Pronounced Cellular Uptake of Pirarubicin versus That of Other

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Pronounced cellular uptake of pirarubicin versus that of other anthracyclines: Comparison of HPMA copolymer conjugates of pirarubicin and doxorubicin Hideaki Nakamura, Eva Koziolová, Petr Chytil, Kenji Tsukigawa, Jun Fang, Mamoru Haratake, Karel Ulbrich, Tomas Etrych, and Hiroshi Maeda Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00697 • Publication Date (Web): 07 Nov 2016 Downloaded from http://pubs.acs.org on November 9, 2016

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Molecular Pharmaceutics

Pronounced cellular uptake of pirarubicin versus that of other anthracyclines: Comparison of HPMA copolymer

conjugates

of

pirarubicin

and

doxorubicin Hideaki Nakamuraa,b, Eva Koziolovác, Petr Chytilc, Kenji Tsukigawab, Jun Fanga,b, Mamoru Haratakeb, Karel Ulbrichc, Tomáš Etrychc, Hiroshi Maedaa* a

Research Institute for Drug Delivery Science, Sojo University, Ikeda 4-22-1, Nishi-ku,

Kumamoto 860-0082, Japan b

Faculty of Pharmaceutical Sciences, Sojo University, Ikeda 4-22-1, Nishi-ku, Kumamoto 860-

0082, Japan c

Institute of Macromolecular Chemistry, The Czech Academy of Sciences, Heyrovsky Sq. 2, 162

06 Prague 6, Czech Republic

*Corresponding author: Research Institute for DDS, Sojo University, Ikeda 4-22-1, Nishi-ku, Kumamoto 860-0082, Japan. Tel.: +81-96-326-4114; fax: +81-96-326-3185. E-mail address: [email protected] (H. Maeda).

Keywords: HPMA polymer conjugate, pirarubicin (THP), doxorubicin (DOX), acid-cleavable linkage, EPR effect

Abstract

ACS Paragon Plus Environment

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Many conjugates of water-soluble polymers with biologically active molecules were developed during last two decades. Although, therapeutic effects of these conjugates are affected by the properties of carriers, the properties of the attached drugs appear more important than the same carrier polymer in this case. Pirarubicin (THP), a tetrahydropyranyl derivative of doxorubicin (DOX), demonstrated more rapid cellular internalization and potent cytotoxicity than DOX. Here, we conjugated the THP or DOX to N-(2-hydroxypropyl)methacrylamide copolymer via a hydrazone bond. The polymeric prodrugs conjugates, abbr. P-THP and P-DOX, respectively, had comparable hydrodynamic sizes and drug loading. Compared with P-DOX, PTHP showed approximately 10 times greater cellular uptake during a 240-min incubation and a cytotoxicity that was more than 10 times higher during a 72-h incubation. A marginal difference was seen in P-THP and P-DOX accumulation in the liver and kidney at 6 h after drug administration, but no significant difference occurred in the tumor drug concentration during 6– 24 h after drug administration. Antitumor activity against xenograft human pancreatic tumor (SUIT2) in mice was greater for P-THP than for P-DOX. To sum up, the present study compared the biological behavior of two different drugs, each attached to an HPMA copolymer carrier, with regard to their uptake by tumor cells, body distribution, and accumulation in tumors, cytotoxicity, and antitumor activity in vitro and in vivo. No differences in the tumor cell uptake of the polymer-drug conjugates P-THP and P-DOX were observed. In contrast, the intracellular uptake of free THP liberated from the P-THP was 25–30 times higher than that of DOX liberated from P-DOX. This finding indicates that proper selection of the carrier, and especially conjugated API are most critical for anticancer activity of the polymer-drug conjugates. THP thus appears more preferable API for polymer conjugation and treatment based on EPR effect target to solid tumors, although both polymer conjugates exhibited similar enhanced permeability and retention (or EPR) effects and drug release profiles in acidic pH.

1. Introduction


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The anthracycline antibiotic daunorubicin was originally isolated from Streptomyces peucetius. Many derivatives, such as doxorubicin (DOX), epirubicin (EPI), pirarubicin (THP), aclarubicin, and idarubicin, were subsequently developed. Anthracycline antibiotics are some of the most widely used anticancer agents for treatment of lymphoma, lung cancer, gastrointestinal cancer, breast cancer, and osteosarcoma 1. However, their indiscriminate accumulation in vital organs causes serious systemic adverse effects including cardiac toxicity and bone marrow suppression, as well as nausea and diarrhea. These adverse effects limit the continuous use of anthracyclines and result in many therapeutic failures.

Therefore, improved methods for

selective delivery of anticancer agents to tumors with minimal adverse effects are needed. Derivatization of parent drugs is one way to control adverse effects and improve antitumor activity. THP was synthesized as a 4ʹ-O-tetrahydropyranyl derivative of DOX in 1979 by Umezawa et al.

2, 3

and Dantchev et al.

2, 3

, and it had less cardiac toxicity and superior

antitumor activity against L-1210 ascites tumor than did the parental DOX. Also, Kunimoto et al. reported that THP manifested a cellular uptake rate that was more than 100 times faster than that of DOX, in parallel with its higher cytotoxicity in mouse lymphoma L5178Y cells

4, 5

. A higher

cytotoxicity of THP compared with that of DOX was also found in many lymphoma and epithelial cancer cell lines. This effect was clear at short incubation times

2, 5-7

. Although THP

causes less cardiac toxicity than does DOX, it still induces serious side effects such as bone marrow suppression, which leads to cessation of therapy. Doxil, which is pegylated (or stealth) liposome-encapsulated DOX, is a successful antitumor drug that suppresses adverse effects by controlling the pharmacokinetics of DOX 8. Similar to Doxil, macromolecular micelles or polymer-drug conjugates are promising drug delivery systems for controlling the pharmacokinetics of cytotoxic drugs and suppressing adverse effects. For example, DaunoXome, Mepact, Myocet, and Genexol-PM are now available on the market, and they are used to treat various solid malignancies. Furthermore, different micellar formulations of anticancer drugs have been developed or are undergoing clinical trials 9. These types of drugs, so-called biocompatible nano-sized drugs or nanomedicines, show prolonged circulation in the blood, and they gradually and selectively accumulate and remain in tumor tissues because of the enhanced permeability and retention (EPR) effect 10-13.


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Antitumor agents that have been encapsulated in macromolecular particles sometimes show antitumor activity regardless of whether their activation or release occurs either inside or outside of cells. Influx into and efflux out of cells, binding affinity for target molecules, and ultimately cytotoxicity of antitumor drugs determine the success or failure of antitumor therapies. Therefore, selection of the proper active pharmaceutical ingredient (API) is one of the most important issues in the design of nanomedicines. Among anthracycline antitumor drugs, DOX is the most widely selected candidate as an API for conjugation to nano-sized carriers or for encapsulation in micelles. As far as we know, very few studies have compared different anthracycline derivatives as nanomedicine APIs. We therefore investigated whether the widely used anthracycline DOX is the best choice for conjugation with macromolecules or nanocarrier encapsulation. Styrenemaleic acid micelles of THP previously demonstrated superior antitumor activity against mouse sarcoma (S-180) compared with DOX-loaded micelles

14

. We also showed that N-(2-

hydroxypropyl)methacrylamide-based copolymers (HPMA copolymers) conjugated with DOX (P-DOX) manifested much greater antitumor activity compared with free DOX with fewer side effects

15, 16

. We also recently reported that the HPMA copolymer-THP conjugate (P-THP)

showed potent antitumor activity against both S-180 tumors and azoxymethane/dextran sodium sulfate-induced colon cancer in mice, without severe adverse effects

17, 18

. However, no

comparison between the antitumor activity of P-DOX and P-THP has yet been reported. Therefore, in this study, we compared the cytotoxicity, cellular uptake, intracellular distribution, and in vivo antitumor activity of DOX and THP conjugated to HPMA copolymers.

2. Materials and Methods 2.1. Chemicals 1-Aminopropan-2-ol,

methacryloyl

chloride,

2,2ʹ-azoisobutyronitrile,

methyl

6-

aminohexanoate hydrochloride, N,Nʹ-dimethylformamide, N,Nʹ- dicyclohexylcarbodiimide, Nethyldiisopropylamine, dimethyl sulfoxide, and 2,4,6-trinitrobenzene-1-sulfonic acid (TNBSA) were purchased from Sigma-Aldrich. THP and DOX were purchased from Meiji Seika, Tokyo,


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Japan. BALB/c nu/nu mice were purchased from Japan SLC, Shizuoka, Japan. Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Nissui Seiyaku, Tokyo, Japan. 1methoxy-5-methylphenazinium methyl sulfate (WST-1) was purchased from Dojindo Chemical Laboratories, Kumamoto, Japan. Fetal calf serum was purchased from Nichirei Bioscience, Tokyo, Japan. SUIT2 (human pancreatic cancer) cells were purchased from RIKEN, Ibaraki, Japan. The BCA Protein Assay Kit was purchased from Thermo Fisher Scientific, Waltham, MA, USA.

2.2. Synthesis of monomers, polymer carriers, and drug conjugates HPMA, melting point (m.p.) 69–70 °C, (C7H13NO2)n (143.19)n: calculated C 58.72, H 9.15, N 9.78; found C 58.98, H 9.18, N 9.82, and 6-methacrylamidohexanohydrazide (Ma-ahNHNH2), m.p. 79–81 °C, (C10H17N3O2)n (213.28)n: calculated C 56.32, H 8.98, N 19.70; found C 56.49, H 8.63, N 19.83, were prepared as described previously via methacrylolation of 1-amino propan-2-ol using K2CO3 as a base 19, 20. 6-Methacrylamidohexanohydrazide (Ma-ah-NHNH2) was prepared in two-step synthesis as described in19 . M.p. 79 - 81 °C; elemental analysis: calc. C 56.32 %, H 8.98 %, N 19.70 %; found C 56.49 %, H 8.63 %, N 19.83 %.

A random copolymer of HPMA with Ma-ah-NHNH2 (P) was prepared by radical copolymerization in methanol (AIBN, 0.8 wt%; monomer concentration 18 wt%; molar ratio HPMA: Ma-ah-NHNH2 93 : 7; 60 °C; 17 h) as described previously

19

. Example of

polymerization: HPMA (2.0 g, 14 mmol), Ma-ah-NHNH2 (227 mg, 1.06 mmol) and AIBN (96 mg, 0.58 mmol) were dissolved in methanol (12.7 mL). The solution was introduced into a polymerization ampoule, bubbled with nitrogen, and sealed. The polymerization was carried out at 60 °C for 17 h. The polymer was isolated by precipitation into ethyl acetate and purified by reprecipitation from methanol solution into ethyl acetate. The polymer was filtered off, washed with ethyl acetate, and dried in vacuum. The yield was 1.76 g (78.5%).


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Polymer conjugates of the polymer precursor P with the anthracyclines THP and DOX were prepared as described previously 18, 19. Briefly: polymer-drug conjugates were prepared by the reaction of random copolymer of HPMA with Ma-ah-NHNH2 containing hydrazide groups with DOX.HCl or THP in methanol in the dark for 16 h with addition of acetic acid (42 µL/mL methanol solution). The polymer-drug conjugates were purified from low-molecular-weight impurities (drug or its degradation products) by gel filtration using a Sephadex LH-20 column and methanol as eluent or by precipitation of polymer conjugates into the ethylacetate.

2.3. Physicochemical characterization of polymer conjugates The molecular weights of the polymers and their conjugates were determined by using a Shimadzu HPLC system equipped with a gel permeation chromatography (GPC) column (TSKgel G3000SWxl, 300 × 7.8 mm; 5 µm), and photodiode array, refractive index OptilabrEX, and multiangle light scattering (DAWN HELEOS II, Wyatt Technology Co., Santa Barbara, CA, USA) detectors with a methanol–sodium acetate buffer (0.3 M; pH 6.5) mixture (80:20 vol%; flow rate 0.5 mL/min). The content of hydrazide group-terminated side chains in the polymer precursor was determined by means of a modified TNBSA assay, as previously described

19

. The molar

absorption coefficient ε500 = 17,200 L·mol-1·cm-1 (λ = 500 nm) estimated for the model reaction of MA-ah-NHNH2 with TNBSA was used. The hydrodynamic diameters of the polymer conjugates were measured by means of dynamic light scattering in a phosphate buffer (1 mg/mL; pH 7.4; 0.1 M with 0.05 M NaCl) with the ELS-Z2 dynamic light-scattering instrument (Otsuka Photal Electronics Co. Ltd., Osaka, Japan).


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2.4. Drug liberation study P-THP or P-DOX was dissolved in 0.1 M sodium acetate buffer (pH 5.5) or in 0.2 M phosphate buffer (pH 6.5 or 7.4) and incubated in the buffers at 37 °C for the indicated time periods. Liberated free drug and polymer-bound drug were separated by using HPLC equipped with an OHpak SB-804 HQ column (Showa Denko, Tokyo, Japan) (300 mm × 8.0 mm), and eluent absorbance was monitored at 488 nm. Eluent was a mixture of 0.1 M sodium phosphate buffer (pH 6.0) and methanol (35:65 vol%).

2.5. Cytotoxicity assay SUIT2 cells were maintained in DMEM supplemented with 10% fetal calf serum in 5% CO2/air atmosphere at 37 °C. SUIT2 cells were then treated with drugs for the indicated time periods, after which the cells were washed with fresh DMEM twice followed by incubation for up to 72 h. Viable cells were quantified by using the WST-1 reagent in accordance with the manufacturer’s instructions.

2.6. Intracellular drug uptake SUIT2 cells were seeded in 9.2-cm2 culture dishes at a cell density of 2 × 105 cells in 2 mL of DMEM. After 24 h, cells were treated with drugs at 5 or 30 µg/mL for the indicated time periods. Culture media were withdrawn and cells were washed with phosphate-buffered saline (PBS) twice followed by cell lysis and drug extraction by sonication (40 W, 30 s) and centrifugation (12,000 g, 10 min) in 1.0% sodium dodecyl sulfate. Liberated drugs were separated from drugs bound to polymers as described above, and eluent was detected by means of fluorescence (excitation wavelength at 488 nm, emission wavelength at 590 nm). The amounts of DOX or THP and P-DOX or P-THP were calculated from the individual standard curves. The protein concentrations in cell extracts were measured by using the BCA Protein Assay method. Bovine serum albumin was used for preparation of a protein standard curve.


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2.7. Confocal laser fluorescence microscopy SUIT2 cells (2 × 105 cells) were suspended in phenol red-free DMEM and seeded in 9.6cm2 glass bottom culture dishes. After 24 h, 5µg/mL DOX or THP, or 30 µg/mL P-DOX or PTHP were applied, and intracellular drugs were visualized by using confocal laser fluorescence microscopy, with the excitation wavelength at 488 nm and emission wavelength at 570–640 nm.

2.8. Intracellular drug uptake in HCT 116 cell spheroid HCT 116 cells (2 × 105 cells) were suspended in DMEM and seeded in 14-cm2 ultra-low cell attachment dishes. After 72h, formed cell spheroids were transferred into 9.6-cm2 glass bottom culture dishes. 30 µg/mL P-DOX or P-THP were applied, and drugs were visualized by using confocal laser fluorescence microscopy, with the excitation wavelength at 488 nm and emission wavelength at 570–640 nm.

2.9. Animal handling and evaluation of antitumor activity in vivo The care and maintenance of animals were undertaken in accordance with the guidelines of the Institutional Animal Care and Use Committee of Sojo University.

2.10. Body distribution of P-THP and P-DOX in SUIT2 tumor-bearing mice SUIT2 cells (5 × 105 cells) were implanted subcutaneously in the dorsal skin of the right and left sides of BALB/c nu/nu mice. Approximately 17 days after tumor cell implantation, when tumors had diameters of more than 5 mm, 15 mg/kg THP or DOX equivalent doses of P-THP or P-DOX were administered via the tail vein. At the indicated time periods, mice were killed, blood samples were withdrawn, and tissues were dissected after perfusion of the vascular void with PBS. Each tissue sample was homogenized after addition of PBS (9 mL/g of tissue). The amounts of total drug in each tissue were measured as described in a previous report 18.


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2.11. In vivo antitumor activity SUIT2 cells (5 × 105 cells) were implanted subcutaneously in the dorsal skin of the right and left sides of BALB/c nu/nu mice. About 17 days after tumor cell implantation, when tumors had diameters of more than 5 mm, 5 or 15 mg/kg THP or DOX equivalent doses of P-THP or PDOX in saline were injected intravenously (i.v.), twice per mouse, at a volume of 0.2 mL as indicated. The tumor volumes and body weights were recorded throughout the experimental period. The tumor volume (mm3) was calculated as (W2 × L)/2, after measuring the length (L) and width (W) of the tumor on the dorsal skin.

2.12. Statistical analysis Data are presented as the mean ± SD. To determine the significance of the obtained result, two-tailed unpaired Student’s t-test was applied. Results were considered statistically significant when P was < 0.05.

3. Results 3.1. Cellular uptake of anthracyclines by SUIT2 cells The absorption and fluorescence spectra of both drugs were analyzed at an excitation wavelength at 488 nm in PBS. Spectroscopic properties of THP, DOX, and EPI were comparable, but those of aclarubicin were not (Fig. S1). We first analyzed the amount of intracellular drug and its subcellular localization by means of confocal laser scanning microscopy. Cellular uptake of free THP was quite rapid; intracellular fluorescent staining was seen at 2 min after treatment with the drug (Fig. 1A). The fluorescence signal increased rapidly and reached saturation within 30 min of incubation in our experimental setting (Fig. 1A). In contrast, uptake of free DOX and that of free EPI by cells were much slower than that of free THP; faint staining was seen at 30 min of incubation, and the fluorescence signals of DOX and


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EPI at 120 min of incubation were still lower than that of free THP at 10 min of incubation (Fig. 1B–C). THP, DOX, and EPI showed nuclear localization (Fig. S2). Rapid cellular uptake of free THP was confirmed by quantifying the intracellular drug amount via HPLC after cell disintegration and extraction with 1% sodium dodecyl sulfate (Fig. 2). Intracellular uptake of THP within 30 min of incubation was approximately 20–28 times higher than that of DOX and EPI (Fig. 2). Almost 100% of the free THP used for treatment was incorporated by the cells within 60 min.

Fig. 1. Microscopy of cellular internalization of anthracycline drugs. SUIT2 cells were treated with (A) free THP, (B) free DOX, or (C) free EPI, each at 5 µg/mL, after which cells were visualized by using confocal laser scanning microscopy under 5 % CO2 at 37°C. Images were standardized to the same setting so as to compare the intracellular drug amounts. The red indicates drug fluorescence. Inset shows high contrast image. Scale bars = 50 µm. These images are representative image of duplicate experiments.


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Fig. 2. Cellular uptake of different anthracyclines. SUIT2 cells were treated with 5 µg/mL THP (gray), DOX (dark gray), or EPI (black) for the indicated time periods.

Intracellular drug amounts were determined by using HPLC (n=3).

Values are means ± SD.

3.2. Cytotoxicity of free anthracyclines Anthracycline cytotoxicity was investigated by using a human pancreatic cancer cell line (SUIT2). Cells were incubated for 72 h in the continuous presence of the drugs in the culture medium. Although all anthracyclines showed potent cytotoxicity, THP had the highest activity among the tested drugs (IC50: THP: 0.0049 µg/mL; DOX: 0.050 µg/mL; EPI: 0.016 µg/mL) (Fig. 3). We also assessed the relationship between incubation time and cytotoxicity of anthracyclines. SUIT2 cells were incubated with each anthracycline for the indicated time (pulse) periods followed by washing the cells and replacing the incubation medium with fresh drug-free medium, after which incubation continued for up to 72 h. The cytotoxicity of all anthracyclines increased with increasing incubation time. In addition, the difference in cytotoxicity was more apparent for the shorter incubation times (e.g. IC50 at 30 min: THP: 0.038 µg/mL; DOX: 1.04 µg/mL; EPI: 0.16 µg/mL) (Fig. 3).


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Fig. 3. Cytotoxicity of free anthracyclines. SUIT2 cells were treated with increasing concentrations of (A) DOX, (B) EPI, or (C) THP for the indicated time periods followed by additional incubation in drug-free medium for up to 72 h. Viable cell numbers were determined by means of the WST-1 assay (n=4). Values are means ± SD. (D) IC50 values obtained for each drug were calculated from the dose-response curves.

3.3. Chemical structures and physicochemical properties of P-THP and P-DOX DOX and THP were conjugated to the HPMA copolymer via a hydrazone bond to form P-DOX and P-THP (see Fig. S3 for their chemical structures). The hydrazone bond was labile in an acidic environment, so liberation of free drug was accelerated at the lower pH of tumor tissue, whereas at pH 7.4 it was greatly decreased. As Fig. S4 shows, free drugs were liberated from PDOX and P-THP at increasing rates as the pH value of the environment decreased. Indeed, no apparent difference in hydrolytic stability was found between P-DOX and P-THP. Table 1


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provides the physicochemical properties of P-DOX and P-THP. No apparent differences were found for the molecular weight, polydispersity, molecular size, and drug loading of P-DOX and P-THP. Table 1 Physicochemical characteristics of P-DOX and P-THP.

Conjug

Drug content (wt%)2

DH (nm)3

(g/mol)1

ate

1

Ð1

MW

P-DOX

39,000

1.97

9.84

8.2 ± 3.1

P-THP

37,800

1.82

9.64

8.1 ± 3.6

Molecular weight (MW) and polydispersity (Ð) were determined via GPC with

multiangle light scattering and refractive index detection. 2

The

drug

content

in

polymer-drug

conjugates

was

determined

spectrophotometrically. 3

Hydrodynamic diameter (DH) was determined by means of dynamic light scattering.

4

Content of free drug in the polymer conjugate was lower than 0.2 wt%.

3.4. Cellular uptake of P-THP and P-DOX The cellular uptake of P-THP and P-DOX was examined via confocal laser scanning microscopy (Fig. 4). Although cells were treated with 6-fold higher THP or DOX equivalent concentrations of P-THP or P-DOX than concentrations of free drugs, staining of cells treated with polymer-drug conjugates was far less than that of cells treated with free drugs under the same conditions. As with the free drugs, cellular uptake of P-THP was faster than that of P-DOX


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(Fig. 4A and B). Accumulation of drugs was observed mainly in the nuclei of SUIT2 cells after incubation with either P-THP or P-DOX (Fig. S5). As seen in Fig. S3, both THP and DOX are linked to the polymer via a hydrazone bond. Therefore, the conjugates liberated THP and DOX effectively in an acidic environment, as Fig. S4 shows. Liberated drug is believed to be the active entity that inhibits cell growth, so we evaluated the liberated free drug content in the cell. Similar to the result seen in Fig. 2, uptake of liberated drug by cells was approximately 10 times higher for P-THP than for P-DOX at incubation times of 30–240 min (Fig. 5). It is interesting that the amount of polymer conjugates present in the cells was almost the same for P-DOX and P-THP (Fig. 5B) for the whole time period. However, the ratio of the amount of intracellular free drug to the amount of total intracellular drug was quite different: 92% of free THP was liberated from the polymer conjugate after 30 min of incubation with P-THP, whereas only 18% of DOX was liberated after 30 min of incubation with P-DOX (Fig. 5C). We next evaluated the involvement of lysosomal cleavage of the hydrazone bond and subsequent liberation of the drug. Bafilomycin A1 is a selective vacuolar type of H+-ATPase inhibitor, so acidification of endosomes is inhibited by treatment of cells with bafilomycin A1 21. The amounts of free drugs in the cells and their ratios did not change after pretreatment with bafilomycin A1, which indicated that the lysosome is not the main site of cleavage of the hydrazone bond in our system (Fig. 5D).

Fig. 4. Fluorescence microscopic images of uptake of P-THP and P-DOX by SUIT2


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cells. SUIT2 cells were treated with 5 µg/mL (A) P-THP or (B) P-DOX or with 30 µg/mL for the indicated time periods, after which cells were visualized by using confocal laser scanning microscopy. The red indicates drug fluorescence. Scale bars = 50 µm. These images are representative image of duplicate experiments.

Fig. 5. Uptake of P-THP and P-DOX by SUIT2 cells. Cells were treated with 30 µg/mL P-THP or P-DOX for the indicated time periods. (A) Intracellular concentration of liberated free drug (n=3). (B) Intracellular concentration of polymer-conjugated drug as quantified via HPLC with fluorescence detection (at 488 nm). (C) Ratio of intracellular liberated free drug to intracellular total drug.

(D) SUIT2 cells were

pretreated with bafilomycin A1 for 1 h or were untreated, followed by P-THP or PDOX treatment for 4 h, after which the amount of intracellular liberated free drug was quantified via HPLC (n=3). The ratio of intracellular free drug to intracellular total drug was calculated from the following equation: Free drug = [(liberated drug amount)/{(liberated drug amount) + (intact drug amount)}] × 100. Values are means ±


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SD.

3.5. Cytotoxicity of P-THP and P-DOX We analyzed the cytotoxicity of P-DOX and P-THP in SUIT2 cells. As seen for other macromolecular drugs, P-THP and P-DOX had approximately 10 times lower cytotoxicity compared with the free drugs (compare Figs. 3 and 6). P-THP manifested a cytotoxicity that was more than 10 times higher than that of P-DOX, and both depended on dose and contact time (Fig. 6). The more potent cytotoxicity of P-THP compared with that of P-DOX correlates well with the cellular uptake rate of the liberated free drugs. A higher cytotoxicity of P-THP compared with that of P-DOX was observed not only for SUIT2 cells but also for various other cancer cells, e.g. non-small cell lung carcinoma, prostate cancer, colorectal cancer, and hepatic cancer (Table S1).


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Fig. 6. Cytotoxicity of P-DOX and P-THP. SUIT2 cells were treated with increasing concentrations of (A) P-THP or (B) P-DOX for the indicated time periods followed by additional incubation in drug-free medium for up to 72 h. The number of viable cells was determined by means of the WST-1 assay (n=4). Values are means ± SD. (C) IC50 values for each drug were calculated from the dose-response curves.

Studies using cultured tumor cells as shown in Figs. 1 and 3 do not reveal 3 dimensional drug penetration in the tissues in vivo. In real in vivo situation, drugs from the circulating blood must extravasate from the blood vessels then penetrate into the stromal tissue matrix, then reach to the cancer cells, although the situation is variable from one tumor to the others. To clarify this point we evaluated the penetration and cellular internalization property of P-THP and P-DOX in the spheroidal cell cultures of HCT 116 cell to mimic the tissue penetration of these polymeric drugs through the tissue matrix. As seen in Fig. 7, P-THP did deeply penetrate into the cell-


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spheroid, and effectively concentrated in this tumor cell. In contrast, this was not seen in P-DOX group. Free THP also showed deeper penetration into the spheroid than free DOX (Fig. S6).

Fig. 7. Penetration and cellular uptake of P-THP and P-DOX in cultured tumor cell spheroid. HCT 116 cell spheroid having approx. 200 µm in diameter was treated with 30 µg/mL of P-THP or P-DOX, and drugs were visualized by confocal laser microscopy at indicated time. Images are taken at the center core of spheroids. Scale bars = 100 µm. These images are representative image of duplicate experiments.

3.6. Body distributions and antitumor effects of P-THP and P-DOX Distributions of P-THP and P-DOX in body and tumor tissues were studied in mice bearing subcutaneously implanted human pancreatic cancer (SUIT2) tumors. THP and DOX equivalent doses (15 mg/kg) of P-THP and P-DOX dissolved in PBS were administered via the tail vein, and total drug concentrations, free drug released plus polymer-drug-conjugate, in each tissue were analyzed by using HPLC after converting the conjugate to free DOX in acid and extraction as total amount of drug. Drug accumulation in the liver and kidney was slightly higher in the P-DOX group than in P-THP group (Fig. 8). The mean intratumor concentrations of PTHP at 6 h and 24 h were 0.61 and 0.46 µg/100 mg of tissue, respectively, whereas mean intratumor P-DOX concentrations at 6 h and 24 h were 0.44 and 0.29 µg/100 mg of tissue, respectively. The results thus showed a tendency of enhanced P-THP tumor accumulation


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compared with tumor accumulation of P-DOX, although no significant difference between them was found (Fig. 8).

Fig. 8. Body distribution of P-THP and P-DOX. Mice with implanted SUIT2 cells were injected with 15 mg/kg P-THP or P-DOX. At the indicated time periods, mice were killed and were perfused transcardially with saline. Each tissue was collected, and the amounts of the drugs after extraction were measured by using HPLC (n=3). See details in the Materials and Methods. Values are means ± SD.

In vivo antitumor activity of P-THP and P-DOX was compared in mice with subcutaneously implanted SUIT2 cells. We previously reported that about 40 mg of THP equivalent/kg was the maximum tolerable dose of P-THP given as an i.v. bolus 18. Therefore, we set the maximum dose at 30 mg/kg to evaluate the therapeutic effect of P-THP and P-DOX. When tumors grew to more than 5 mm in diameter, mice were given two treatments, at 5-day intervals, of 5 or 15 mg/kg (THP or DOX equivalent) of P-THP or P-DOX. All treated groups manifested suppressed tumor growth (Fig. 9). Among all treatments, 2 × 15 mg (THP equivalent)/kg of P-THP was the most effective compared with the same dose of P-DOX (Fig.


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9). Groups treated with 15 mg/kg P-THP or P-DOX lost approximately 5% of body weight (Fig. S7).

Fig. 9. Antitumor activity in SUIT2 tumor-bearing mice. Mice were treated with 5 or 15 mg/kg P-THP or P-DOX twice at the indicated times (arrows). Tumor volumes were measured as described in Materials and Methods, and the relative tumor volumes each day were normalized according to the tumor volume at day 0 (n=11-12). Values are means + SD. *P < 0.05 and **P

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