Correlation of Subcellular Compartmentalization of HPMA Copolymer-Mce6 Conjugates with Chemotherapeutic Activity in Human Ovarian Carcinoma Cells

Purpose. Intracellular targets sensitive to oxidized damage generated by photodynamic therapy (PDT) utilizing N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-mesochlorin e₆ monoethylenediamine (Mce₆) conjugates was explored to aid in the design of second-generation PDT delivery systems. Methods. Low temperature, metabolic inhibitor, and nuclear localization sequences (NLS(FITC)) were used to achieve desired subcellular localization that was evaluated by confocal analysis and subcellular fractionation. Mce₆ was bound to HPMA copolymer conjugates via non-degradable dipeptide linkers (P-GG-Mce₆, P-NLS(FITC)-GG-Mce₆) or lysosomally degradable tetrapeptide spacers (P-GFLG-Mce₆, P-NLS(FITC)-GFLG-Mce₆). Chemotherapeutic efficacy was assessed by the concentration that inhibited growth by 50% (IC₅₀), cell associated drug concentration (CAD) and confocal microscopy. Results. P-GFLG-Mce₆ possessed enhanced chemotherapeutic activity compared to P-GG-Mce₆ indicating enzymatically released Mce₆ was more active than copolymer-bound Mce₆. Lysosomes appeared less sensitive to photodamage as observed by a higher IC₅₀. Nuclear-directed HPMA copolymer-Mce₆ conjugates (P-NLS(FITC)-GG-Mce₆ P-NLS(FITC)-GFLG-Mce₆) possessed enhanced chemotherapeutic activity. However, control cationic HPMA copolymer-Mce₆ conjugates containing a scrambled NLS (P-scNLS(FITC)-GG-Mce₆) or amino groups (P-NH₂-GG-Mce₆) also displayed increased chemotherapeutic activity. Conclusions. Nuclear delivery was observed for P-NLS(FITC)-GG-Mce₆ and P-NLS(FITC)-GFLG-Mce₆ indicating NLS was a feasible approach for nuclear delivery. Due to the cationic nature of NLS, increased membrane binding of PDT systems incorporating cationic nuclear targeting moieties must be addressed.     

Preparation of Fab' from Murine IgG2a for Thiol Reactive Conjugation

Lysyl endopeptidase (LE) from Achromobacter lyticus M497-1 (EC 3.4.21.50) was utilized to prepare F(ab')₂ fragments from mouse anti-P-glycoprotein IgG2a obtained from the UIC2 hybridoma. This report describes a novel single step purification procedure for F(ab')₂ fragments that eliminates residual LE activity responsible for secondary cleavage of F(ab')₂ to Fab fragments. The purification of F(ab')₂ and F_c fragments was accomplished utilizing protein G affinity chromatography and either gradient or step changes in the pH/ionic strength for elution of the F_c and F(ab')₂ fragments. Residual LE was eluted from the protein G column with buffer containing 200 mM L-lysine prior to elution of F(ab')₂ and F_c fragments. The activity of LE was monitored using the fluorogenic substrate Boc-Val-Leu-Lys-7-amido 4-methyl coumarin. A similar purification procedure for F(ab')₂ fragments produced following pepsin digestion of IgG2a is also outlined. The ability of Fab' fragments, from reduced F(ab')₂ fragments following LE digestion of IgG2a, to conjugate to thiol reactive groups was demonstrated using N-(2-hydroxypropyl)methacrylamide (HPMA) copolymermeso chlorin eg mono (N-2-aminoethylamide) (Mce₆) conjugates containing reactive maleimide groups. The biological activity of the Fab' targeted HPMA copolymer-Mce₆ conjugates was tested against the P-glycoprotein expressing human ovarian carcinoma A2780/AD cell line utilizing a cell survival assay. Fab' targeted HPMA copolymer-Mce₆ conjugate demonstrated significantly higher cytotoxicity than either a monoclonal antibody (mAb) targeted HPMA copolymer-Mce₆ conjugate or a non-targeted HPMA copolymer-Mce₆ conjugate, p < 0.05.     

Biorecognition of HPMA Copolymer-Adriamycin Conjugates by Lymphocytes Mediated by Synthetic Receptor Binding Epitopes

Purpose. The EDPGFFNVE nonapeptide (NP) was recognized as the CD21 (CR2) binding epitope of the Epstein-Barr virus (EBV) gp350/ 220 envelope glycoprotein which mediates the virus attachment to human B lymphocytes (Nemerow et al., Cell 56:369-377, 1989). Here we evaluated the targeting potential of a synthetic receptor binding epitope (NP) covalently attached to a water-soluble polymeric drug carrier. In particular, the biorecognition of N-(2-hydroxypropyl) metha-crylamide (HPMA) copolymer-NP conjugates by B- and T-cells and the cytotoxicity of HPMA copolymer-NP-adriamycin (ADR) conjugates toward B-cells, T-cells, and peripheral blood lymphocytes (PBL) were evaluated. Methods. HPMA copolymer-NP and optionally ADR conjugates varying in the NP density and mode of NP attachment were incubated with Raji B-cells (human Burkitt's lymphoma), CCRF-CEM T-cells (acute human lymphoblastic leukemia), and CCRF-HSB-2 T-cells (human lymphoblastic leukemia). The kinetics of binding was studied, the Langmuir adsorption isotherms analyzed, binding constants calculated, and IC₅₀ doses determined. Results. Flow cytometry studies revealed that binding was homogeneous to both cell types. The apparent binding constants to T-cells were about two times higher when compared to B-cells. The binding and cytotoxicity increased with increased amount of epitopes per polymer chain. Attachment of the NP via a GFLG spacer resulted in increased biorecognition when compared with conjugates containing NP bound via a GG spacer. HPMA copolymer-NP-ADR conjugates possessed specific cytotoxicity to T- and B-malignant cells. Concentrations, which were lethal to the latter, were not toxic for PBL. Conclusions. The data obtained seem to indicate the potential of the HPMA copolymer-NP conjugates as polymer anticancer drug carriers targetable to immunocompetent cells.     

Targetable HPMA Copolymer–Aminohexylgeldanamycin Conjugates for Prostate Cancer Therapy

Purpose  This study focuses on the synthesis and characterization of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer–cyclo-RGD (Arg-Gly-Asp) conjugates for delivery of geldanamycin to prostate tumors. Materials and Methods  HPMA copolymers containing aminohexylgeldanamycin (AH-GDM) with and without the targeting peptide RGDfK were synthesized and characterized. Drug release from copolymers was evaluated using cathepsin B. Competitive binding of copolymer conjugates to α_vβ₃ integrin was evaluated in prostate cancer (PC-3) and endothelial (HUVEC) cell lines and in vitro growth inhibition was assessed. The maximum tolerated dose for single i.v. injections of free drug and the conjugates was established in nude mice. Results  HPMA copolymers containing AH-GDM and RGDfK showed active binding to the α_vβ₃ integrin similar to that of free peptide. Similarly, growth inhibition of cells by conjugates was comparable to that of the free drug. Single intravenous doses of HPMA copolymer–AH-GDM–RGDfK conjugates in mice were tolerated at 80 mg/kg drug equivalent, while free drug caused morbidity at 40 mg/kg. No signs of toxicity were present in mice receiving HPMA copolymer-AH-GDM-RGDfK over the 14-day evaluation period. Conclusion  Results of in vitro activity and in vivo tolerability experiments hold promise for the utility of HPMA copolymer–AH-GDM–RGDfK conjugates for treatment of prostate cancer with greater efficacy and reduced toxicity.     

Comparison of active and passive targeting of doxorubicin for somatostatin receptor 2 positive tumor models by octreotide-modified HPMA copolymer-doxorubicin conjugates

Somatostatin receptor 2 (SSTR2), specifically over-expressed on many tumor cells, is a potential receipt for active targeting in cancer therapy. In the present study, octreotide (Oct), which had high affinity to SSTR2, was attached to N-(2-hydroxypropyl) methacrylamide (HPMA) polymeric system to enhance the antitumor efficiency of the anticancer drug doxorubicin (DOX). Two kinds of cell lines (HepG2 and A549), which overexpress SSTR2, were chosen as cell models. Compared with non-modified conjugates, Oct-modified conjugates exhibited superior cytotoxicity and intracellular uptake on both HepG2 and A549 cell lines. This might be due to the mechanism of receptor-mediated endocytosis. Subsequently, the in vivo biodistribution and antitumor activity evaluations showed that Oct modification significantly improved the tumor accumulation and antitumor efficacy of HPMA copolymer conjugates in SSTR2 over-expressed Kunming mice bearing H₂₂ tumor xenografts. In summary, Oct-modified HPMA polymer-DOX conjugates might be a promising system for the treatment of SSTR2 over-expressed cancers.     

Noninvasive Visualization of Pharmacokinetics,Biodistribution and Tumor Targeting of Poly[<Emphasis Type="BoldItalic">N</Emphasis>-(2-hydroxypropyl)methacrylamide] in Mice Using Contrast Enhanced MRI

Purpose To study a non-invasive method of using contrast enhanced magnetic resonance imaging (MRI) to visualize the real-time pharmacokinetics, biodistribution and tumor accumulation of paramagnetically labeled poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) copolymer conjugates with different molecular weights and spacers in tumor-bearing mice. Materials and Methods Paramagnetically labeled HPMA copolymer conjugates were synthesized by free radical copolymerization of HPMA with monomers containing a chelating ligand, followed by complexation with Gd(OAc)₃. A stable paramagnetic chelate, Gd-DO3A, was conjugated to the copolymers via a degradable spacer GlyPheLeuGly and a non-degradable spacer GlyGly, respectively. The conjugates with molecular weights of 28, 60 and 121 kDa and narrow molecular weight distributions were prepared by fractionation with size exclusion chromatography. The conjugates were injected into athymic nude mice bearing MDA-MB-231 human breast carcinoma xenografts via a tail vein. MR images were acquired before and at various time points after the injection with a 3D FLASH sequence and a 2D spin-echo sequence at 3T. Pharmacokinetics, biodistribution and tumor accumulation of the conjugates were visualized based on the contrast enhancement in the blood, major organs and tumor tissue at various time points. The size effect of the conjugates was analyzed among the conjugates. Results Contrast enhanced MRI resulted in a real-time, three-dimensional visualization of blood circulation, pharmacokinetics, biodistribution and tumor accumulation of the conjugates, and the size effect on these pharmaceutical properties. HPMA copolymer conjugates with high molecular weight had a prolonged blood circulation time and high passive tumor targeting efficiency. Non-biodegradable HPMA copolymers with molecular weights higher than the threshold of renal filtration demonstrated higher efficiency for tumor drug delivery than biodegradable poly(L-glutamic acid). Conclusions Contrast enhanced MRI is an effective method for non-invasive visualization of in vivo properties of the paramagnetically labeled polymer conjugates in preclinical studies.     

Water-soluble HPMA copolymer—prostaglandin E1 conjugates containing a cathepsin K sensitive spacer

A novel bone targeting, N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer based, prostaglandin E₁ (PGE₁) delivery system was designed, synthesized and characterized. PGE₁ was bound to the polymer backbone via a spacer, composed of a cathepsin K sensitive tetrapeptide (Gly-Gly-Pro-Nle) and a self-eliminating 4-aminobenzyl alcohol structure. The HPMA copolymer conjugates were prepared by photo–initiated free radical copolymerization of HPMA, PGE₁-containing macromonomer, and optionally a comonomer containing a reactive p-nitrophenyl ester group. The latter group was used as attachment points for the d-aspartic acid octapeptide targeting moieties. Incubation of the PGE₁-containing macromonomer and HPMA copolymer-PGE₁ conjugates with cathepsin K resulted in release of unmodified PGE₁. The rate of release depended on the composition of the conjugate. The higher the PGE₁ content in the conjugate, the slower the PGE₁ release. This appeared to be the result of association of hydrophobic side-chains in aqueous media, which rendered the formation of the enzyme substrate complex more difficult. The data seems to indicate that HPMA copolymer-PGE₁ conjugates have a potential in the treatment of osteoporosis and other bone diseases.     

HPMA Copolymer-1,5-Diazaanthraquinone Conjugates as Novel Anticancer Therapeutics

1,5-Diazaanthraquinones (DAQs) are promising anticancer drugs, however, their clinical potential is limited due to poor solubility. Conjugation of anticancer agents to hydrophilic water-soluble polymers can overcome this problem and has already been used to generate conjugates with demonstrated clinical benefit. Here a library of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates containing a novel amino-functionalised 1,5-diazaanthraquinone derivative (amino-DAQ) have been synthesised. The conjugates were fully characterised by UV, HPLC, SEC, FT-Raman and NMR spectroscopy. Conjugation to HPMA copolymers improved amino-DAQ aqueous solubility (>7-fold). The HPMA copolymer-amino-DAQ conjugates were slightly less haemolytic than the parent compound (2% Hb released in 1 h for conjugate HPMA copolymer-GFLG (5 mol%)-amino-DAQ conjugate compared to 13% obtained with amino-DAQ). When conjugates were incubated with isolated rat liver lysosomal enzymes (Tritosomes) the rate of amino-DAQ release was influenced by both drug loading and the composition of the peptidyl side chain used to link the drug to the carrier. The higher the drug loading the lower the rate of drug release. Whereas the GG linker did not release amino-DAQ, up to 26% of the amino-DAQ was released from a GFLG linker over 24 h. The in vitro cytotoxicity of these conjugates was evaluated against two different cell lines, B16F10 murine melanoma and MCF-7 human breast cancer cells. HPMA copolymer-amino-DAQ conjugates, which are internalised by cells by the endocytic pathway, showed much lower in vitro cytotoxicity (IC₅₀ for HPMA copolymer-GFLG (5 mol%)-amino-DAQ conjugate > 397 μM drug-equiv.) than the free drug (the IC₅₀ for amino-DAQ was 12.6 and 2.8 μM against the B16F10 murine melanoma and the MCF-7 breast cancer cell line, respectively). Nonetheless, the observed lysosomal activation of the HPMA copolymer-GFLG-amino-DAQ conjugates, suggests that evaluation of the antitumour potential in vivo is warranted.     

Molecular imaging of HPMA copolymers: Visualizing drug delivery in cell,mouse and man

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers and their drug conjugates are some of the most intensively investigated drug delivery systems for over 30 years. Some of the HPMA copolymer drug conjugates have entered clinical trials. Various molecular imaging technologies have been used to investigate the mechanism of drug delivery with HPMA copolymers. Fluorescence imaging has been used for the study of the process of intracellular drug delivery, including cell binding, subcellular trafficking and intracellular fate, of HPMA copolymers and drug conjugates. Magnetic resonance imaging and nuclear medicine, including γ-scintigraphy, SPECT and PET, have been used for the non-invasive visualization of pharmacokinetics, biodistribution and drug targeting efficiency of HPMA copolymers in animal models. γ-Scintigraphy has been used to study HPMA copolymer drug conjugates in human patients. The application of imaging technologies in the study of HPMA copolymers and properties of the copolymers demonstrated by imaging is summarized in this review.     

Water-soluble polymers for targeted drug delivery to human squamous carcinoma of head and neck

Human squamous cell carcinoma of the head and neck (SCCHN) is characterized by over expression of a tumor cell surface-specific receptor namely Hsp47/CBP2 that makes it a favorable candidate for targeted delivery of anticancer drugs. Several synthetic peptides have been identified as effective ligands for binding to CBP2. The purpose of this study is to investigate the potential of water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (Dox) conjugates containing a Hsp47/CBP2 binding peptide sequence, namely WHYPWFQNWAMA for targeted delivery to SCCHN. An HPMA copolymer containing Dox and CBP2 targeting peptide conjugated via lysosomally degradable glycylphenylalanylleucylglycine (GFLG) spacer was synthesized by free radical precipitation copolymerization. A control polymer without targeting moiety was also synthesized. The conjugates were characterized for drug content, peptide content, molecular weight and molecular weight distribution. The uptake of polymeric conjugates by both drug resistant and drug sensitive SCCHN cells were determined in vitro by flow cytometry using FACS scan analysis. Cytotoxicity of the conjugates towards drug sensitive as well as multidrug resistant SCCHN cells were evaluated by a clonal survival assay and compared to free Dox. The cytotoxicity of the free peptide was similarly evaluated. The internalization and subcellular fate of the conjugates in drug sensitive SCCHN cells was monitored using confocal microscopy. The new targetable copolymer contained 0.16 mmole peptide/g polymer. Studies on drug sensitive SCCHN cells demonstrated lesser uptake of both targeted and non-targeted conjugates compared to free Dox suggesting a slower endocytic mechanism of uptake for the conjugates as opposed to rapid diffusion of free Dox. At higher Dox equivalent concentrations (>20 μM) the targeted conjugate showed significantly higher uptake (p≤0.028) than the non-targeted conjugate. The uptake of the targeted conjugate was inhibited in the presence of an anti Hsp47 antibody suggesting the involvement of active receptor mediated endocytosis in cell entry of the conjugate. Compared to free Dox, the targeted and non-targeted conjugates caused marginally lower inhibition (p≤0.01) of the drug sensitive SCCHN cells. In contrast, the same conjugates showed significantly higher uptake (p≤0.004) by drug resistant SCCHN cells and caused significantly higher inhibition (p≤0.02) of drug resistant SCCHN cells when compared to free Dox. Results suggest that the polymeric conjugates were able to overcome drug resistance. Confocal microscopy studies demonstrated the uptake of the polymeric conjugates, followed by internalization, intralysosomal localization and subsequent release of Dox. HPMA copolymer-Dox-peptide conjugates targeted to SCCHN cells were able to overcome drug resistance and increase efficacy in vitro. The results suggest that targetable polymeric conjugates have potential to improve systemic head and neck cancer chemotherapy by increasing tumor localization and reducing dose-limiting toxicity.     

Binding and Cytotoxicity of HPMA Copolymer Conjugates to Lymphocytes Mediated by Receptor-Binding Epitopes

Purpose. Studies on the recognition of epitopes presented on a template peptide showed the potential of nonapeptide-related sequences to act as biorecognition sites for the B-cell CD21 receptor. This study was intended to evaluate the capability of three epitope sequences to mediate specific cell binding and to enhance the cytotoxicity of HPMA copolymer conjugates. Methods. HPMA copolymer conjugates were synthesized containing three different epitopes at various contents and either a fluorescent marker or doxorubicin (DOX). The binding and cytotoxicity of the conjugates to CD21⁺ Raji B cells and CD21^– HSB-2 T cells were evaluated. Results. The epitope-containing conjugates were found to bind to Raji cells at different apparent affinities depending on epitope structure and content. The conjugates generally possessed higher affinities for Raji cells than for HSB-2 cells. Targeted HPMA copolymer-DOX conjugates exhibited higher cytotoxicities than the nontargeted conjugate, likely indicative of enhanced internalization by receptor-mediated endocytosis. HSB-2 cells were more sensitive to both free and polymer-bound DOX than Raji cells; however, the enhancement of cytotoxicity of the conjugates by incorporation of epitopes was more pronounced for Raji cells. Conclusions. The results verified the concept of using receptor-binding epitopes as targeting moieties in HPMA copolymer conjugates for the delivery of anticancer drugs to lymphoma cells.     

HPMA copolymer–aminoglutethimide conjugates inhibit aromatase in MCF-7 cell lines

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer–doxorubicin (Dox) has already shown clinical activity in breast cancer patients. Moreover, we have recently found that an HPMA conjugate containing a combination of both Dox and the aromatase inhibitor aminoglutethimide (AGM) shows significantly increased anti-tumour activity in vitro. To better understand the mechanism of action of HPMA copolymer–AGM conjugates several models were used here to investigate their effect on cell growth and aromatase inhibition. Cytotoxicity of HPMA copolymer conjugates containing AGM, Dox and also the combination AGM–Dox was determined by MTT assay in MCF-7 and MCF-7ca cells. Androstenedione (5 × 10^{? 8} M) stimulates the growth of MCF-7ca cells. Both free AGM and polymer-bound AGM (0.2–0.4 mg/ml) were shown to block this mitogenic activity. When MCF-7ca cells were incubated [³H]androstenedione both AGM and HPMA copolymer–GFLG–AGM (0.2 mg/ml AGM-equiv.) showed the ability to inhibit aromatase. Although, free AGM was able to inhibit isolated human placental microsomal aromatase in a concentration dependent manner, polymer-bound AGM was not, suggesting that drug release is essential for activity of the conjugate. HPMA copolymer conjugates containing aromatase inhibitors have potential for the treatment of hormone-dependant cancers, and it would be particularly interesting to explore further as potential therapies in post-menopausal women as components of combination therapy.     

The Role of Galactose,Lactose, and Galactose Valency in the Biorecognition of N-(2-Hydroxypropyl)Methacrylamide Copolymers by Human Colon Adenocarcinoma Cells

Purpose. To examine the -galactoside and -lactoside binding capacity of three human colon-adenocarcinoma cell lines and their sugar specificity, using N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates of galactosamine, lactose, and triantennary galactose. Methods. Three types of HPMA copolymers containing the saccharide epitopes galactosamine (P-Gal), lactose (P-Lac), or triantennary galactose (P-TriGal) were synthesized. The relationship between the content of the saccharide moieties, the valency of the galactose residues, and their biorecognition by the cell lines (Colo-205, SW-480, and SW-620) was investigated using flow cytometry and confocal fluorescence microscopy analysis. Results. The binding of the glycoconjugates to the human colon-adenocarcinoma cell lines was dependent on the type and the number of bound sugar residues per macromolecule. The higher the sugar contents in the HPMA copolymers, the higher the extent of binding. Although introduction of galactoside residues into the HPMA copolymer resulted in a significant increase in the binding of the copolymers to the cells, low biorecognition of the lactoside-containing HPMA copolymers by all cell lines used was observed. The trivalent galactoside-containing HPMA copolymers did not yield a notable glycoside cluster effect for the -galactoside-binding lectin expressed on human colon-adenocarcinoma cells. Among the various cell lines, little differences in the extent of binding of the glycopolymers to the cells were observed. The data on the internalization of HPMA copolymer conjugates obtained by confocal fluorescence microscopy correlated well with the flow cytometry analysis of their biorecognition by target cells. Conclusions. The lectin-mediated endocytosis of the HPMA-glycoconjugates in human colon cancer cell lines suggests their potential use as targeting tools of cytotoxic drugs to colon adenocarcinoma.     

Combination chemotherapy and photodynamic therapy with fab' fragment targeted HPMA copolymer conjugates in human ovarian carcinoma cells

The biological activities of sequential combinations of anticancer drugs, SOS thiophene (SOS) and mesochlorin e 6 monoethylenediamine (Mce 6), in the form of free drugs, nontargeted N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-drug conjugates, P-GFLG-Mce 6 and P-GFLG-SOS (P is the HPMA copolymer backbone and GFLG is the glycylphenylalanylleucylglycine spacer), and Fab'-targeted HPMA copolymer-drug conjugates, P-(GFLG-Mce 6)-Fab' and P-(GFLG-SOS)-Fab' (Fab' from OV-TL16 antibodies complementary to CD47), were evaluated against human ovarian carcinoma OVCAR-3 cells. Mce 6, SOS, P-GFLG-Mce 6, P-GFLG-SOS, P-(GFLG-Mce 6)-Fab', and P-(GFLG-SOS)-Fab', when used as single agents or in binary combination, exhibited cytotoxic activities against OVCAR-3 cells, as determined using a modified MTT assay. The binding and internalization of P-(GFLG-Mce 6)-Fab' and P-(GFLG-SOS)-Fab' by OVCAR-3 cells were visualized by confocal microscopy and flow cytometry. The results confirmed an enhanced biorecognition by OVCAR-3 cells of Fab'-targeted HPMA copolymer conjugates over nontargeted conjugates. The median-effect analysis and the determination of the combination index (CI) were used to describe the drug interaction and quantify the synergism, antagonism, or additivity in anticancer effects. The sequential combinations of SOS+Mce 6 and P-GFLG-SOS+P-GFLG-Mce 6 displayed very strong synergism to synergism in the entire range of cell inhibition levels ( f a = 0.5 - 0.95). The P-(GFLG-SOS)-Fab'+P-(GFLG-Mce 6)-Fab' exhibited a strong synergism for f a values up to about 0.85, but showed synergistic effect and nearly additive effect at f a = 0.9 and 0.95, respectively. These observations support the continuation of in vivo investigations of these conjugates for the treatment of ovarian cancer.     

HPMA copolymer-anticancer drug-OV-TL16 antibody conjugates. 3. The effect of free and polymer-bound adriamycin on the expression of some genes in the OVCAR-3 human ovarian carcinoma cell line.

The effect of an N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-Adriamycin-OV-TLl6 antibody conjugate [P(GFLG)-ADR-Ab] on OVCAR-3 human ovarian carcinoma cells was studied. A nontargeted HPMA copolymer-ADR conjugate (P(GFLG)-ADR) and free ADR were the controls. The IC(50) doses were 0.65, 3.0, and 65 microM for free ADR, targeted P(GFLG)-ADR-Ab conjugate, and nontargeted P(GFLG)-ADR conjugate, respectively. These differences reflect the different mechanisms of cell entry of the compounds evaluated. Free ADR and HPMA copolymer-ADR conjugates had different impacts on the expression of MDR1, MRP, c-fos, c-jun, and bcl-2 genes which encode the P-glycoprotein (MDR1) and the multidrug resistance-associated protein (MRP) efflux pumps, and play an important role in cell death signaling pathways (c-fos, c-jun, and bcl-2). Whereas high doses of free ADR induced MDR1 gene expression, HPMA copolymer-bound ADR appeared to be without effect. On the contrary, expression of the MRP gene was not influenced by free ADR, whereas HPMA copolymer-ADR conjugates seemed to suppress the gene expression in a concentration-dependent manner. There were differences in the expression of c-fos, c-jun, and bcl-2 genes after the incubation of OVCAR-3 cells with free and HPMA copolymer-bound ADR indicating differences in activation of cell death signaling pathways.     

Influence of the Structure of Drug Moieties on the in Vitro Efficacy of HPMA Copolymer–Geldanamycin Derivative Conjugates

Purpose To optimize the structure of geldanamycin (GDM) derivative moieties attached to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers via an enzymatically degradable spacer. Methods HPMA copolymers containing different AR-GDM (AR = 3-aminopropyl (AP), 6-aminohexyl (AH), and 3-amino-2-hydroxy-propyl AP(OH)) were synthesized and characterized. Their cytotoxicity towards the A2780 human ovarian carcinoma cells was evaluated. Results The cytotoxic efficacy of HPMA copolymer-AR-GDM conjugates depended on the structure of AR-GDM. Particularly, HPMA copolymer-bound AH-GDM, which possessed the longest substituent at the 17-position, demonstrated the highest efficacy among the polymer-bound GDM derivatives; however the activity of free AH-GDM was lower than that of the other free AR-GDMs. The relative increase of the activity of macromolecular AH-GDM when compared to AP-GDM or AP(OH)-GDM correlated with the enhanced recognition of AH-GDM terminated oligopeptide side-chains by the active site of the lysosomal enzyme, cathepsin B Drug stability and further stabilization upon binding to HPMA copolymer also contributed to the observed phenomena. Conclusion AH-GDM was found to be a suitable GDM derivative for the design of a drug delivery system based on HPMA copolymers and enzymatically-degradable spacers.     

Spacer length impacts the efficacy of targeted docetaxel conjugates in prostate-specific membrane antigen expressing prostate cancer

Abstract

Combination of targeted delivery and controlled release is a powerful technique for cancer treatment. In this paper, we describe the design, synthesis, structure validation and biological properties of targeted and non-targeted N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-docetaxel conjugates. Docetaxel (DTX) was conjugated to HPMA copolymer via a tetrapeptide spacer (–GFLG-). 3-(1,3-dicarboxypropyl)-ureido]pentanedioic acid (DUPA) was used as the targeting moiety to actively deliver DTX for treatment of Prostate-Specific Membrane Antigen (PSMA) expressing prostate cancer. Short and long spacer DUPA monomers were prepared, and four HPMA copolymer – DTX conjugates (non-targeted, two targeted with short spacer of different molecular weight and targeted with long spacer) were prepared via Reversible Addition-Fragmentation Chain Transfer (RAFT) copolymerization. Following confirmation of PSMA expression on C4-2 cell line, the DTX conjugates’ in vitro cytotoxicity was tested against C4-2 tumor cells and their anticancer efficacies were assessed in nude mice bearing s.c. human prostate adenocarcinoma C4-2 xenografts. The in vivo results show that the spacer length between targeting moieties and HPMA copolymer backbone can significantly affect the treatment efficacy of DTX conjugates against C4-2 tumor bearing nu/nu mice. Moreover, histological analysis indicated that the DUPA-targeted DTX conjugate with longer spacer had no toxicity in major organs of treated mice.     

Development of HPMA copolymer–anticancer conjugates: Clinical experience and lessons learnt

The concept of polymer–drug conjugates was proposed more than 30 years ago, and an N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugate of doxorubicin covalently bound to the polymer backbone by a Gly-Phe-Leu-Gly peptidyl linker (FCE28068) became the first synthetic polymer-based anticancer conjugate to enter clinical trial in 1994. This conjugate arose from rational design attempting to capitalise on passive tumour targeting by the enhanced permeability and retention effect and, at the cellular level, lysosomotropic drug delivery to improve therapeutic index. Early clinical results were promising, confirming activity in chemotherapy refractory patients and the safety of HPMA as a new polymer platform. Subsequent Phase I/II trials have investigated an HPMA copolymer-based conjugate containing a doxorubicin and additionally galactose as a targeting moiety to promote liver targeting (FCE28069), and also HPMA copolymer conjugates of paclitaxel (PNU 166945), camptothecin (PNU 166148) and two platinates (AP5280 and AP5346- ProLindac™). The preclinical and clinical observations made in these, and clinical studies with other polymer conjugates, should shape the development of next generation anticancer polymer therapeutics.     

Targeted Delivery of Doxorubicin by HPMA Copolymer-Hyaluronan Bioconjugates

Purpose. Overexpression of hyaluronan (HA) receptors on cancer cells results in enhanced endocytotic uptake of the drug conjugate. An N-(2-hydroxypropyl)methacrylamide (HPMA)-HA polymeric drug delivery system was used for targeted delivery of doxorubicin to cancer cells. Methods. HA-doxorubicin (DOX) bioconjugates (HA-DOX), and HPMA copolymer-DOX conjugates containing HA as a side chain (HPMA-HA-DOX) were synthesized. The cytotoxicity of the polymer-drug conjugate was evaluated via in vitro cell culture. The internalization of the conjugate was visualized by fluorescence microscopy. Results. Cytotoxicity of HPMA-HA-DOX targeted bioconjugate was higher against human breast cancer (HBL-100), ovarian cancer (SKOV-3), and colon cancer (HCT-116) cells when compared to the non-targeted HPMA-DOX conjugate. Fluorescence confocal microscopy revealed that the targeted HPMA-HA-DOX conjugates were internalized more efficiently by cancer cells relative to the non-targeted HPMA-DOX conjugate. Both HPMA-DOX and HPMA-HA-DOX showed minimal cytotoxicity toward mouse fibroblast NIH 3T3 cells. The internalization of polymer conjugates was correlated with their cytotoxicity. Conclusions. Selective delivery of anti-cancer agents to cancer cells was achieved by biochemical targeting. The HA-modified HPMA copolymer showed improved toxicity due to receptor-mediated uptake of the macromolecular drug.     

Biodistribution and pharmacokinetic studies of bone-targeting N-(2-hydroxypropyl)methacrylamide copolymer-alendronate conjugates

The biodistribution and pharmacokinetics of bone-targeting N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-alendronate conjugates were evaluated following intravenous administration of radioiodinated conjugates to young healthy BALB/c mice. The synthesis of a polymerizable and cathepsin K cleavable alendronate derivative, N-methacryloylglycylglycylprolylnorleucylalendronate, enabled the preparation of HPMA copolymer-alendronate conjugates with varying composition. Using the RAFT (reversible addition-fragmentation chain transfer) polymerization technique, four conjugates with different molecular weight and alendronate content and two control HPMA copolymers (without alendronate) with different molecular weight were prepared. The results of biodistribution studies in mice demonstrated a strong binding capacity of alendronate-targeted HPMA copolymer conjugates to bone. Conjugates with low (1.5 mol%) alendronate content exhibited a similar bone deposition capacity as conjugates containing 8.5 mol % of alendronate. The molecular weight was an important factor in the biodistribution of the HPMA copolymer conjugates. More conjugate structures need to be evaluated, but the data suggest that medium molecular weights (50-100 kDa) might be effective drug carriers for bone delivery.