transient blood pool retention time, do not render them ideal for cardiovascular imaging. These agents can rapidly extravasate out of blood vessels into the extracellular matrix, introducing difficulties for a quantitative diagnosis that force the application of complex mathematical models (1). In addition, the relaxation time of these molecules is relatively slow, necessitating a higher dose of extravascular contrast agent to achieve signal enhancement comparable to that of a macromolecular agent (2). In order for macromolecular Gd complexes to be safe and effective it must be large enough to remain in the blood stream, yet small enough to be excreted after the exam. Previous attempts in the design of macromolecular blood pool agents have been hampered by the potential toxicity of the agents due to the slow clearance of the Gd complexes. Current blood pool agents based on macromolecular Gd(III) complexes from polymers, dendrimers, polypeptides, and polysaccharides have a less complete elimination than their low molecular weight counter* To whom correspondence should be addressed. Dr. ZhengRong Lu, 421 Wakara Way, Suite 318, Salt Lake City, UT 84108. Phone: 801 587-9450. Fax: 801 585-3614. E-mail: [email protected]. † Department of Pharmaceutics and Pharmaceutical Chemistry, University of Utah. ‡ Department of Radiology, LDS Hospital. § Department of Radiology, University of Utah.

parts, resulting in the possible accumulation of toxic metabolic byproducts (3-6). The size limitation to macromolecular Gd(III) complexes presents a significant barrier for their application. In response, polymeric Gd(III) chelate conjugates with a cleavable disulfide spacer or polydisulfide-based biodegradable macromolecular Gd complexes were introduced to circumvent the problem of long-term toxic Gd3+ accumulation (7, 8). The disulfide bond is cleaved by endogenous or exogenous thiols via the disulfide-thiol exchange reaction to release Gd(III) chelates from the conjugates or break down the macromolecular complexes into smaller complexes to facilitate the clearance of Gd(III) complexes. The plasma concentration of free thiols in humans is approximately 15 µM, allowing for gradual, yet sufficient breakdown of the macromolecules into smaller Gd complexes for complete elimination of the agent after the MRI examination (9, 10). Previously, we reported the first polydisulfide-based macromolecular Gd(III) complexes, Gd-DTPA cystamine copolymers. This agent resulted in more significant and prolonged blood pool contrast enhancement than a low molecular weight control, Gd-(DTPA-BMA). The agent was then rapidly degraded and extravasated from the blood pool (8). Further structural modification of the agent is needed to optimize its physicochemical and pharmacokinetic properties. Here we report a modified biodegradable polydisulfide-based macromolecular Gd(III) complex, poly(GdDTPA-co-L-cystine) grafted with monomethoxy-poly(ethylene glycol)amine (MPEG-NH2), PEG-g-poly(GdDTPA-co-L-cystine). Poly(ethylene glycol) is a nontoxic, nonantigenic and biocompatible polymer that has a large hydrodynamic radius rendering itself ideal for biomedical applications. With respect to MRI, PEG has been used to stabilize paramagnetic vesicles (11), used in formulation with an oral contrast agent to enhance the distal ileum (12), incorporated into the

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A Biodegradable Macromolecular MRI Contrast Agent

backbone of copolymeric contrast agents (13-15), and grafted onto polymeric contrast agents (16, 17). Preliminary results show significant contrast enhancement in the blood pool of mice for a longer period of time with the PEGylated polydisulfide agent compared to the clinically used low molecular weight agent, Gd-(DTPABMA), while still showing acceptable clearance. EXPERIMENTAL PROCEDURES

DTPA was purchased from J. T. Baker (Phillipsburg, NJ). L-Cystine was purchased from Sigma (St. Louis, MO). Gd(OAc)3 was purchased from Alfa Aesar (Ward Hill, MA). Monomethoxy-poly(ethylene glycol)-amine (MPEG-NH2, MW ) 2000) was synthesized according to the literature (18) as was DTPA dianhydride (19). Synthesis of Poly(GdDTPA-co-L-cystine). Poly(DTPA-co-L-cystine) (I) was first synthesized with the following procedure. L-Cystine (2.40 g, 10 mmol), deionized water (10 mL), and Na2CO3 (4.2 g) were stirred for 15 min. DTPA-dianhydride (3.93 g, 11 mmol) was then added in portions to the mixture with stirring, and the mixture was stirred overnight at room temperature. The copolymers were dialyzed against deionized water for 24 h using a regenerated cellulose membrane (MWCO ) 6000-8000 Da) and were then concentrated in vacuo to dryness. The molecular weights of the copolymers were determined by size exclusion chromatography (SEC) on an AKTA FPLC system with a Superose 12 (10/300 GL) column and UV and refractive index detectors. The molecular weights were calibrated with poly[N-(2hydroxypropyl)methacrylamide] standards. The number average (Mn) and weight average (Mw) molecular weights of the copolymers were 10.2 and 11.4 kDa, respectively. Proton NMR (ppm, D2O): 2.94 (m, 8H, NCH2CH2N), 3.18 (s, 4H, NCH2CONH), 3.25 (d, 2H, NHCHCH2S), 3.32 (s, 4H, NCH2COOH), 3.48 (s, 2H, NCH2COOH), 4.42 (m, 1H, NHCHCH2S). Complexation was achieved by reacting poly(DTPA-coL-cystine) with Gd(OAc)3 in 8 mL of deionized water. Xylenol orange indicator was added and 1 N HCl was added dropwise until pH 5-5.5 was reached. Gd(III) acetate was added to the mixture until the color became pink, indicating an excess of free Gd3+ ions. Excess Gd(OAc)3 was removed by eluting the solution through a Sephadex G-25 desalting column (Pharmacia). The purified poly(GdDTPA-co-L-cystine) (II, GDCP) was concentrated to dryness in vacuo and the molecular weights of the copolymers were determined by SEC, Mn ) 10.0 kDa, Mw ) 10.1 kDa. Gd content was 862 µmol Gd/g polymer as determined by ICP-OES (Perkin-Elmer, Norwalk, CT, Optima 3100XL). Synthesis of PEG-g-poly(GdDTPA-co-L-cystine) (A). Poly(GdDTPA-co-L-cystine) (200 mg, 0.26 mmol cystine) was dissolved in 4 mL deionized water, and N-hydroxysuccinimide (270 mg, 2.4 mmol) was then added with stirring. An excess of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (700 mg, 3.65 mmol) was slowly added, and the resulting yellow solution was stirred for 15 min. MPEG-NH2 (MW ) 2000 Da, 262 mg) was added in portions, and the reaction mixture was stirred overnight. White precipitate was removed by filtration. Unreacted PEG and salts were removed by ultrafiltration using a Centricon-10 concentrator (membrane MWCO ) 10 000 Da). The resulting PEGylated poly(GdDTPA-co-L-cystine) (PEGa-GDCP) was characterized by SEC and ICP-OES as described above. PEGaGDCP: Mn ) 21.0 kDa, Mw ) 24.5 kDa; 720 µmol Gd/g polymer.

Synthesis of PEG-g-poly(GdDTPA-co-L-cystine) (B). PEG-g-poly(GdDTPA-co-L-cystine) (PEGb-GDCP) with high PEG content was prepared similarly with a high PEG to poly(GdDTPA-co-L-cystine) ratio. Briefly, poly(GdDTPA-co-L-cystine) (200 mg, 0.26 mmol cystine) was dissolved in 4 mL deionized water, and N-hydroxysuccinimide (270 mg, 2.4 mmol) and 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (700 mg, 3.65 mmol) were then added with stirring. MPEG-NH2 (524 mg) was added to the reaction and the mixture was stirred overnight. Unreacted PEG and salts were removed by ultrafiltration using a Centricon-10 concentrator (membrane MWCO ) 10 000 Da). PEGb-GDCP: Mn ) 19.9 kDa kDa, Mw ) 22.9 kDa; 449 µmol Gd/g polymer. Relaxivity of PEG-g-poly(GdDTPA-co-L-cystine). The T1 of different PEGylated complexes was determined on a Siemens Trio 3T scanner. T1 relaxation times for three concentrations of the polymeric contrast agents were determined, along with reference values for water and Gd-(DTPA-BMA) by sequential application of a standard inversion-recovery (IR) pulse sequence with TR ) 5000 ms, TE ) 17 ms for inversion times TI ) 22, 25, 30, 35, 40, 50, 100, 200, 300, 400, 500, 600, 700, and 800 ms. Net magnetization amplitude data for each sample were determined from the appropriate ROI and fit using the Marquardt-Levenberg algorithm for multiparametric nonlinear regression analysis to simultaneously determine T1 and M0. Regressions were individually inspected for convergence. Parameters and standard errors were determined using ANOVA as provided in the Mathematica Statistics Toolbox. Slope of the plot of 1/T1 vs [Gd(III)] then gave the relaxivity R1. Degradation of PEG-g-poly(GdDTPA-co-L-cystine). PEGylated polymers PEGa-GDCP and PEGbGDCP and un-PEGylated GDCP were separately incubated in an aqueous cysteine solution (100 µM) at 37°C. SEC was performed on the samples at 0, 3, and 24 h after incubation to demonstrate the time dependent degradation of the disulfide bonds by looking at the molecular weight change of the copolymers. MALDI-TOF mass spectrometry was then performed on the reaction mixture to observe the degradation products of PEGa-GDCP, PEGb-GDCP, and GDCP. In Vivo MR Imaging. In vivo contrast enhanced MR imaging with the macromolecular agents was investigated in CD-1 mice (Charles River Laboratories). The animals were cared for under an approved protocol and the guidelines of the University of Utah Institutional Animal Care and Use Committee. The mice were anesthetized by the intramuscular administration of a mixture of ketamine (80 mg/kg) and xylazine (12 mg/kg). Contrast-enhanced images of mice were obtained on a Siemens Trio 3T MR scanner with a wrist coil pre- and postcontrast using the following imaging parameters: TE ) 1.74 ms, TR ) 4.3 ms, 25° RF tip angle, 3D acquisition with 128 slices/slab, 120 mm FOV, and 1.6 mm coronal slice thickness. Either Gd-(DTPA-BMA) (0.1 mmol Gd/ kg) or a polymeric agent GDCP, PEGa-GDCP, or PEGbGDCP (0.03 mmol Gd/kg for all polymeric agents) was injected via a tail vein into anesthetized mice and images were acquired at 1, 5, 15, 30, and 60 min postinjection of contrast agent. Each agent was investigated in a group of four mice. RESULTS

Synthesis of PEG-g-poly(GdDTPA-co-L-cystine). The synthetic procedure of PEG-g-poly(GdDTPA-co-Lcystine) is described in Scheme 1. Poly(DTPA-co-L-

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Scheme 1. Synthesis of PEG-g-poly(GdDTPA-co-L-cystine)a

a

(i) Na2CO3, H2O, rt, overnight; (ii) Gd(OAc)3, pH 5.0-5.5, rt, 2 h; (iii) NHS, EDC, MPEG-NH2, H2O, pH 7-8, rt, overnight

cystine) was first synthesized by the copolymerization of DTPA dianhydride with L-cystine. L-Cystine is sparingly soluble in organic solvents and the copolymerization was performed in Na2CO3 aqueous conditions. The weak base Na2CO3 was used to maintain a relatively basic pH and to minimize the hydrolysis of the dianhydride. The molecular weight of poly(DTPA-co-L-cystine) was relatively low due to partial hydrolysis of the dianhydride, limiting chain growth of the copolymers. The monomethoxy-PEG amine with an average molecular weight of approximately 2000 Da was used in the modification of GDCP. The content of PEG in PEG-gpoly(GdDTPA-co-L-cystine) was controlled by the molar ratio of MPEG-NH2 and GDCP. PEG-g-poly(GdDTPA-coL-cystine) with two different degrees of PEG modification were prepared. The molar ratio of PEG to the Gd-DTPA monomer was 0.33 and 0.76 for conjugates PEGa-GDCP and PEGb-GDCP, respectively, calculated from the Gd content per gram of polymer sample before and after PEGylation as determined by ICP-OES. The ratio of PEG grafted to cystine residues in the GDCP backbone is lower than the stoichiometric ratios used in the reaction, possibly due to the spatial restriction caused by the large hydrodynamic radius of PEG blocking the conjugation of more PEG molecules to the adjacent cystine residues. PEGylation increased the hydrodynamic volume of the copolymers. However, the increasing PEG content did not result in further increase of the hydrodynamic volume of the copolymers when comparing PEGa-GDCP (Mw ) 24.5 kDa) with PEGb-GDCP (Mw ) 22.9 kDa). In Vitro Degradation. The in vitro degradation study was performed by incubating the macromolecular agent with L-cysteine to verify the degradability of the agents via the disulfide-thiol exchange reaction. The results showed the expected decrease for the high molecular weight fraction and an increase in PEGylated degradation products of the agents in the incubation mixture. SEC analysis of the incubation mixtures showed that there was a significant shift of molecular weight distribution to lower molecular weight, representing a breakdown in the most prominent polymer species. The timedependent molecular weight distribution of PEGa-GDCP in the incubation is shown as an example in Figure 1. The initial polymer peak at 27.4 min completely dis-

appeared after 24 h of incubation. Consequently, there is a rise in signal intensity at 31.4 and 34.8 min over the same time period representing an increase in the PEGylated and unPEGylated degradation units, respectively. The low molecular weight degradation complexes IV, V, and VI in Scheme 2 were identified in the MALDITOF mass spectrum acquired on the reaction mixture at the end of the incubation period. Relaxivity. Both PEGylated polymers, PEGa-GDCP and PEGb-GDCP, had a higher relaxivity than GDCP (5.5 mM-1 s-1), while PEGa-GDCP had a higher relaxivity than PEGb-GDCP, 16.3 and 12.7 mM-1 s-1, respectively. It is likely that PEG offers some steric limitation to the movement of the polymer, which increases the rotational correlation coefficient of the copolymers and in turn the relaxivity (20). However, PEGb-GDCP with high PEG content possessed lower relaxivity than PEGa-GDCP with low PEG content. In Vivo MR Imaging. Figure 2 shows the coronal MR images of the heart of mice contrast enhanced with PEGaGDCP, PEGb-GDCP, GDCP, and Gd-(DTPA-BMA) at various time points. Strong contrast enhancement was observed in the heart with the macromolecular agents at a dose of 0.03 mmol-Gd/kg 1 min postcontrast and the signal intensity gradually decreased over the period of observation. Significant contrast enhancement was still visible in the heart at 60 min for the polymeric agents. The low molecular weight control agent, Gd-(DTPABMA), produced visible contrast enhancement at a dose of 0.1 mmol/kg 1 min postinjection and little to no enhancement afterward. The macromolecular agents also resulted in significant contrast enhancement in mouse blood vessels with the same trend. Figure 3 shows the coronal images of the descending aorta and common iliac arteries at various time points. The macromolecular agents clearly revealed the blood vessels 1 min post-contrast while little contrast enhancement was observed with the control agent. The blood vessels with PEGylated agents were more visible 5 min postinjection than those with the un-PEGylated polymeric agent. This demonstrates that PEGylation contributes to increased vascular retention, possibly by increasing the size of the agent.

A Biodegradable Macromolecular MRI Contrast Agent

Bioconjugate Chem., Vol. 15, No. 6, 2004 1427

Figure 1. The effect on the molecular weight distribution of PEGa-GDCP with incubation of L-cysteine in H2O at 37 °C. Scheme 2. Degradation Products from the Incubation of PEG-g-poly(GdDTPA-co-L-cystine) by the Incubation of L-Cysteine

DISCUSSION

Polydisulfide-based macromolecular Gd complexes have been designed to facilitate the clearance of Gd complexes after the MRI examination. Previously, we have demonstrated the feasibility of contrast-enhanced MRI with the first polydisulfide-based contrast agent, (Gd-DTPA)cystamine copolymers (8). The copolymers resulted in significant blood pool contrast enhancement in rats in the early period postcontrast, approximately 2 min at a high dose (0.1 mmol-Gd/kg). However, less significant

blood pool contrast enhancement was observed with the agent at a low dose (0.03 mmol-Gd/kg). PEGylated GDCP was designed and prepared to modify the physicochemical properties and in vivo contrast enhancement of biodegradable macromolecular agents. PEGylation increased the molecular weight and hydrodynamic size of the macromolecular agent. It is interesting to note that the amount of MPEG-NH2 grafted on the GDCP did not significantly affect on the hydrodynamic volume of the grafted agent. The increase of

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Figure 2. Coronal MR images of mouse heart preinjection (a) and 1 (b), 5 (c), 15 (d), 30 (e), and 60 min (f) postintravenous injection of PEGa-GDCP (A), PEGb-GDCP (B), GDCP (C), and Gd(DTPA-BMA) (D). Polymeric agents were given at a dose of 0.03 mmol-Gd/ kg, and Gd(DTPA-BMA) was given at the standard clinical dose of 0.1 mmol/kg.

Figure 3. Coronal MR images of descending aorta (solid arrow) and common iliac arteries (hollow arrow) of the mouse preinjection (a) and 1 (b), 5 (c), 15 (d), 30 (e), and 60 min (f) postintravenous injection of PEGa-GDCP (A), PEGb-GDCP (B), GDCP (C), and Gd(DTPA-BMA) (D). Polymeric agents were given at a dose of 0.03 mmol-Gd/kg, and Gd(DTPA-BMA) was given at the standard clinical dose of 0.1 mmol/kg.

hydrodynamic volume of PEGb-GDCP with a high PEG to Gd ratio (approximately 0.76:1) was less appreciable than that of PEGa-GDCP with a low ratio (approximately 0.33:1). Although this seems counterintuitive because of PEG’s large hydrodynamic radius, it is plausible that when more PEG is added, the density of PEG molecules on the polymer chain with a fixed length increases and it does not extend the length of the copolymers. The higher PEG density along the polymer chain may increase intermolecular interaction between the PEG chains and, probably, diminish their contribution to the hydrodynamic volume of the macromolecules. The graft of PEG also increased the relaxivity of GDCP, most likely due to steric limitation on the rotational movement of the polymer increasing the rotational correlation coefficient and in turn the relaxivity (20). However, a further increase of PEG in PEGb-GDCP resulted in decreased relaxivity. These results are con-

sistent with the literature. Raymond et al. suggested that this is due to the possible association of water to PEG (21). As more PEG is added, more water is associated to PEG via hydrogen bonding, effectively reducing the local concentration of water proximal to the Gd3+ ions. This causes a probable decrease in the water-ion exchange rate, which increases the residence time of the coordinated water, τΜ, resulting in a decreased relaxivity, as shown in the following equation:

Ris 1p )

1 c×q × H 55.6 T1M + τM

where Ris 1p is the inner coordination sphere relaxation of bulk water surrounding the paramagnetic ion, c is the concentration of the ion, q is the number of coordinated water molecules to the metal ion, and TH 1M is the longitudinal relaxation time of the coordinated water (22).

A Biodegradable Macromolecular MRI Contrast Agent

In addition to increased relaxivity, the other advantage of PEGylation is the reduced tissue uptake of the macromolecular agents by the reticuloendothelial system (RES) and nonspecific binding. PEG is commonly used in the modification of biological and physiochemical properties of biological systems to minimize bioadhesion and immunological response (23). The incorporation of PEG in drug delivery systems significantly reduces the liver uptake of the systems and prolongs their circulation half-life. Kobayashi and co-workers have also reported that when the PEG content is increased on dendrimeric contrast agents, less contrast agent accumulates in the liver, spleen and kidneys leaving a larger percent in the blood pool, accounting for the increased signal intensity (17). The PEGylation of polydisulfide-based biodegradable macromolecular Gd complexes, GDCP, resulted in high relaxivity and relatively large size of the modified agent and improved blood pool contrast enhancement in mice at a relatively low dose. The size of GDCP was relatively small due to the limitation of the current synthetic method. The blood pool contrast enhancement with the PEGylated agent may be further improved if GDCP with a larger size is used. Detailed investigations on the novel biodegradable macromolecular agents with different sizes are ongoing. The studies are focusing on the size impact of the PEGylated biodegradable macromolecular agent with respect to its physicochemical properties and in vivo contrast enhancement, pharmacokinetics, clearance, and long-term tissue Gd(III) accumulation. CONCLUSION

Biodegradable PEGylated macromolecular Gd(III) complexes, PEG-g-poly(GdDTPA-co-L-cystine), were prepared and tested as a blood pool contrast agent in mice. The PEGylated macromolecular contrast agent demonstrated superior contrast enhancement in the heart and blood vessels as compared to the low molecular weight agent, Gd-(DTPA-BMA). An in vitro study demonstrated the time-dependent and complete degradation of PEGg-poly(GdDTPA-co-L-cystine). MALDI-TOF mass spectrometry confirmed the degradation products. PEG-gpoly(GdDTPA-co-L-cystine) shows promise as a blood pool MRI contrast agent because of its increased signal intensity and retention time as well as its controllable degradation characteristics. ACKNOWLEDGMENT

This research is supported in part by NIH grant EB00489. We thank Dr. Matthias Schabel for technical assistant in T1 measurement and MR image acquisition. Mass spectral data were acquired at the University of Utah Mass Spectrometry Facility. LITERATURE CITED (1) Paetsch, I., Gebker, R., Fleck, E., and Nagel, E. (2003) Cardiac magnetic resonance (CMR) imaging: A noninvasive tool for functional and morphological assessment of coronary artery disease. J. Interven. Cardiol. 16, 457-463. (2) Li, D., Zheng, J., and Weinmann, H.-J. (2001) Contrastenhanced MR imaging of coronary arteries: comparison of intra- and extravascular contrast agents in swine. Radiology 218, 670-678. (3) Bogdanov, A. A., Weissleder, R., Frank, H. W., Bogdanova, A. V., Nossif, N., Schaffer, B. K., Tsai, E., Papisov, M. I., and Brady, T. J. (1993) A new macromolecule as a contrast agent

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