Antisense oligonucleotides delivered to the lysosome escape and actively inhibit the hepatitis B virus

The subcellular fate and activity in inhibiting the hepatitis B virus of free and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-phosphorothioate oligonucleotides were studied. Their internalization and subcellular fate were monitored with confocal microscopy. A fraction of the internalized free oligonucleotides escaped into the cytoplasm and nucleus of Hep G2 cells but were not active antiviral agents. Covalently attaching the oligonucleotides to the HPMA copolymers via nondegradable dipeptide GG spacers resulted in sequestering the oligonucleotides in vesicles after internalization. Conjugation of the oligonucleotides to an HPMA copolymer via a lysosomally cleavable tetrapeptide GFLG spacer resulted in release of the oligonucleotide in the lysosome and subsequent translocation into the cytoplasm and nucleus of the cells. The HPMA copolymer-oligonucleotide conjugate possessed antiviral activity, indicating that phosphorothioate oligonucleotides released from the carrier in the lysosome were able to escape into the cytoplasm and nucleus and remain active. The Hep G2 cells appeared to actively internalize the phosphorothioate oligonucleotides as oligonucleotide-HPMA copolymer conjugates were internalized to a greater extent than unconjugated polymers.     

Evaluation of protein-N-(2-hydroxypropyl)methacrylamide copolymer conjugates as targetable drug carriers. 1. Binding, pinocytic uptake and intracellular distribution of transferrin and anti-transferrin receptor antibody conjugates

The transferrin receptor of human skin fibroblasts was studied as an in vitro model target antigen receptor for interaction with protein-polymer conjugates having potential for targeted drug delivery. Pinocytic uptake of 125I-labelled N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugated to monoclonal antibody B3/25 (specific for the transferrin receptor) or transferrin was up to 9-fold greater than uptake of the parent HPMA copolymer. The ability of these conjugates to bind specifically was confirmed by Scatchard analysis. Pinocytic internalisation was dependent on the molecular mass of the conjugate. Intracellular routing following internalisation was evaluated using density-gradient centrifugation. Unmodified HPMA copolymer was transferred via the endosomal compartment into secondary lysosomes, where, being resistant to degradation, it accumulated. Although the majority of endocytosed transferrin is recycled via the endosome, it was shown that any transferrin reaching the lysosomes was rapidly degraded and low-molecular-weight degradation products were released. Monoclonal antibody B3/25 showed a subcellular distribution consistent with prolongation on the cell surface, followed by internalisation and subcellular trafficking, via endosomes, into the lysosomal compartment, with subsequent degradation. Conjugation of protein to HPMA copolymer increased lysosomal accumulation of polymer up to 9-fold, with no detectable degradation of conjugate. The data presented here have implications regarding clinical potential of protein-HPMA copolymer conjugates designed for lysosomotropic drug delivery.     

Synthesis of bioadhesive lectin-HPMA copolymer-cyclosporin conjugates

An amino group containing cyclosporin A (CsA) derivative has been synthesized and conjugated to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer via an aromatic azo bond, which can be specifically cleaved by azoreductase activity in colon to release the drug for the treatment of colon diseases. Lectins, peanut (Arachis hypogea) agglutinin (PNA) and wheat germ agglutinin (WGA), have been conjugated to HPMA copolymer-CsA derivative conjugates (PCsA), respectively, to give bioadhesive conjugates. The PNA and WGA are the targeting proteins that can bind to diseased colon tissue and healthy tissue, respectively. There were on average four P(CsA) copolymer chains attached on one WGA molecule with a drug content of 16.0 wt % and five P(CsA) copolymer chains attached on one PNA molecule with a drug content of 11.5 wt %. The incubation of a P(CsA) copolymer with the rat cecal contents resulted in the cleavage of the azo bond and release of the cyclosporin derivative. The biological evaluation of the conjugates is under way.     

Tat-conjugated synthetic macromolecules facilitate cytoplasmic drug delivery to human ovarian carcinoma cells

We have synthesized N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-cell penetrating peptide Tat conjugates and evaluated their subcellular distribution in A2780 human ovarian carcinoma cells by confocal fluorescence microscopy and subcellular fractionation. Our data indicate the transport of these conjugates by a single Tat molecule to both the cytoplasm and nucleus via a nonendocytotic and concentration independent process. The uptake was observed to occur within 3 min, as confirmed by live cell microscopy. In contrast, HPMA copolymers lacking the Tat peptide were internalized solely by endocytosis. For the first time, Tat-mediated cytoplasmic delivery of a polymer bound anticancer drug, doxorubicin, was also demonstrated. These findings establish the feasibility of overcoming major cellular and subcellular obstacles to intracellular macromolecular delivery and hold great promise for the development of polymer-based systems for the cytoplasmic delivery of therapeutic molecules.     

N-(2-hydroxypropyl)methacrylamide copolymer-6-(3-aminopropyl)-ellipticine conjugates. Synthesis, in vitro, and preliminary in vivo evaluation

Ellipticine derivatives have potential as anticancer drugs. Their clinical use has been limited, however, by poor solubility and host toxicity. As N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-anticancer conjugates are showing promise in early clinical trials, a series of novel HPMA copolymer conjugates have been prepared containing the 6-(3-aminopropyl)-ellipticine derivative (APE, NSC176328). Drug was linked to the polymer via GFLG or GG peptide side chains. To optimize biological behavior, HPMA copolymer-GFLG-APE conjugates with different drug loading (total APE: 2.3-7% w/w; free APE: <0.1% w/w) were synthesized. Conjugation of APE to HPMA copolymers considerably increased its aqueous solubility (>10-fold). HPMA copolymer-GG-APE did not liberate drug in the presence of isolated lysosomal enzymes (tritosomes), but HPMA copolymer-GFLG-APE released APE to a maximum of 60% after 5 h. The rate of drug release was influenced by drug loading; lower loading led to greater release. Whereas free APE (35 microg/mL) caused significant hemolysis (50% after 1 h), HPMA copolymer-APE conjugates were not hemolytic up to 300 microg/mL (APE-equiv). As would be expected from its cellular pharmacokinetics, HPMA copolymer-GFLG-APE was >75 times less cytotoxic than free drug (IC(50) approximately 0.4 microg/mL) against B16F10 melanoma in vitro. However, in vivo when tested in mice bearing s.c. B16F10 melanoma, HPMA copolymer-GFLG-APE (1-10 mg/kg single dose, APE-equiv) given i.p. was somewhat more active (highest T/C value of 143%) than free APE (1 mg/kg) (T/C =127%). HPMA copolymer-APE conjugates warrant further evaluation as potential anticancer agents.     

Enhanced biorecognition and internalization of HPMA copolymers containing multiple or multivalent carbohydrate side-chains by human hepatocarcinoma cells.

N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing pendant saccharide moieties (galactosamine, lactose, and triantennary galactose) were synthesized. The relationship between the content of saccharide moieties and three-dimensional arrangement of galactose residues and their biorecognition and internalization by human hepatocarcinoma HepG2 cells was investigated. The results obtained clearly indicated preferential binding of the trivalent galactose and the lactose-containing copolymers to these cells. The higher the saccharide moieties content in HPMA copolymers, the higher the levels of binding. The biorecognition of the glycosylated HPMA copolymers by HepG2 cells was inhibited by free lactose. The data on the internalization and subcellular trafficking of HPMA copolymer conjugates obtained by confocal fluorescence microscopy correlated well with the flow cytometric analysis of their biorecognition by target cells. Structural features of the glycosides responsible for the specific recognition of the HPMA copolymers have been identified. The results underline the potential of glycosylated HPMA copolymers for delivery of pharmaceutical agents to hepatocarcinoma cells.     

Macromolecular HPMA-Based Nanoparticles with Cholesterol for Solid-Tumor Targeting: Detailed Study of the Inner Structure of a Highly Efficient Drug Delivery System

We report a rigorous investigation into the detailed structure of nanoparticles already shown to be successful drug delivery nanocarriers. The basic structure of the drug conjugates consists of an N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer bearing the anticancer drug doxorubicin (Dox) bound via a pH-sensitive hydrazone bond and a defined amount of cholesterol moieties that vary in hydrophobicity. The results show that size, anisotropy, and aggregation number N(aggr) of the nanoparticles grows with increasing cholesterol content. From ab initio calculations, we conclude that the most probable structure of HPMA copolymer-cholesterol nanoparticles is a pearl necklace structure, where ellipsoidal pearls mainly composed of cholesterol are covered by a HPMA shell; pearls are connected by bridges composed of hydrophilic HPMA copolymer chains. Using a combination of techniques, we unambiguously show that the Dox moieties are not impregnated inside a cholesterol core but are instead uniformly distributed across the whole nanoparticle, including the hydrophilic HPMA shell surface.     

Identification of CD21-binding peptides with phage display and investigation of binding properties of HPMA copolymer-peptide conjugates

Cancer targeting with peptides has become promising with the emergence of combinatorial peptide techniques such as phage display. Using phage display under stringent screening conditions, we selected five distinct peptides that specifically recognized the CD21 receptor, a cell surface marker of malignant B cell lymphoma. Two highly hydrophobic sequences were excluded (RLAYWCFSGLFLLVC and PVAAVSFVPYLVKTY). The binding affinity toward CD21 of the other three selected peptides (RMWPSSTVNLSAGRR, PNLDFSPTCSFRFGC, and GRVPSMFGGHFFFSR) was analyzed with fluorescence quenching. Their dissociation constants were determined to be within the micromolar range. On the basis of the results of phage ELISA, competitive phage ELISA, and fluorescence quenching, the binding sites of the three selected peptides were found to reside within the first four short consensus repeats of CD21 (SCR1-4). The peptide RMWPSSTVNLSAGRR (P1) was bound to the N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer, a potential drug carrier for chemotherapeutic agents, and the surface binding properties of HPMA copolymer-P1 conjugates were investigated. Specific interactions were observed between HPMA copolymer-P1 conjugates and surface-bound receptor. Binding of HPMA copolymer-P1 conjugates was directly related to the amount of surface (MaxiSorp plate) bound receptor, and the binding of the conjugates could be inhibited by the application of a 3-4 orders-of-magnitude excess of free peptide over the peptide concentration in conjugates. The enhanced binding of polymer-bound peptide was ascribed to multivalent interactions between the HPMA copolymer-P1 conjugate and the surface-bound CD21 receptor.     

Antiproliferative effect of a lectin- and anti-Thy-1.2 antibody-targeted HPMA copolymer-bound doxorubicin on primary and metastatic human colorectal carcinoma and on human colorectal carcinoma transfected with the mouse Thy-1.2 gene

The aim of this study was to compare the potential of two plant lectins [peanut agglutinin (PNA) and wheat germ agglutinin (WGA)], monoclonal antibody (anti-Thy-1.2), its F(ab')(2) fragments, and galactosamine as targeting moieties bound to the polymer drug carrier to deliver a xenobiotic, doxorubicin, to selected cancer cell lines. We have used primary (SW 480, HT 29) and metastatic (SW 620) human colorectal cancer cell lines and a transfectant, genetically engineered SW 620 cell line with mouse gene Thy-1.2 (SW 620/T) to test the possibility of marking human cancer with xenogeneic mouse gene and use it for effective site-specific targeting. The targeting moieties and doxorubicin were conjugated to a water-soluble copolymer based on N-(2-hydroxypropyl)methacrylamide (HPMA) acting as a carrier responsible for controlled intracellular release of the targeted drug. FACS analysis showed a strong binding of WGA-FITC to all tested cell lines. Binding of PNA-FITC was considerably weaker. The in vitro antiproliferative effect of lectin-targeted HPMA carrier-bound doxorubicin evaluated as [(3)H]TdR incorporation reflected both the intensity of the binding and the different sensitivity of the tested cancer cells lines to doxorubicin. The antiproliferative effect of conjugates targeted with WGA was comparable to that with the conjugates targeted with the anti-Thy-1.2 monoclonal antibody or their F(ab')(2) fragments. The magnitude of the cytotoxic effect of HPMA-doxorubicin targeted with PNA was lower in all tested cell lines. While the conjugates with WGA were more cytotoxic, the conjugates with PNA were more specific as their binding is limited to cancer cells and to the sites of inflammation. Noncytotoxic conjugates with a very low concentration of doxorubicin and targeted with PNA, anti-Thy-1.2, or their F(ab')(2) fragments exerted in some lines (SW 480, SW 620) low mitogenic activity. The Thy-1.2 gene-transfected SW 620 metastatic colorectal cancer cell line was sensitive to the antiproliferative effect of Thy-1.2-targeted doxorubicin as was shown for the Thy-1. 2(+) EL4 cell line and for Thy-1.2(+) concanavalin A-stimulated mouse T lymphocytes. These results represent the first indication of the suitability of transfection of human cancer cells with selected targeting genes for site-specific therapy of malignancies.     

Polymerizable Fab' antibody fragments for targeting of anticancer drugs.

We have designed a new pathway for the synthesis of targeted polymeric drug delivery systems, using polymerizable antibody Fab' fragments (MA-Fab'). The targeted systems can be directly prepared by copolymerization of the MA-Fab', N-(2-hydroxypropyl)methacrylamide (HPMA) and drug-containing monomers. Both MA-Fab' and the Fab'-targeted copolymers can effectively bind to target cells. An MA-Fab' (from OV-TL 16 Ab) targeted HPMA copolymer containing mesochlorin e6 (Mce6) was synthesized by copolymerization of MA-Fab', HPMA, and MA-GFLG-Mce6. The targeted copolymer exhibited a higher cytotoxicity toward OVCAR-3 human ovarian carcinoma cells than the nontargeted Mce6-containing copolymer or free Mce6. The targeted copolymer was internalized more efficiently by OVCAR-3 cells than the nontargeted copolymer.     

Prostate-cancer-targeted N-(2-hydroxypropyl)methacrylamide copolymer/docetaxel conjugates

Biodistribution, pharmacokinetics, and efficacy of prostate-cancer-targeted HPMA copolymer/DTX conjugates are evaluated in nude mice bearing prostate cancer C4-2 xenografts. PSMA-specific monoclonal antibodies 3F/11 are used as the targeting moiety. Control conjugates tumor accumulation to total background organs (heart, lung, kidney, liver, spleen and blood) accumulation increase substantially with time for the targeted conjugate, and the ratio at 48 h is 7-fold higher than that at 6 h. Preliminary evaluation of the efficacy of the conjugates in vivo show tumor growth inhibition for all HPMA copolymer/DTX conjugates.     

P-glycoprotein-mediated colchicine resistance in different cell lines correlates with the effects of colchicine on P-glycoprotein conformation

The multidrug transporter P-glycoprotein (Pgp) is an ATPase efflux pump for multiple cytotoxic agents, including vinblastine and colchicine. We have found that resistance to vinblastine but not to colchicine in cell lines derived from different types of tissues and expressing the wild-type human Pgp correlates with the Pgp density. Vinblastine induces a conformational change in Pgp, evidenced by increased reactivity with a conformation-sensitive monoclonal antibody UIC2, in all the tested cell lines. In contrast, colchicine increases the UIC2 reactivity in only some of the cell lines. In those lines where colchicine alone did not affect UIC2 reactivity, this drug was, however, able to reverse the vinblastine-induced increase in UIC2 reactivity. The magnitude of the increase in UIC2 reactivity in the presence of saturating concentrations of colchicine correlates with the relative ability of Pgp to confer colchicine resistance in different cell lines, suggesting the existence of some cell-specific factors that have a coordinate effect on the ability of colchicine to induce conformational transitions and to be transported by Pgp. Colchicine, like vinblastine, reverses the decrease in UIC2 reactivity produced by nonhydrolyzable nucleotides, but unlike vinblastine, it does not reverse the effect of ATP at a high concentration. Colchicine, however, decreases the Hill number for the effect of ATP on the UIC2 reactivity from 2 to 1. Colchicine increases the UIC2 reactivity and reverses the effect of ATP in ATPase-deficient Pgp mutants, but not in the wild-type Pgp expressed in the same cellular background, suggesting that ATP hydrolysis counteracts the effects of colchicine on the Pgp conformation.     

Two-step fluorescence screening of CD21-binding peptides with one-bead one-compound library and investigation of binding properties of N-(2-hydroxypropyl)methacrylamide copolymer-peptide conjugates

Using the one-bead one-compound (OBOC) combinatorial method, four heptapeptide ligands of CD21 receptor, a cell surface marker of malignant B cell lymphoma, were identified with an innovative two-step fluorescence screening method to overcome the limitation caused by autofluorescence of TentaGel resin. The binding affinities of selected peptides, YILIHRN (B1), PTLDPLP (B2), and LVLLTRE (B3), were in the micromolar region as determined by a fluorescence quenching assay. Peptide B1 was conjugated to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer via spacers of different lengths, composed of one to four repeats of the 8-amino-3,6-dioxaoctanoic acid (A) group. The evaluation of the biorecognizability of HPMA copolymer-B1 conjugates by the CD21 receptor revealed that increasing the number of repeats of A in the spacer from one to three resulted in continuous improvements in the biorecognition by the CD21 receptor; the increase from three to four repeats showed no significant effect. This work showed the potential of the OBOC combinatorial approach to select peptide ligands as targeting moieties for CD21 specific polymeric drug carriers.     

Star structure of antibody-targeted HPMA copolymer-bound doxorubicin: a novel type of polymeric conjugate for targeted drug delivery with potent antitumor effect

The aim of this study was to compare the properties and antitumor potential of a novel type of antibody-targeted N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound doxorubicin conjugates with star structure with those of previously described classic antibody-targeted or lectin-targeted HPMA copolymer-bound doxorubicin conjugates. Classic antibody-targeted conjugates were prepared by aminolytic reaction of the multivalent HPMA copolymer containing side-chains ending in 4-nitrophenyl ester (ONp) reactive groups with primary NH(2) groups of the antibodies. The star structure of antibody-targeted conjugates was prepared using semitelechelic HPMA copolymer chains containing only one reactive N-hydroxysuccinimide group at the end of the backbone chain. In both types of conjugates, B1 monoclonal antibody (mAb) was used as a targeting moiety. B1 mAb recognizes the idiotype of surface IgM on BCL1 cells. The star structure of the targeted conjugate had a narrower molecular mass distribution than the classic structure. The peak in the star structure was around 300-350 kDa, while the classic structure conjugate had a peak around 1300 kDa. Doxorubicin was bound to the HPMA copolymer via Gly-Phe(D,L)-Leu-Gly spacer to ensure the controlled intracellular delivery. The release of doxorubicin from polymer conjugates incubated in the presence of cathepsin B was almost twice faster from the star structure of targeted conjugate than from the classic one. The star structure of the targeted conjugate showed a lower binding activity to BCL1 cells in vitro, but the cytostatic activity measured by [(3)H]thymidine incorporation was three times higher than that seen with the classic conjugate. Cytostatic activity of nontargeted and anti-Thy 1.2 mAb (irrelevant mAb) modified HPMA copolymer-bound doxorubicin was more than hundred times lower as compared to the star structure of B1 mAb targeted conjugate. In vivo, both types of conjugates targeted with B1 mAb bound to BCL1 cells in the spleen with approximately the same intensity. The classic structure of the targeted conjugate bound to BCL1 cells in the blood with a slightly higher intensity than the star structure. Both types of targeted conjugates had a much stronger antitumor effect than nontargeted HPMA copolymer-bound doxorubicin and free doxorubicin. The star structure of targeted conjugate had a remarkably higher antitumor effect than the classic structure: a single intravenous dose of 100 microg of doxorubicin given on day 11 completely cured five out of nine experimental animals whereas the classic structure of targeted conjugate given in the same schedule only prolonged the survival of experimental mice to 138% of control mice. These results show that the star structure of antibody-targeted HPMA copolymer-bound doxorubicin is a suitable conjugate for targeted drug delivery with better characterization, higher cytostatic activity in vitro, and stronger antitumor potential in vivo than classic conjugates.     

Improved mitochondrial targeting effect of HPMA copolymer by SS20 peptide mediation and nonendocytosis pathway

Mitochondrion plays an important role in executing cell programmed death pathway. Therefore, drugs designed to target mitochondria are supposed to make superior contributions to cancer therapy. However, the problem that drugs or drug delivery systems being sequestrated in endosomes/lysosomes needs to be solved for effective drug delivery. Here, mitochondrial targeting and nonendocytic cell entry peptide SS20 modified HPMA copolymer (P‐FITC‐SS20) was synthesized. With SS20 peptide modification, the uptake behavior of HPMA copolymers changed remarkably compared with unmodified ones. The internalization of P‐FITC‐SS20 was not influenced by endocytic inhibitors and temperature. Further, the internalized copolymers were not trapped in endosomes/lysosomes. Although cellular uptake of HPMA copolymer was decreased after SS20 peptide modification, SS20 peptide significantly improved mitochondrial accumulation of HPMA copolymers due to its outstanding mitochondrial targeting ability. Moreover, owing to lower susceptibility to macrophagocyte in blood, P‐SS20‐Cy5 showed longer blood circulation time and enhanced tumor accumulation. The current study validated that SS20 peptide modification is a promising strategy for mitochondrial targeting drug delivery systems and can be further applied to mitochondria associated diseases to improve therapeutic efficacy.     

Backbone degradable multiblock N-(2-hydroxypropyl)methacrylamide copolymer conjugates via reversible addition-fragmentation chain transfer polymerization and thiol-ene coupling reaction

Telechelic water-soluble HPMA copolymers and HPMA copolymer-doxorubicin (DOX) conjugates have been synthesized by RAFT polymerization mediated by a new bifunctional chain transfer agent (CTA) that contains an enzymatically degradable oligopeptide sequence. Postpolymerization aminolysis followed by chain extension with a bis-maleimide resulted in linear high molecular weight multiblock HPMA copolymer conjugates. These polymers are enzymatically degradable; in addition to releasing the drug (DOX), the degradation of the polymer backbone resulted in products with molecular weights similar to the starting material and below the renal threshold. The new multiblock HPMA copolymers hold potential as new carriers of anticancer drugs.     

Dynamic light scattering study of self-assembly of HPMA hybrid graft copolymers

The time course of self-assembly of a hybrid hydrogel system was investigated using dynamic light scattering (DLS) techniques. The self-assembling system consisted of a hydrophilic synthetic N-(2-hydroxypropyl)methacrylamide (HPMA) polymer backbone and a pair of oppositely charged peptide grafts (CCE and CCK). These two distinct pentaheptad peptides were anticipated to act as physical cross-linkers by the formation of antiparallel coiled-coil heterodimers. Equimolar mixture of HPMA graft copolymers CCE-P and CCK-P solutions (where P is the HPMA copolymer backbone) with total concentration from 1.25 to 10 mg/mL were measured at a scattering angle 90 degrees and room temperature. A critical extension of average relaxation time was observed with increasing concentration and incubation time. To reveal the role of coiled-coil grafts in the self-assembly process, a pair of modified random coil peptides, CCEw and CCKy, was designed. The DLS evaluation of HPMA copolymer conjugates (CCEw-P and CCKy-P) at total concentration of 10 mg/mL demonstrated that no association occurred after 28 h of incubation. Moreover, addition of a competing peptide (CCK) or a denaturant (guanidium chloride, GndHCl) to the self-assembled CCE-P/CCK-P hydrogels resulted in partial disassembly or collapse of the hydrogel clusters. These results correlated to changes in the secondary structure of peptides (grafts) as measured by circular dichroism spectroscopy (CD). These investigations supported the hypothesis that the self-assembly of CCE-P/CCK-P into hybrid hydrogels is mediated by the formation of coiled-coil heterodimers.     

Using small-angle neutron scattering to study the solution conformation of N-(2-hydroxypropyl)methacrylamide copolymer-doxorubicin conjugates

Our past research developed two N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (Dox) conjugates that became the first synthetic polymer-anticancer conjugates to be evaluated clinically. The first, FCE28068, contained Dox bound to the polymeric carrier via a tetrapeptidic linker (glycine-phenylalanine-leucine-glycine (GFLG)) (Mw approximately 30,000 g/mol; approximately 8 wt % drug), and the second, FCE28069, contained additionally galactosamine (Gal) (Mw approximately 30,000 g/mol; approximately 7.5 wt % Dox) again bound by a GFLG linker. Galactosamine was included to promote hepatocyte/hepatoma targeting via the asialoglycoprotein receptor. Both conjugates showed antitumor activity and were clinically less toxic than free Dox (2-5 fold). However, despite their similar chemical characteristics, the conjugates displayed a significantly different maximum-tolerated dose (MTD) in patients. The aim of this study, therefore, was to use small-angle neutron scattering (SANS) to explore the solution behavior of a small library of HPMA polymer conjugates including FCE28068, FCE28069, and their pharmaceutical formulations, plus as reference compounds HPMA copolymer-GFLG conjugates containing aminopropanol (Ap) or galactosamine (Gal) alone (i.e., without Dox). The SANS data obtained showed that HPMA copolymer-GFLG-Ap conjugates (containing 5 and 10 mol % side chains) showed evidence of polymer aggregation, however, no indication of aggregation was observed for FCE28068 and FCE28069 over the concentration range studied (2.5-50 mg/mL). Clear differences in the scattering behavior for the two conjugates were observed at equivalent concentration. Data were best fitted by a model for polydisperse Gaussian coils, and the HPMA copolymer-Dox conjugate with Gal (FCE28069) exhibited a larger radius of gyration (Rg) (by approximately 2.5 nm) compared to FCE28068. In conclusion, we have shown that SANS will be a valuable tool to elucidate conformation-performance relationships for polymer-drug conjugates.     

The Strong In Vivo Anti-Tumor Effect of the UIC2 Monoclonal Antibody Is the Combined Result of Pgp Inhibition and Antibody Dependent Cell-Mediated Cytotoxicity

P-glycoprotein (Pgp) extrudes a large variety of chemotherapeutic drugs from the cells, causing multidrug resistance (MDR). The UIC2 monoclonal antibody recognizes human Pgp and inhibits its drug transport activity. However, this inhibition is partial, since UIC2 binds only to 10–40% of cell surface Pgps, while the rest becomes accessible to this antibody only in the presence of certain substrates or modulators (e.g. cyclosporine A (CsA)). The combined addition of UIC2 and 10 times lower concentrations of CsA than what is necessary for Pgp inhibition when the modulator is applied alone, decreased the EC₅₀ of doxorubicin (DOX) in KB-V1 (Pgp⁺) cells in vitro almost to the level of KB-3-1 (Pgp^-) cells. At the same time, UIC2 alone did not affect the EC₅₀ value of DOX significantly. In xenotransplanted severe combined immunodeficient (SCID) mice co-treated with DOX, UIC2 and CsA, the average weight of Pgp⁺ tumors was only ∼10% of the untreated control and in 52% of these animals we could not detect tumors at all, while DOX treatment alone did not decrease the weight of Pgp⁺ tumors. These data were confirmed by visualizing the tumors in vivo by positron emission tomography (PET) based on their increased ¹⁸FDG accumulation. Unexpectedly, UIC2+DOX treatment also decreased the size of tumors compared to the DOX only treated animals, as opposed to the results of our in vitro cytotoxicity assays, suggesting that immunological factors are also involved in the antitumor effect of in vivo UIC2 treatment. Since UIC2 binding itself did not affect the viability of Pgp expressing cells, but it triggered in vitro cell killing by peripheral blood mononuclear cells (PBMCs), it is concluded that the impressive in vivo anti-tumor effect of the DOX-UIC2-CsA treatment is the combined result of Pgp inhibition and antibody dependent cell-mediated cytotoxicity (ADCC).     

P-Glycoprotein conformational changes detected by antibody competition.

Conformational changes accompanying P-glycoprotein (Pgp) mediated drug transport are reflected by changes in the avidity of certain monoclonal antibodies (mAbs). More of the UIC2 mAb binds to Pgp-expressing cells in the presence of substrates or modulators [Mechetner, E.B., Schott, B., Morse, S.B., Stein, W., Druley, T., Dvis, K.A., Tsuruo, T. & Roninson, I.B. (1997) Proc. Natl Acad. Sci. USA 94, 12908-12913], while the binding of other mAbs (e.g. MM12.10, MRK16, 4E3) is not conformation sensitive. Pre-staining of Pgp+ cells with UIC2 decreased the subsequent binding of MM12.10 mAb by about 30-40%, suggesting that there are Pgp molecules available for both UIC2 and MM12.10, and others accessible only for MM12.10. In the presence of certain substrates/modulators such as vinblastin, cyclosporin A or valinomycin, the MM12.10 reactivity was completely abolished by preincubation with UIC2. However, verapamil, Tween-80 and nifedipine did not influence the ratio of bound mAbs significantly. This is the first assay to our knowledge, sharply distinguishing two classes of modulators. The conformational changes accompanying the mAb competition phenomenon appear to be closely related, though not identical to those accompanying the UIC2-shift, as suggested by the simultaneous assessment of the two phenomena.