Supporting Information - PNAS · Supporting Information Sur et al. 10.1073/pnas.1324135111 SI...

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Supporting Information Sur et al. 10.1073/pnas.1324135111 SI Materials and Methods General Method for Synthesis of Ionizable β-Cyclodextrins. β-Cyclo- dextrin (Sigma) was monotosylated with a 0.9 molar equivalent of tosyl chloride in pyridine at the primary 6-hydroxyl group to afford the corresponding tosylate, which was converted to the iodo derivative by treatment with a large excess of sodium iodide in acetone. The iodo derivative was converted to the desired aminated cyclodextrin by heating at 80 °C for 812 h with a fivefold molar excess of the appropriate amine (1). 6-Mono- succinyl-β-cyclodextrin was synthesized by treatment of parent β-cyclodextrin with a 0.9 molar equivalent of succinic anhydride in dimethylformamide (DMF) (2). The product was precipitated in acetone and purified by HPLC before use. 6,6,6,6,6,6,6- Heptakis-succinyl-β-cyclodextrin was synthesized from β-cyclo- dextrin by treatment with excess succinic anhydride in DMF and precipitated with acetone. Fractional crystallization afforded the desired compound at 85% purity. Dansylated cyclodextrins I, IV, and V were synthesized from commercially available β-cyclodextrin and compounds II and III, respectively, by treatment with a 0.9 molar equivalent of dansyl chloride in pyridine. Each intermediate and the final product were purified by HPLC using a preparative C18 column and linear gradients of 095% solvent B (acetonitrile) in solvent A (water). All cyclo- dextrins were characterized by 1 H NMR and electrospray ioni- zation MS and matched with previously published literature references. 6,6,6,6,6,6,6-Heptakis-amino-β-cyclodextrin was purchased from CTD Holdings and used without further purification. BI-2536 (3) and PD-0325901 (4) were synthesized and formu- lated for in vivo experiments as previously described. General Procedure for the Preparation of Liposomes. Hydrogenated egg phosphatidylcholine (Avanti Polar Lipids), cholesterol, and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG 2000) (Avanti Polar Lipids) (molar ratios 50:45:5) were dissolved in chloroform (20 mL). The solvent was removed in vacuo to give a thin lipid film, which was hydrated by shaking in the appropriate buffer (PBS, pH 7.4; 200 mM citrate, pH 4.0; or 80 mM Arg·Hepes, pH 9.0) at 50 °C for 2 h. The vesicle suspension was sonicated for 30 min and then extruded successively through 0.4-, 0.2-, and 0.1-μm polycarbonate membranes (Whatman; Nuclepore Track- Etched Membrane) at 50 °C to obtain the final liposomes with low polydispersity at the desired size. The transmembrane gra- dient was then created by equilibrium dialysis of the liposomes against 300 mM sucrose or PBS overnight. The average size and polydispersity index were then measured by dynamic light- scattering experiments on a Zetasizer Nano ZS90 (Malvern In- struments) at a wavelength of 633 nm and a 90° detection angle. Protocol for Passive Loading of Liposomes. Method 1: Encapsulation of BI-2536 in the lipid layer. Hydrogenated egg phosphatidylcholine (Avanti Polar Lipids), cholesterol, and DSPE-PEG2000 (Avanti Polar Lipids) (molar ratios 50:45:5) were dissolved in chloroform (20 mL). Ten milligrams of BI-2536 (in 1 mL chloroform) was added and the solvent was evaporated to generate a thin film. One milliliter of PBS (pH 7.4) was added to hydrate the lipid layer, and the mixture was shaken at 50 °C for 2 h as previously described. The vesicle suspension was sonicated for 30 min and then ex- truded successively through 0.4-, 0.2-, and 0.1-μm polycarbonate membranes (Whatman; Nuclepore Track-Etched Membrane) at 50 °C to obtain the final liposomes with low polydispersity at the desired size. The liposomes were then dialyzed in PBS overnight to remove unentrapped drug. The average size and polydispersity index were then measured by dynamic light-scattering experiments on a Malvern ZS90. Drug content was calculated by rupturing the liposomes with an equal volume of methanol and measuring the UV-vis absorbance on a NanoDrop 1000. Method 2: Hydration of the lipid layer with an aqueous formulation of BI-2536. Hydrogenated egg phosphatidylcholine (Avanti Polar Lipids), cholesterol, and DSPE-PEG2000 (Avanti Polar Lipids) (molar ratios 50:45:5) were dissolved in chloroform (20 mL), and the solvent was evaporated in vacuo to generate a thin film. One milliliter of aqueous BI-2536 (4 mg; pH 5.5) was added to hydrate the lipid layer, and the mixture was shaken at 50 °C for 2 h as previously described. The vesicle suspension was sonicated for 30 min and then extruded successively through 0.4-, 0.2-, and 0.1-μm polycarbonate membranes (Whatman; Nuclepore Track- Etched Membrane) at 50 °C to obtain the final liposomes with low polydispersity at the desired size. The liposomes were then dialyzed against PBS overnight to remove unentrapped drug. The average size and polydispersity index were then measured by dynamic light-scattering experiments on a Malvern ZS90. Drug content was calculated by rupturing the liposomes with an equal volume of methanol and measuring the UV-vis absorbance on a NanoDrop 1000. General Procedure of Preparation of Encapsulated Complexes. Equi- molar quantities of the drug (0.1 mmol; 3050 mg) and ap- propriate cyclodextrin (0.11 mmol; 110185 mg) were dissolved separately in methanol (nearly saturated 12 mL) and deionized water (1020 mg/mL), respectively. The methanolic solu- tion of the drug was then added dropwise into the cyclodextrin solution with agitation, ensuring a uniform suspension. This suspension was then shaken at 55 °C for 3648 h using an Eppendorf Thermomixer R. The solution was filtered to re- move particulate matter and flash-frozen in a dry ice/acetone bath followed by lyophilization. The lyophilized complex was stored at 20 °C until further use. General Protocol for Remote Loading of Liposomes. The lyophilized powder complex previously described was pulverized and in- cubated with appropriate liposomal solutions (3040 mg drug equivalent in 6 mL liposomal solution, to achieve loading ratios of 58 mg/mL concentrations) for 1 h at 65 °C. They were centrifuged at 1,000 × g for 3 min to remove particulate matter and then dialyzed against 300 mM sucrose or commercial PBS solution (pH 7.4) overnight to remove material that had not been loaded into the liposomes. The size distributions of the liposomal formulations were characterized using a Malvern ZS90. Concentrations of BI-2536 and PD-0325901 in liposomes were measured in triplicate using a NanoDrop 1000 after disruption of the liposomal solutions with equal volumes of methanol at 367 nm for BI-2536 and 277 nm for PD-0325901. Tissue Biodistribution Study. Coumarin 334 was used as a drug surrogate to assess biodistribution and pharmacokinetics of cy- clodextrin-encapsulated liposomes. Coumarin 334 (3 mg) was dissolved in methanol (6 mL) and added dropwise to an aqueous solution of cyclodextrin VI (14 mg in 20 mL water). The solution was shaken at 55 °C for 48 h and lyophilized. The lyophilized powder was incubated with citrate liposomes (internal pH 4.0) at 65 °C for 1 h. The liposomal solution was dialyzed against PBS Sur et al. www.pnas.org/cgi/content/short/1324135111 1 of 4

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Page 1: Supporting Information - PNAS · Supporting Information Sur et al. 10.1073/pnas.1324135111 SI Materials and Methods General Method for Synthesis of Ionizable β-Cyclodextrins. β-Cyclo-

Supporting InformationSur et al. 10.1073/pnas.1324135111SI Materials and MethodsGeneral Method for Synthesis of Ionizable β-Cyclodextrins. β-Cyclo-dextrin (Sigma) was monotosylated with a 0.9 molar equivalentof tosyl chloride in pyridine at the primary 6′-hydroxyl group toafford the corresponding tosylate, which was converted to theiodo derivative by treatment with a large excess of sodium iodidein acetone. The iodo derivative was converted to the desiredaminated cyclodextrin by heating at 80 °C for 8–12 h witha fivefold molar excess of the appropriate amine (1). 6′-Mono-succinyl-β-cyclodextrin was synthesized by treatment of parentβ-cyclodextrin with a 0.9 molar equivalent of succinic anhydridein dimethylformamide (DMF) (2). The product was precipitatedin acetone and purified by HPLC before use. 6′,6′,6′,6′,6′,6′,6-Heptakis-succinyl-β-cyclodextrin was synthesized from β-cyclo-dextrin by treatment with excess succinic anhydride in DMF andprecipitated with acetone. Fractional crystallization afforded thedesired compound at ∼85% purity.Dansylated cyclodextrins I, IV, and V were synthesized from

commercially available β-cyclodextrin and compounds II and III,respectively, by treatment with a 0.9 molar equivalent of dansylchloride in pyridine.Each intermediate and the final product were purified by

HPLC using a preparative C18 column and linear gradients of0–95% solvent B (acetonitrile) in solvent A (water). All cyclo-dextrins were characterized by 1H NMR and electrospray ioni-zation MS and matched with previously published literaturereferences.6′,6′,6′,6′,6′,6′,6′-Heptakis-amino-β-cyclodextrin was purchased

from CTD Holdings and used without further purification.BI-2536 (3) and PD-0325901 (4) were synthesized and formu-

lated for in vivo experiments as previously described.

General Procedure for the Preparation of Liposomes. Hydrogenatedegg phosphatidylcholine (Avanti Polar Lipids), cholesterol, and1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DSPE-PEG 2000) (Avanti PolarLipids) (molar ratios 50:45:5) were dissolved in chloroform(20 mL). The solvent was removed in vacuo to give a thin lipidfilm, which was hydrated by shaking in the appropriate buffer(PBS, pH 7.4; 200 mM citrate, pH 4.0; or 80 mM Arg·Hepes,pH 9.0) at 50 °C for 2 h. The vesicle suspension was sonicatedfor 30 min and then extruded successively through 0.4-, 0.2-, and0.1-μm polycarbonate membranes (Whatman; Nuclepore Track-Etched Membrane) at 50 °C to obtain the final liposomes withlow polydispersity at the desired size. The transmembrane gra-dient was then created by equilibrium dialysis of the liposomesagainst 300 mM sucrose or PBS overnight. The average sizeand polydispersity index were then measured by dynamic light-scattering experiments on a Zetasizer Nano ZS90 (Malvern In-struments) at a wavelength of 633 nm and a 90° detection angle.

Protocol for Passive Loading of Liposomes. Method 1: Encapsulation ofBI-2536 in the lipid layer. Hydrogenated egg phosphatidylcholine(Avanti Polar Lipids), cholesterol, and DSPE-PEG2000 (AvantiPolar Lipids) (molar ratios 50:45:5) were dissolved in chloroform(20 mL). Ten milligrams of BI-2536 (in 1 mL chloroform) wasadded and the solvent was evaporated to generate a thin film. Onemilliliter of PBS (pH 7.4) was added to hydrate the lipid layer, andthe mixture was shaken at 50 °C for 2 h as previously described.The vesicle suspension was sonicated for 30 min and then ex-truded successively through 0.4-, 0.2-, and 0.1-μm polycarbonatemembranes (Whatman; Nuclepore Track-Etched Membrane) at

50 °C to obtain the final liposomes with low polydispersity at thedesired size. The liposomes were then dialyzed in PBS overnightto remove unentrapped drug. The average size and polydispersityindex were then measured by dynamic light-scattering experimentson a Malvern ZS90. Drug content was calculated by rupturingthe liposomes with an equal volume of methanol and measuringthe UV-vis absorbance on a NanoDrop 1000.Method 2: Hydration of the lipid layer with an aqueous formulation ofBI-2536. Hydrogenated egg phosphatidylcholine (Avanti PolarLipids), cholesterol, and DSPE-PEG2000 (Avanti Polar Lipids)(molar ratios 50:45:5) were dissolved in chloroform (20 mL), andthe solvent was evaporated in vacuo to generate a thin film. Onemilliliter of aqueous BI-2536 (4 mg; pH 5.5) was added to hydratethe lipid layer, and the mixture was shaken at 50 °C for 2 h aspreviously described. The vesicle suspension was sonicated for30 min and then extruded successively through 0.4-, 0.2-, and0.1-μm polycarbonate membranes (Whatman; Nuclepore Track-Etched Membrane) at 50 °C to obtain the final liposomes withlow polydispersity at the desired size. The liposomes were thendialyzed against PBS overnight to remove unentrapped drug.The average size and polydispersity index were then measuredby dynamic light-scattering experiments on a Malvern ZS90.Drug content was calculated by rupturing the liposomes with anequal volume of methanol and measuring the UV-vis absorbanceon a NanoDrop 1000.

General Procedure of Preparation of Encapsulated Complexes. Equi-molar quantities of the drug (0.1 mmol; 30–50 mg) and ap-propriate cyclodextrin (0.11 mmol; 110–185 mg) were dissolvedseparately in methanol (nearly saturated ∼1–2 mL) and deionizedwater (∼10–20 mg/mL), respectively. The methanolic solu-tion of the drug was then added dropwise into the cyclodextrinsolution with agitation, ensuring a uniform suspension. Thissuspension was then shaken at 55 °C for 36–48 h using anEppendorf Thermomixer R. The solution was filtered to re-move particulate matter and flash-frozen in a dry ice/acetonebath followed by lyophilization. The lyophilized complex wasstored at −20 °C until further use.

General Protocol for Remote Loading of Liposomes. The lyophilizedpowder complex previously described was pulverized and in-cubated with appropriate liposomal solutions (30–40 mg drugequivalent in 6 mL liposomal solution, to achieve loading ratiosof 5–8 mg/mL concentrations) for 1 h at 65 °C. They werecentrifuged at 1,000 × g for 3 min to remove particulate matterand then dialyzed against 300 mM sucrose or commercial PBSsolution (pH 7.4) overnight to remove material that had notbeen loaded into the liposomes. The size distributions of theliposomal formulations were characterized using a Malvern ZS90.Concentrations of BI-2536 and PD-0325901 in liposomes weremeasured in triplicate using a NanoDrop 1000 after disruptionof the liposomal solutions with equal volumes of methanol at 367nm for BI-2536 and 277 nm for PD-0325901.

Tissue Biodistribution Study. Coumarin 334 was used as a drugsurrogate to assess biodistribution and pharmacokinetics of cy-clodextrin-encapsulated liposomes. Coumarin 334 (3 mg) wasdissolved in methanol (6 mL) and added dropwise to an aqueoussolution of cyclodextrin VI (14 mg in 20 mL water). The solutionwas shaken at 55 °C for 48 h and lyophilized. The lyophilizedpowder was incubated with citrate liposomes (internal pH 4.0)at 65 °C for 1 h. The liposomal solution was dialyzed against PBS

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overnight. To assess loading efficiency, 100 μL liposomes wasbroken with 100 μL methanol and analyzed for fluorescence. Theloading efficiency was found to be 90%. Female athymic BALB/cnude mice bearing HCT116 subcutaneous xenografts were usedin the study following a modified protocol (5). When the tumorvolumes reached 400–600 mm3, 12 mice were treated i.v. with200 μL cyclodextrin-encapsulated, liposomal (CYCL-)coumarin334 (0.5 mg/mL). Posttreatment, four mice were euthanized attime points 2, 24, and 48 h and tumor, spleen, liver, kidneys,heart, and lungs were excised and weighed. Blood was also col-lected, and plasma was separated and stored at 4 °C. Except forplasma, each frozen tissue was homogenized and sonicated in0.9% saline [3× volume (μL) of tissue mass (mg)]. Methanol wasadded to a final volume of 33% (vol/vol) with vortexing. Thesamples were centrifuged (6,000 × g, 10 min) and the fluo-rescence in the supernatants was measured by a CytoFluor IIfluorescence multiwall plate reader (Applied Biosystems) usingexcitation 485 nm/emission 530 nm. As a control for tissue au-tofluorescence, tumor-bearing animals treated with equivalentvolumes of empty liposomes were euthanized and their tissueand plasma were harvested.

Studies in Mice. All animal experiments were designed in accor-dance with the National Institute of Health’s Guide for the Careand Use of Laboratory Animals (6) and were approved by The

Johns Hopkins University’s Institutional Animal Care and UseCommittee.Five million HCT116 (p53−/−), HCT116 (p53+/+), or RKO

cells were injected s.c. into the flanks of female athymic BALB/cnude mice and allowed to grow for 3 wk, reaching 300–400 mm3

in volume. In the case of CYCL-BI-2536, the animals were thenrandomly segregated into four arms. In all cases, liposomalformulations (CYCL-drug) have been reported as equivalents offree drug. Over the course of 2 wk, the first arm received emptyliposomes; the second arm received a single dose of a 100 mg/kgformulation of the free drug twice using a formulation reportedin literature (at days 0 and 7); the third and fourth arms received100 and 400 mg/kg, respectively, of the CYCL-BI-2536 liposomalformulation at the same time points. Tumor volume was re-corded every 48 h. The average tumor size for each respectivegroup was normalized to the tumor volume on the first day oftreatment. In the case of CYCL-PD-0325901, the first arm wastreated twice with empty liposomes at days 0 and 8, whereasthe other two arms received two doses of free PD-0325901 andCYCL-PD-0325901, respectively, at the same time points. Forclarity of the experimental outcome, the data are presented asthe average tumor size of each group normalized to the tumorvolume on day 0. The tumor regression experiments in eachcase were terminated and the animals were euthanized when thetumors on the control animals reached 2,000 mm3.

1. Tang W, Ng SC (2008) Facile synthesis of mono-6-amino-6-deoxy-alpha-, beta-, gamma-cyclodextrin hydrochlorides for molecular recognition, chiral separation and drugdelivery. Nat Protoc 3(4):691–697.

2. Cucinotta V, et al. (2005) The 6-derivative of beta-cyclodextrin with succinic acid: A newchiral selector for CD-EKC. J Pharm Biomed Anal 37(5):1009–1014.

3. Hoffmann M, et al. (2004) Dihydropteridinones, method for the production and usethereof in the form of drugs (WO Patent 2,004,076,454).

4. Warmus JS, et al. (2008) 2-Alkylamino- and alkoxy-substituted 2-amino-1,3,4-oxadiazoles-O-alkyl benzohydroxamate esters replacements retain the desired inhibition and selectivityagainst MEK (MAP ERK kinase). Bioorg Med Chem Lett 18(23):6171–6174.

5. MacDiarmid JA, et al. (2007) Bacterially derived 400 nm particles for encapsulationand cancer cell targeting of chemotherapeutics. Cancer Cell 11(5):431–445.

6. Committee on Care and Use of Laboratory Animals (1985) Guide for the Care and Useof Laboratory Animals (Natl Inst Health, Bethesda), DHHS Publ No (NIH) 85–23.

Citrate liposomes

PBS liposomes

Citrate liposomes

PBS liposomes

Cyclodextrin IV Dansylated I

Fig. S1. Incorporation of dansylated cyclodextrins into citrate liposomes. Fluorescence analyses in relative fluorescence units (RFU) of dansylated I andcompound IV in citrate liposomes and control (PBS) liposomes are presented.

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N O O

O

O--- III:C314 --- I:C314--- C314

Fig. S2. Ability of various cyclodextrins to transfer coumarin 314 into citrate liposomes. Fluorescence of uncomplexed coumarin 314 and coumarin 314 complexedwith III (ionizable mono-6′-ethylenediamino-6′-deoxy-cyclodextrin) and I (unionizable β-cyclodextrin) followed by remote loading into citrate liposomes.

N

N N

ON

HNO

NH

O

N

BI-2536

HN

F

I

OHN

OOH

HO

FF

PD-0325901

Compound BI-2536 PD-0325901

Solubility in water at 25 ºC at pH 7.4 0.5 mg/ml 0.01mg/ml

IC50 against HCT 116 0.01nM 0.3nM

Dose limiting toxicity Neutropenia Retinal vein occlusion

Fig. S3. Structure and physical properties of BI-2536 and PD-0325901.

-0.20

0.00

0.20

0.40

0.60

0.80

1.00

1.20

tumor heart lung spleen kidney liver blood

g/m

g ss

ue

2h24h48h

Fig. S4. Tissue biodistribution of CYCL-coumarin 334 at 2, 24, and 48 h time points (data is presented as mean and SD).

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0

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100

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300

20 22 24 26 28 30 32 34 36

Rel

ativ

e tu

mor

vol

ume

(%)

Days

HCT116 p53 -/- xenografts

UntreatedFree PD-0325901 (150 mg/kg)CYCL-PD-0325901 (250 mg/kg)

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20 22 24 26 28 30 32 34 36 38

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UntreatedFree BI-2536 (100 mg/kg)CYCL-BI-2536 (400 mg/kg)

Fig. S5. Antitumor activity of CYCL-BI-2536 and CYCL-PD-0325901 in a second xenograft model. Liposomal formulations have been reported as equivalents offree drug; relative tumor volume and SD of each experimental arm are shown.

No R

II mono 6’-amino

III mono 6’-ethylene diamino

IV mono 6’-dansylamido

V mono 6’-dansylamido ethylene amino

VI 6’-per amino

VII 6’-per ethylene diamino

VIII 6’-succinyl

IX 6’-per succinyl

O

OH

HO

OHO

OHO

HOOH

O

OHO

OH

OH

O

O

HO OH

HO

O

OOH

OHHO O

O OH

HO

HO

O

O

OHHO

OH

OO

OH

HO

OHO

OHO

HOO

OHO

OH

OH

O

O

HO OH

HO

O

OOH

OHHO O

O OH

HO

HO

O

O

OHHO

OH

O

chemical modification

R

II-VIIII

Scheme S1. Table of synthesized ionizable cyclodextrins.

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