Gene Ther Mol Biol Vol 10, 113-122, 2006

Using N-(2-hydroxypropyl) methacrylamide copolymer drug bioconjugate as a novel approach to deliver a Bcl-2-targeting compound HA14-1 in vivo

Research Article

Matthew Oman¹, Jihua Liu¹, Jun Chen¹, David Durrant¹, Hung-Sheng Yang¹, Yongwen He¹, Pavla Kopecková², Jindrich Kopecek² and Ray M. Lee^1,*

¹Huntsman Cancer Institute, Department of Pharmaceutics and

²Pharmaceutical Chemistry, University of Utah, Salt Lake City, UT 84112

__________________________________________________________________________________

*Correspondence: Ray M. Lee, Huntsman Cancer Institute at the University of Utah, Salt Lake City, Utah 84112, USA

Current address: Massey Cancer Center, Virginia Commonwealth University, 1101 E. Marshall St. P.O. Box 980230, Richmond, VA 23298; E mail: rlee5@vcu.edu

Key words: apoptosis, HPMA copolymer bioconjugate, HA14-1, xenograft

Abbreviations: ethyl 2-amino-6-bromo-4-(1-cyano-2-ethoxyl-2-oxoethyl)-4H-chromene-3-carboxylate, (HA14-1); N-(2-hydroxypropyl)methacrylamide, (HPMA); Fluorescein-5- isothiocyanate, (FITC)

Received: 1 July 2005; Accepted: 31 January 2006; electronically published: March 2006

Summary

Bcl-2 plays a critical role in regulation of apoptosis and tumor pathogenesis; thus it’s a good therapeutic target for cancer. Small compounds blocking Bcl-2 have been identified but their efficacy in vivo has hardly been demonstrated. We developed water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing a Bcl-2 targeting compound HA14-1. Their efficacy was confirmed in cell lines, and tested in a tumor xenograft model. Intraperitoneal injected copolymer HA14-1 bioconjugates suppressed tumor growth by 50%. Using FITC as a marker to trace biodistribution, we demonstrated that the concentration of the copolymer was sufficient to induce apoptosis. This was confirmed by the presence of activated caspase 9 in tumor treated with the copolymer HA14-1 bioconjugate, but not in normal organs or tumor treated with a control polymer. No toxicity was observed in liver and kidney, where copolymers are excreted. The HPMA copolymer is thus a promising strategy for in vivo delivery of Bcl-2-targeting compounds to solve their poor solubility problem and to enhance tumor selectivity.

I. Introduction

Overexpression of Bcl-2 family apoptotic inhibitors contributes to tumor pathogenesis and drug resistance (Cory and Adams, 2002; Cory et al, 2003; Gross et al, 1999). Bcl-2 is a good therapeutic target for the development of novel cancer therapy (O'Neill and Hockenbery, 2003). One way to down-regulate Bcl-2 is by an antisense oligonucleotide that is currently in clinical trials (Pepper et al, 2001). Another approach is to develop small molecular compounds fitting into a hydrophobic pocket of Bcl-2 that interacts with the BH3 domain of pro-apoptotic members of the Bcl-2 family (Muchmore et al, 1996; Aritomi et al, 1997). Based on a structure-based molecular screen, HA14-1 was first identified (Wang et al, 2000). Subsequently, several other compounds were also found to bind and inhibit Bcl-2, including antimycin, BH3Is, gossypol, chelerythrine and polyphenols (Degterev et al, 2001; Tzung et al, 2001; Chan et al, 2003; Kitada et al, 2003; Zhang et al, 2003). They induce apoptosis in many cell lines with IC₅₀ mostly in mM range (Wang et al, 2000; Chen et al, 2002; An et al, 2004). The most potent Bcl-2 inhibitor currently is ABT-737, which is two to three orders of magnitude more potent than other Bcl-2 inhibitors (Oltersdorf et al, 2005). Many potential strategies of using Bcl-2 inhibitors have been developed using HA14-1. Synergistic effects have been shown between HA14-1 and many other compounds, including chemotherapeutic agent cytarabine (Lickliter et al, 2003), proteasome inhibitor bortezomib (Pei et al, 2003), TRAIL, (Hao et al, 2004), MEK inhibitor PD184352 (Milella et al, 2002), and peripheral benzodiazepine receptor inhibitor PK11195 (Chen et al, 2002). Similar to other Bcl-2 inhibitors, the most potent agent, ABT-737, is not active in inducing cell death by itself, but synergizes chemotherapeutic agents (Oltersdorf et al, 2005).

Before developing small molecule Bcl-2 inhibitors into a clinically useful drug, there are three major hurdles that need to be solved. One is its poor water solubility that makes drug formulation difficult. This is likely a universal problem for all Bcl-2 inhibitors because they fit into the hydrophobic pocket of Bcl-2. The second is lacking tumor selectivity, and the third is its low potency, but now overcome by the availability of ABT-737. These three problems could be solved by conjugating Bcl-2 inhibitor such as HA14-1 to HPMA copolymers. Macromolecular therapeutics derived from HPMA copolymer-drug bioconjugates are novel approaches to deliver chemotherapeutic agents into tumors (Jensen et al, 2001, 2002; Kasuya et al, 2001; Kopecek et al, 2001; Lu et al, 2002; Luo et al, 2002; Peterson et al, 2003). HPMA copolymer drug bioconjugates are internalized by endocytosis and remain in endosomes/lysosomes (Kopecek et al, 2001). Depending on the linkage to the side chains of biopolymers, conjugated drugs can be either non-cleavable or cleavable in lysosomes. If cleavable, the compound attached to the side chain termini can be cleaved by a protease and released from lysosomes to other organelles (Jensen et al, 2001). Doxorubicin, geldanamycin, mesochlorin e₆, antisense oligonucleotides, and anti-angiogenic compound TNP-470 have been delivered into tumor cells successfully using HPMA copolymer drug bioconjugates (Kasuya et al, 2001; Jensen et al, 2002; Lackner et al, 2003; Nishiyama et al, 2003; Peterson et al, 2003; Satchi-Fainaro et al, 2004). In mouse tumor models, the HPMA copolymer doxorubicin bioconjugate was more effective than free doxorubicin in an ovarian cancer xenograft that expressed a multiple drug resistance (MDR) gene (Minko et al, 2000). The copolymer-drug bioconjugate also has an advantage of preferential accumulation in solid tumors, known as the enhanced permeability and retention (EPR) effect (Langer, 1998; Moses et al, 2003). Based on these favorable characteristics, we developed HPMA copolymer HA14-1 bioconjugates which potentially move HA14-1 or other insoluble Bcl-2-targeting compounds closer to clinical application.

II. Materials and methods

A. Materials

A2780 cells were grown in the RPMI supplemented with 10% fetal bovine serum, penicillin-streptomycin-glutamine and insulin (10 mg/ml) (Life Technology Inc.). HA14-1 was from Calbiochem (San Diego, CA). FITC was from Molecular Probes (Eugene, OR). TUNEL assay kits were from Roche (Penberg, Germany). MTT assays were from Chemicon International Corp. (Temecula, CA). Antibodies against activated caspases 9 were from Cell Signaling Technology Inc. (Cambridge, MA).

B. Synthesis and characterization of HPMA copolymer bound HA14-1

The bioconjugates were prepared via a two-step procedure. First, polymer precursors (Kopecek et al, 2001; Lu et al, 2002)were prepared by radical precipitation copolymerization of comonomers: (a) HPMA, (b) N-methacryloylglycylglycine p-nitrophenyl ester (non-degradable spacer) or N-methacryloylglycylphenylalanylleucylglycine p-nitrophenyl ester (degradable spacer), and optionally (c) methacryloylated FITC (3-methacryloylaminopropyl thioureidyl fluorescein). The molar ratio of monomer units in the polymerization mixture were 94: 5: 1. The polymer precursors were characterized by size exclusion chromatography (to determine the molecular weight), the content of p-nitrophenyl ester (ONp) groups and FITC moieties.

In the second step, the HA14-1 was bound to the polymer precursors by aminolysis of p-nitrophenyl ester groups at polymer side-chain termini by amine group of HA14-1. Example of binding procedure: 100 mg (0.030 mmol ONp groups) of polymer precursor (copolymer of HPMA and N-methacryloylglycylphenylalanylleucylglycine p-nitrophenyl ester) was dissolved in 0.75 ml dimethylsulfoxide and 18 mg HA14-1 (0.045 mmol) was added while stirring. After dissolution the dimethylaminopyridine catalyst (8 mg, 0.03 mmol) was added and the reaction mixture was stirred 4 h at room temperature. The polymer was precipitated into acetone: diethylether (3:1) mixture, filtered off, washed, and dried. The conjugate was purified by 24 h dialysis at 4 ^oC, first in ethanol/H₂O, and then in H₂O, using dialyzing tubing with 6-8 kDa cut off. The yield after dialysis was 70 mg. The content of HA14-1 was determined by UV spectroscopy using the extinction coefficient e 9300 M^-1cm^-1in MeOH, l_max 273 nm. The composition of copolymers is shown in Table 1.

C. Mouse xenograft model

Nude mice (BALB/cAnNCr-nu/nu, NCI Developmental Therapeutics Program) were injected with 5 x 10⁶ A2780 cells at both flanks subcutaneously on day 1. Tumors became visible after 13 days. Intraperitoneal injection of the copolymer-drug bioconjugates or normal saline was performed twice a week for five doses. Paclitaxel and free HA14-1 was dissolved in DMSO before injection. The largest diameter of the tumor was measured in three dimensions, twice a week, before each injection. The size of the tumor was calculated by the formula V = (4/3) x p x R₁ x R₂x R₃, where R₁, R₂, R₃ are the largest radii of the tumor in three dimensions.

D. Analysis of the biodistribution of the copolymer drug bioconjugate

All major organs and tumors were dissected, cut into small pieces and dounced with loose and tight pistons 10 times in a buffer containing 50 mM Tris (pH7.4), 100 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 1 mM PMSF. The homogenates (100 ml) were transferred to a 96-well plate and the fluorescence of FITC was determined using a microplate reader (Bio-Tek Instrument Inc., Winooski, VT), and converted to fmole using a standard curve established by the cleavable copolymer. Protein concentrations of the homogenates were determined by the Bio-Rad protein assay.

E. Histological examination and TUNEL staining

Pieces of normal organs and tumors were fixed in formalin and processed by the Department of Pathology at the University of Utah Hospital for regular sections and H & E staining. Unstained sections were also analyzed by TUNEL staining according to the manufacturer’s protocol.

III. Results

A. Induction of apoptosis by the copolymer HA14-1 bioconjugate in cell lines

Two HPMA copolymer HA14-1 bioconjugates were designed to evaluate the requirement of lysosomal cleavage. One is non-cleavable in the lysosomes with a Gly-Gly linker, and the other is cleavable due to the presence of a Gly-Phe-Leu-Gly linker (Figure 1a). The linkage of HA14-1 to the copolymer is stable in the blood stream; thus the release of HA14-1 only happens intracellularly. The cleavable conjugate also contains FITC (Figure 1a) to trace its biodistribution. A third polymer with FITC was used as a control copolymer.

We tested the copolymer HA14-1 bioconjugates with HL60 leukemia cells and found that the cleavable bioconjugate was more effective than the non-cleavable (Figure 1b). The non-cleavable bioconjugate required up to 150 mM to achieve killing; whereas 50-100 mM of the cleavable bioconjugate was sufficient (Figure 1c). Monomeric HA14-1 had a dose-dependent apoptotic effect as described (Chen et al, 2002). TUNEL assays also confirmed that cell death was due to apoptosis (Figure 1c). Although the HPMA copolymer appeared to require a higher concentration, we did not consider that a problem due to significantly advantage of the HPMA copolymer HA14-1 conjugate due to the advantages of copolymers, including water solubility, the route of uptake through endocytosis, and the preferential accumulation in tumor (Langer, 1998; Moses et al, 2003). The high effective concentration and the failure of releasing free HA14-1 in blood are two safety features and advantages of the copolymer to avoid toxicity.

Studies with A2780 ovarian cancer cells showed a similar result in that the non-cleavable copolymer was less effective than the cleavable form (Figure 2). When cells were treated with 50, 100 and 150 mM of the cleavable copolymer HA14-1 bioconjugate for 24 hours, the percentages of apoptotic cells, determined by TUNEL assays, were 6.44%, 11.43%, and 19.35%, respectively. A2780 cells were more resistant to free HA14-1 than HL60 cells and required a concentration up to 50 mM to achieve significant apoptosis (Figure 2). Because the non-cleavable copolymer did not show any activity in cell lines, we focused on the cleavable HPMA copolymer HA14-1 conjugate to test their efficacy by mouse xenograft model in vivo.

B. Biodistribution of the cleavable HPMA copolymer HA14-1 bioconjugate

We then studied the biodistribution of the HPMA copolymer HA14-1 bioconjugate using mice with established subcutaneous tumors. After being injected intraperitoneally with the bioconjugate, the mice were sacrificed at 6, 24, 48, 72 and 168 hours. The fluorescence of FITC was quantified and compared with a standard curve established by the cleavable HA14-1 bioconjugate. Because the cleavable copolymer bioconjugate contains both FITC and HA14-1, we measured the fluorescence activity of FITC, converted to fmole per mg of protein extracted from the organ. This information represented the accumulation of the copolymer in normal tissues and tumors. The concentration (fmole/mg) was highest in the kidney and liver, likely related with the metabolism or excretion of the copolymer in these two organs. The spleen and bowel also contained high concentrations, which was apparently due to intraperitoneal injection. Tumor contained a level comparable to the spleen and bowel, but much higher than organs outside of the peritoneal cavity, such as lung, heart and brain (Figure 3a). We also calculated the total amount of FITC in normal organs and tumors by multiplying the concentration of FITC with the total amount of protein. The liver and kidney contained the most FITC, whereas tumor had a level comparable to bowel. Other organs contained very little FITC (Figure 3b), supporting the preferential accumulation of the copolymer in tumor.

To determine whether the concentration of the copolymer in tumor was sufficient to induce apoptosis, we treated A2780 cells with 50-150 mM of the cleavable bioconjugate in vitro and analyzed the concentration of FITC (right panel, Figure 3c). The concentration of FITC in tumor was comparable to that detected in vitro at 100 mM (left panel, Figure 3c), which induced about 11% of apoptosis by TUNEL assays (Figure 2).

C. Testing copolymer HA14-1 bioconjugates in a mouse xenograft model

We then used the same mouse xenograft model to test the efficacy in vivo. Tumors become visible after 13 days, and mice were injected intraperitoneally with normal saline (group 1), FITC-only copolymer (group 2), cleavable HA14-1 copolymer (group 3) and free HA14-1 in DMSO (group 4) at days 13, 17, 20, 23 and 27. Mice treated with paclitaxel in DMSO at 10 mg/kg were used as positive controls (group 5). The amount of copolymer HA14-1 bioconjugates injected in Group 3 was equivalent to 5 mg/kg of free HA14-1 (group 4). The amount of bioconjugates in Groups 2 and 3 were equal in FITC to ensure that the accumulation of FITC did not contribute to the decrease of tumor size. After 5 injections, we observed

Figure 3. Biodistribution of the cleavable copolymer HA14-1 bioconjugate. (a) The concentration of FITC (fmole/mg of protein) in each organ and tumor. Mice with established tumors were injected with the cleavable copolymer HA14-1 bioconjugate and sacrificed at 6, 24, 48, 72 and 168 hours (two mice each), and visceral organs were removed and total protein extracted. The fluorescence of FITC was measured by a microplate reader in triplicates, and converted into fmole based on a standard curve established by the cleavable copolymer HA14-1 bioconjugate. The concentrations of FITC per milligram of protein were calculated. (b) The total amount of FITC in each organ and tumor was calculated by multiplying the concentration with the total protein. (c) The concentrations of FITC (fmole/mg) accumulated in tumor (left panel) were compared to those of A2780 cells treated in vitro with the cleavable copolymer HA14-1 bioconjugate (right panel). Treatment in vitro was done with 50, 100, and 150uM for 24 hours before cells were harvested for the determination of the concentration of FITC.

Figure 4. Effect of HPMA copolymer HA14-1 bioconjugates in a mouse xenograft model. (a) BALB/cAnNCr-nu/nu mice were injected with A2780 cells and tumors became visible after 13 days. Copolymers was injected intraperitoneally at days 13, 17, 20, 23, 27 and tumor volumes were measured. Each group were injected with 0.1 c.c. normal saline, the FITC control copolymer, the cleavable HPMA copolymer HA14-1 bioconjugate, free HA14-1 or paclitaxel positive control. (b) Photographs of representative mice treated with the copolymer HA14-1 bioconjugate (top mouse) and the control copolymer (bottom mouse).

Figure 5. H & E staining of liver, kidney, bowel and tumor from mice treated with the HPMA copolymer HA14-1 bioconjugate. The two left lower panels show different distribution of FITC in tumor and liver. No FTIC was seen in the hepatocytes but high activity was in the liver sinusoids.

E. Presence of apoptotic markers in tumors treated with HA14-1-containing polymers but not in normal organs

To demonstrate apoptosis in tumor after treatment with the copolymer, we analyzed protein extracts from each organ and tumor. Because HA14-1-induced apoptosis is associated with release of cytochrome c to the cytoplasm for activation of the apoptosome complex and caspase 9 (Li et al, 1997), we investigated whether tumor extracts contained activated caspases. Immunoblotting with the antibody specific to activated caspase 9 (37 kD) was positive in tumor samples but not in extracts from normal organs (Figure 6a). This result was consistent with the normal histological sections of liver and kidney shown in the previous section, and it also established tumor killing by the copolymer HA14-1 bioconjugate. We also performed TUNEL assays to demonstrate apoptosis in tumor treated with the copolymer HA14-1 conjugates. The tumor treated with the FITC control copolymer rarely exhibited positive TUNEL cells; whereas tumor treated with the copolymer HA14-1 bioconjugate had more TUNEL positive cells (Figure 6b), confirming that apoptosis was only present in the tumor treated with the copolymer HA14-1 bioconjugate.

IV. Discussion

Bcl-2 is a good target to develop novel cancer therapies. Targeting Bcl-2 has become potentially feasible by the identification of small molecular compounds that fit into a hydrophobic pocket occupied by the BH3 peptide. However, due to the nature of binding, all small molecular compounds identified so far have poor water solubility. Besides, most pro-apoptotic agents may not have the desired tumor selectivity due to ubiquitous expression of Bcl-2. The new agent ABT-737, although significantly more potent than other Bcl-2 inhibitor, still has the same problem of poor solubility that needs to be solve before its clinical application. We used a commercially available Bcl-2-targeting compound, HA14-1, and proved that Bcl-2-targeting compounds can be made water-soluble and thus suitable for intravenous administration. The accumulation of copolymer in tumor may also solved the problem of non-selectivity.

The HPMA copolymers, due to their molecular weight, could not diffuse through the plasma membrane. They enter cells through endocytosis and remain in endosomes that eventually fuse with lysosomes. The HPMA copolymers we synthesized to contain two kinds of linkers between HA14-1 and the backbone of the polymer. One (GFLG) is lysosomal cleavable, and the other (GG) is non-cleavable. As expected, the lysosomal cleavable form was more effective than the non-cleavable form, indicating that the release of free HA14-1 is essential for its activity.

This finding can be explained by the fact that the amino group of HA14-1 used for conjugation fits in the hydrophobic pocket (Wang et al, 2000), and the polymer precludes the incorporation of conjugated-HA14-1 into the pocket.

In the mouse xenograft model, tumor growth was suppressed by the HPMA copolymer HA14-1 bioconjugate by about 50%. The appearance of activated caspase 9 suggested that apoptosis occurred in tumor treated with the HA14-1-containing copolymer. However, merely 50% of tumor growth suppression reflects that HA14-1 itself is not very potent to A2780 cells as shown in Figure 2. H & E section of the copolymer-treated tumor revealed a high mitotic index, indicating that cells still proliferate fast despite the 50% growth suppression by the copolymer HA14-1 bioconjugate. This finding provides the rationale to combine the HPMA copolymer HA14-1 bioconjugate with chemotherapeutic agents to enhance the therapeutic efficacy. Combination with other agents synergistic with HA14-1, such as chemotherapeutic drug cytarabine, bortezomib, or other compounds currently in development may help to solve this problem (Chen et al, 2002; Milella et al, 2002; Lickliter et al, 2003; Pei et al, 2003; Hao et al, 2004).

We included FITC in the cleavable bioconjugate to analyze the biodistribution of the copolymer and examined the enhanced permeability and retention effect of the copolymer conjugates. The kinetics study revealed that accumulation of FITC in tumor reached a plateau at 6 hours. This study is based on the consensus that the linker for HA14-1 is not degradable in circulation and that the backbone along with the non-cleavable FITC is retained in cells (Jensen et al, 2001, 2002; Kasuya et al, 2001; Kopecek et al, 2001; Lu et al, 2002; Luo et al, 2002; Peterson et al, 2003). This is very important for copolymer drug delivery because a rapid release of free drug in circulation will certainly cause significant toxicity. Although we detected high concentrations of FITC in liver and kidney, which is likely due to a normal excretion of the copolymer, it was comforting not to detect any evidence of toxicity in these two organs by histological staining and immunoblotting analysis.

In conclusion, we have developed a feasible approach to convert a water-insoluble Bcl-2 targeting compound to a soluble copolymer-drug bioconjugate. This strategy eliminates the major hurdles for development of Bcl-2-targeting compounds. Although HA14-1 is not the ideal compound for further clinical development, our proof-of-principle study confirmed that HPMA copolymer drug conjugate is suitable to carry Bcl-2 inhibitors into tumor to induce apoptosis and suppress tumor growth in vivo without inducing toxicity in normal organs. This reagent will allow us to test combination strategies and to address how targeting Bcl-2 may enhance available chemotherapeutic agents in vivo.

Acknowledgements

We thank Dr. Joseph Holden for helping the analysis of histological sections, Dr. Wayne Green for the flow cytometry study. This work is supported by the Marsha Rivkin Ovarian Cancer Research Foundation (R.M.L.) and NIH grant support (J.K.).

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Conjugate #	Oligopeptide spacer	HA14-1 wt%^a	FITC wt%^b	M_w^c, kDa
1	-GlyGly-	10.1	-	25
2	-GlyPheLeuGly-	8.7	1.3	28
3	N/A	-	1.0	25