Properties of HPMA copolymer-doxorubicin conjugates with pH-controlled activation: effect of polymer chain modification.

Various conjugates of anticancer drug doxorubicin (DOX) covalently attached via hydrolytically degradable hydrazone bond to water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer drug carriers were synthesized. Three types of precursors containing either positively or negatively charged groups or a hydrophobic substituent were employed. In vitro incubation of the conjugates in buffers showed relative stability at pH 7.4 (modelling blood) and a fast DOX release at pH 5 (modelling intracellular environment). The presence of carboxylic groups in the copolymer structure resulted in an increase in the DOX release rate of 15-20% while no effect of the introduction of positively charged groups was observed if compared with the unmodified conjugate. Self-assembling of the oleoyl groups-containing conjugate led into formation of polymeric micelles with high apparent molecular weight (M(w)=170,000) in aqueous solution and resulted in a decrease in the DOX release rate of approximately 20%. The cytostatic activity of the conjugates tested on several cancer cell lines was comparable with that of free DOX.HCl, depending on the sensitivity of a particular cell line to DOX. All the conjugates showed a much higher antitumour activity in vivo than the free drug tested in mice bearing EL4 T-cell lymphoma and treated using the therapeutic regime of drug administration. The highest activity (100% long-term survivors) exhibited polymer-DOX conjugate containing negatively charged GFLG sequences.     

HPMA copolymer-bound doxorubicin targeted to tumor-specific antigen of BCL1 mouse B cell leukemia.

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer carrier containing the anticancer drug doxorubicin and targeted with B1 monoclonal antibody (mAb) to BCL1 leukemia cells was synthesised and tested in vitro and in vivo. BCL1 leukemia growing in syngenic Balb/c mice was selected as a tumor model system. B1 mAb recognising the idiotype of surface IgM on BCL1 cells was used as a targeting moiety. Both B1 mAb and doxorubicin were conjugated to HPMA copolymer carrier by aminolysis through a tetrapeptidic Gly-Phe(D,L)-Leu-Gly spacer to ensure the intracellular delivery and controlled release of the drug. B1 mAb-targeted conjugate was shown to possess strictly tumor-specific binding capacity to target BCL1 cells in vitro. A similar conjugate, but containing human nonspecific Ig (HuIg) instead of B1 mAb, failed to bind to BCL1 cells. In vitro, B1 mAb-targeted conjugate demonstrated 40-fold higher cytotoxic effect than nontargeted or human nonspecific Ig-containing HPMA copolymer-bound doxorubicin. Conjugate targeted with B1 mAb was also shown to bind to target BCL1 cells in vivo. B1 mAb-targeted conjugate was shown to be more efficient in the treatment of established BCL1 leukemia than free doxorubicin, nontargeted and human nonspecific Ig-containing conjugate. Antibody-targeted polymeric drugs are thus promising conjugates for cancer treatment.     

Doxorubicin bound to a HPMA copolymer carrier through hydrazone bond is effective also in a cancer cell line with a limited content of lysosomes.

We have synthesized conjugates containing doxorubicin (DOX) bound to oligopeptide side chains (GlyGly or GlyPheLeuGly) of a water-soluble copolymer carrier based on poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA) either through proteolytically (PK1 conjugates) [Synthetic polymeric drugs. U.S. Patent 5,037,883 (1991)] or hydrolytically cleavable bond (HC conjugates). Pharmacological efficacy of PK1 and HC conjugates was compared in vitro on murine: T-cell lymphoma EL4, B-cell leukemia BCL1, B-cell lymphoma 38C13, leukemia P388 and Con A-stimulated A/Ph splenocytes and on human: primary (SW480) and metastatic (SW620) colorectal cancer cell lines parent and transfected with Thy 1.2 gene [2] and on erythromyeloid leukemia cell line K 562. Inhibition of proliferation determined by 3[H]-thymidine incorporation revealed that the cytostatic effect of HC conjugates is up to two orders of magnitude higher compared to PK1 conjugates. In some cancer cell lines (SW 620/T, SW 480) the pharmacological activity of HC conjugates is in vitro comparable with the activity of the free drug. Unlike PK1 conjugates, HC conjugates with a lysosomally degradable spacer (GlyPheLeuGly) are less effective compared to HC conjugates containing lysosomally non-degradable spacer (GlyGly). Moreover, HC conjugates exert pronounced anti-proliferative activity also in erythroblastoid leukemia cell line K 562 with a limited content of lysosomes.     

Cytostatic and immunomobilizing activities of polymer-bound drugs: experimental and first clinical data.

An N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer carrier containing doxorubicin and human immunoglobulin as an actively/passively targeting moiety was used in four patients with generalized breast cancer resistant to standard cytotoxic chemotherapy. The dose and time schedule were deduced from a Phase I clinical trial in which doxorubicin bound to HPMA copolymer carrier (PK1) was tested. It was confirmed that the Dox-HPMA-HuIg conjugate is stable and doxorubicin remains in the peripheral blood with a small amount also in the urine, mostly in its polymer-bound form. More than 116 biochemical, immunological and hematological parameters were determined for blood samples taken from patients 24 h, 48 h, 72 h and 1 to 11 weeks after treatment. Depending on the patient, some parameters decreased permanently or temporarily to the normal level (CRP, C3, CA 72-4, beta(2)-microglobulin, ferritin, CEA, CA 125, CD4, CD8, CE19, CD16(+)56(+), leu, ery) and some moved markedly towards physiological values (AST, ALT, ALP, GMT, CA 15-3, NSE, AFP). While the number of peripheral blood reticulocytes was significantly decreased after treatment with the classical free drug, their number was not affected or was even elevated after treatment with Dox-HPMA-HuIg. Increased absolute numbers of CD16(+)56(+) and CD4(+) cells in the peripheral blood and activation of NK and LAK cells in all patients support data obtained in experimental animals, pointing to a dual, i.e. cytostatic and immunomobilizing character of Dox-HPMA conjugates containing a targeting immunoglobulin moiety.     

Acquired and specific immunological mechanisms co-responsible for efficacy of polymer-bound drugs.

We present data providing new evidence that poly[N-(2-hydroxypropyl)methacrylamide] (PHPMA)-bound drugs, unlike free drugs, have both cytostatic and immunomobilizing activity (CIA). Immediately after injection, due to the high level of the drug, the main activity of the polymeric conjugate is cytotoxic and cytostatic. Later on, long-term circulating PHPMA-bound drug, at concentrations lower than its minimal inhibitory levels, mobilizes the defense mechanisms of the host. Cytotoxic and cytostatic effects of drug-PHPMA were repeatedly confirmed. The following data support the concept of the immunomobilizing activity of the N-(2-hydroxypropyl)methacrylamide (HPMA) conjugates: (a) pre-treatment with free drugs (doxorubicin, cyclosporin A) accelerates the appearance of EL4 mouse T-cell lymphoma while a similar pre-treatment with doxorubicin-PHPMA induces limited but definitive mobilization of the host's defense mechanisms; (b) mice cured of EL4 mouse T-cell lymphoma, BCL1 mouse B-cell leukemia and 38C13 mouse B-cell lymphoma by injection of doxorubicin-PHPMA conjugate targeted with monoclonal antibodies (anti-Thy 1.2 for EL4, anti-B1 for BCL1 and anti-CD71 for 38C13) and re-transplanted with a lethal dose of the same cancer cells survive without any treatment considerably longer than control mice; (c) increased NK activity and anti-cancer antibody was detected only in animals treated with doxorubicin-PHPMA conjugate; and (d) considerably increased NK and LAK activity was seen in a human patient treated for generalized breast carcinoma with doxorubicin-PHPMA-IgG.     

HPMA copolymers with pH-controlled release of doxorubicin: in vitro cytotoxicity and in vivo antitumor activity.

Data on the synthesis, physicochemical characterisation and in vitro and in vivo biological properties of the new, nontargeted or antibody-targeted polymer-doxorubicin conjugates designed as anticancer drugs are presented. In the conjugates, the anticancer drug doxorubicin (DOX) is attached to the polymer carrier via a simple hydrolytically labile spacer containing either a hydrazone bond or cis-aconitic acid residue. In vitro incubation of the conjugates in buffers led to a fast DOX release from the polymer at pH 5 (modelling intracellular environment) while at pH 7.4 (modelling blood) the conjugates are relatively stable. Cytotoxicity of the conjugates to T cell lymphoma EL4 depended on the detailed structure of the spacer and the method used for antibody attachment and was much higher compared with the effect of similar classic conjugates (DOX attached to the polymer via enzymatically degradable spacer). In both protective and therapeutic regimes of drug administration, the in vivo anti-tumor activity of the hydrazone conjugates containing only DOX was significantly enhanced (T cell lymphoma EL4, C57BL/10 mice) in comparison with free DOX or classic PK1, the PHPMA-DOX conjugate clinically tested at present. Increasing the molecular weight of the polymer carrier resulted in a more pronounced in vivo antitumor effect. Antibody-targeted conjugates with DOX bound via hydrazone bond exhibited even more extensive inhibition of the tumor growth with some long-term survivors. No survivors were observed after treatment of mice with free DOX or the nontargeted PHPMA-DOX conjugate.     

New HPMA copolymers containing doxorubicin bound via pH-sensitive linkage: synthesis and preliminary in vitro and in vivo biological properties.

In this paper we describe the synthesis, physico-chemical characteristics and results of tests of biological activity of polymer drugs based on conjugates of anti-cancer drug doxorubicin (Dox) with water-soluble polymer drug carriers, N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers. In the conjugates the drug is attached to the polymer backbone via a spacer stable under physiological conditions (pH 7.4) and hydrolytically degradable in mild acidic environment (e.g., endosomes, pH approximately 5). This enables designing polymer drugs with long blood circulation and release and specific activation of the active compound in endosomes of target cells. Two types of Dox conjugates differing in the length and structure of the oligopeptide spacer were synthesised (GG and GFLG). In both types, the linkage susceptible to hydrolytic cleavage was formed by the reaction of the carbonyl group of Dox with the hydrazide group terminating the oligopeptide side chains of the polymer. In vitro incubation of conjugates in buffers resulted in much faster release of Dox from the polymer at pH 5 than at pH 7.4 (more than 10 times) the rate being higher for the conjugate containing GG spacer. The presence of cathepsin B in incubation media increased the rate of Dox release from the conjugate with GFLG spacer, Dox release from conjugate with GG spacer remained unchanged. Cytotoxicity of conjugates for T-splenocytes and mouse EL-4 T cell lymphoma cells was much higher compared with the effect of similar 'classic' conjugates bearing Dox attached via amide bond. In vivo anti-tumor activity of conjugates containing hydrolytically sensitive linkage was also significantly improved in mouse EL4 T cell lymphoma.     

Polymeric drugs based on conjugates of synthetic and natural macromolecules. II. Anti-cancer activity of antibody or (Fab')(2)-targeted conjugates and combined therapy with immunomodulators.

We provide data on in vivo targeting of the Thy 1.2 (CDw90) cell surface receptor expressed on neoplastic T cells, mouse EL4 T cell lymphoma. The targeting antibody and the anticancer drug, doxorubicin (DOX) 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 conjugated drug. The in vivo therapeutic efficacy of HPMA copolymer-bound DOX targeted with anti-EL4 antibody, polyclonal anti-thymocyte globulin (ATG), monoclonal anti-Thy 1.2 antibody or its F(ab')(2) fragment was compared with the efficacy of DOX conjugated to HPMA copolymer containing nonspecific IgG or bovine serum albumin (BSA). Anti-EL4 antibody-targeted conjugate caused a significant retardation of tumor growth and an extension of the life span of treated mice. The effect was comparable with that of HPMA copolymer-bound DOX targeted with ATG, anti-Thy 1.2 antibody or its F(ab')(2) fragment. However, considerable antitumor effect was seen also in conjugates targeted instead of specific antibodies with syngeneic nonspecific IgG or BSA. Patients with advanced cancer are often immunocompromised due to dysfunction of their immune system induced by cancer and cytotoxic drugs. A significant decrease of unwanted side-effects of targeted drugs against a number of vital organs was already documented. In this study we have compared immunotoxic effects of free DOX with those of its antibody-targeted form on NK cells and cytolytic T lymphocytes (CTLs) isolated from C57BL/10 mice bearing EL4 T cell lymphoma. In the same model we have tested the combination therapy with immunomodulators (beta-glucan or AM-2) injected together with targeted daunomycin. We have observed a significant protective effect of targeted DOX against NK cells and CTLs. Moreover, the data revealed that combination therapy considerably enhances antitumor efficacy of the targeted anticancer drug.     

Investigating the mechanism of enhanced cytotoxicity of HPMA copolymer-Dox-AGM in breast cancer cells.

Recently we have described an HPMA copolymer conjugate carrying both the aromatase inhibitor aminoglutethimide (AGM) and doxorubicin (Dox) as combination therapy. This showed markedly enhanced in vitro cytotoxicity compared to the HPMA copolymer-Dox (FCE28068), a conjugate that demonstrated activity in chemotherapy refractory breast cancer patients during early clinical trials. To better understand the superior activity of HPMA copolymer-Dox-AGM, here experiments were undertaken using MCF-7 and MCF-7ca (aromatase-transfected) breast cancer cell lines to: further probe the synergistic cytotoxic effects of AGM and Dox in free and conjugated form; to compare the endocytic properties of HPMA copolymer-Dox-AGM and HPMA copolymer-Dox (binding, rate and mechanism of cellular uptake); the rate of drug liberation by lysosomal thiol-dependant proteases (i.e. conjugate activation), and also, using immunocytochemistry, to compare their molecular mechanism of action. It was clearly shown that attachment of both drugs to the same polymer backbone was a requirement for enhanced cytotoxicity. FACS studies indicated both conjugates have a similar pattern of cell binding and endocytic uptake (at least partially via a cholesterol-dependent pathway), however, the pattern of enzyme-mediated drug liberation was distinctly different. Dox release from PK1 was linear with time, whereas the release of both Dox and AGM from HPMA copolymer-Dox-AGM was not, and the initial rate of AGM release was much faster than that seen for the anthracycline. Immunocytochemistry showed that both conjugates decreased the expression of ki67. However, this effect was more marked for HPMA copolymer-Dox-AGM and, moreover, only this conjugate decreased the expression of the anti-apoptotic protein bcl-2. In conclusion, the superior in vitro activity of HPMA copolymer-Dox-AGM cannot be attributed to differences in endocytic uptake, and it seems likely that the synergistic effect of Dox and AGM is due to the kinetics of intracellular drug liberation which leads to enhanced activity.     

HPMA copolymer conjugates with reduced anti-CD20 antibody for cell-specific drug targeting. I. Synthesis and in vitro evaluation of binding efficacy and cytostatic activity

Synthesis, physicochemical and biological properties and preliminary anticancer activity of new star-shaped polymer–doxorubicin (DOX) conjugates targeted with anti-CD20 monoclonal antibody were investigated. Mild reduction of antibody (Ab) with dithiothreitol (DTT) resulted in introduction of thiol groups into Ab. Polymer precursors used for the synthesis of the conjugates were based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers with a functional group at the polymer chain end. The copolymers were linked to the thiol groups of the reduced Ab via one-point attachment forming a star-shaped structure with central antibody surrounded by hydrophilic polymer chains. Neither reduction nor polymer modification of Ab influenced binding activity of the Ab to its specific cancer cell membrane antigen as it was confirmed in vitro by standard flow cytometry. The anticancer drug DOX was attached to the HPMA copolymer chain in an Ab–polymer system via a pH–labile hydrazone linkage or via an oligopeptide sequence degradable by lysosomal enzymes. Such Ab–polymer–DOX conjugates were fairly stable in aqueous solution at pH 7.4 and the drug was readily released in mildly acid environment at pH 5–5.5 by hydrolysis of hydrazone bond or more slowly by enzymolysis with lysosomal enzymes. The cytostatic activity of the anti-CD20 monoclonal Ab-targeted conjugates tested on several CD20-positive or negative human and mouse cancer cell lines confirmed considerable targeting capacity of the monoclonal Ab after its binding to the polymer carrier. New method of synthesis of star antibody-targeted polymer–drug conjugates with pH-controlled drug release described in this paper opens new perspectives for development of new therapeutics intended for cancer therapy.     

Time- and concentration-dependent apoptosis and necrosis induced by free and HPMA copolymer-bound doxorubicin in human ovarian carcinoma cells.

A2780 sensitive and A2780/AD doxorubicin (DOX) resistant human ovarian carcinoma cells were exposed to different concentrations (0.25, 0.5, 1, 5 and 10xIC(50)) of free and HPMA copolymer-bound DOX for 12, 24, 36, 48, 60 and 72 h. Apoptosis and necrosis were evaluated using the FITC-conjugated annexin V and propidium iodide staining. The data obtained showed that the induction of apoptosis and necrosis by both free DOX and HPMA copolymer-bound DOX were time- and concentration-dependent. The data also showed significant differences between the drugs. It was found that: (i) under the action of HPMA copolymer-bound doxorubicin the alterations in the plasma membrane permeability preceded disturbances in cellular metabolism; (ii) HPMA copolymer-bound doxorubicin kills the cells mainly by necrosis; (iii) HPMA copolymer-bound doxorubicin is a more effective anticancer drug than free doxorubicin.     

Preparation and biological evaluation of polymerizable antibody Fab' fragment targeted polymeric drug delivery system.

A new polymerizable antibody Fab' fragment with a PEG spacer (MA-PEG-Fab') was prepared from OV-TL 16 antibody, specific against the OA-3 antigen expressed on most human ovarian carcinomas. The MA-PEG-Fab' possessed a higher reactivity in the copolymerization with N-(2-hydroxypropyl)methacrylamide (HPMA) than the polymerizable Fab' fragment MA-Fab' with a short spacer. The MA-PEG-Fab' was copolymerized with HPMA and MA-Gly-Phe-Leu-Gly-Mce(6) producing an Fab' targeted HPMA copolymer-Mce(6) conjugate. The number and weight average molecular weights of the copolymer were 164000 and 271000 Da, respectively. About two MA-PEG-Fab' fragments per chain were incorporated in the copolymer conjugates. Preliminary in vivo antitumor studies indicated that the Fab' targeted conjugates showed a higher efficacy of tumor growth inhibition in nude mice than the non-targeted conjugate.     

Synthesis and characterization of HPMA copolymer-aminopropylgeldanamycin conjugates.

Geldanamycin (GDM) is a benzoquinone ansamycin antibiotic with anticancer activity. The use of drug delivery systems based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing lysosomally degradable oligopeptide (GFLG) spacers results in an increased therapeutic efficacy of anticancer drugs. The objective of this study was to synthesize HPMA copolymer-GDM conjugates with anticancer activity and reduced toxic side-effect of the compound. 17-(3-Aminopropylamino)-17-demethoxygeldanamycin (AP-GDM) was synthesized and converted into a polymerizable GDM derivative, N-methacryloylglycylphenylalanylglycyl-17-(3-aminopropylamino)-17-demethoxygeldanamycin [MA-GFLG-(AP-GDM)]. The structures of AP-GDM and MA-GFLG-(AP-GDM) were validated by mass spectroscopy, elemental analysis, and two-dimensional nuclear magnetic resonance. MA-GFLG-(AP-GDM) was copolymerized with HPMA and N-methacryloyglycylglycine p-nitrophenylester by radical precipitation polymerization. Water-soluble HPMA copolymer-AP-GDM conjugates (M(r)=16 kDa) were obtained. Monoclonal antibody OV-TL16, which recognizes the OA-3 antigen expressed on the OVCAR-3 human ovarian carcinoma cell line, was optionally attached to the HPMA copolymer-AP-GDM conjugate. Cytotoxicity of polymer-bound AP-GDM (both targeted and non-targeted) was determined using OVCAR-3 and another human ovarian carcinoma cell line, A2780. The HPMA copolymer-AP-GDM conjugate was cytotoxic toward A2780 cells. Attachment of OV-TL16 antibody enhanced cytotoxicity of the conjugate toward OVCAR-3 cells.     

Interactions of diuretics with the peripheral-type benzodiazepine receptor in rat kidney

The peripheral-type benzodiazepine receptor (PBR) has been autoradiographically localized to the thick ascending limb and early distal tubule. To elucidate further the role of this receptor in kidney function, we have examined the effects of all classes of diuretics on the binding of labeled PBR-specific ligands (R05-4864, PK 11195) to rat kidney membranes (13,000 X g X 10 min). Drugs capable of inhibiting R05-4864 binding by 50% at less than 200 microM included: metolazone (IC50 = 1 microM), indacrinone (IC50 = 42 microM), indapamide (IC50 = 58 microM), hydrochlorothiazide (HCTZ; IC50 = 117 microM) and trichloromethiazide (IC50 = 175 microM). Conversely, diuretics of the loop (e.g., furosemide), K+-sparing (e.g., triamterene), and carbonic anhydrase inhibitor (e.g., acetazolamide) classes exerted no significant effects on R05-4864 binding (IC50S greater than or equal to 1 mM). Inhibition by indacrinone was stereoselective. Thiazide-like compounds inhibited R05-4864 binding with a rank-order of potencies similar to that for their enhancement of in vivo natriuresis (metolazone greater than HCTZ approximately equal to trichlormethiazide greater than chlorothiazide). Scatchard analysis revealed that metolazone, indacrinone, indapamide and HCTZ inhibited R05-4864 binding by reducing Kd, with no effect on maximum binding. The apparent Kd of metolazone for the renal PBR was 3.8 X 10(-7) M. IC50 values of 14 metolazone derivatives for inhibition of R05-4864 binding correlated well (r = .71, P less than .01) with their natriuretic efficacies. PK 11195 binding to digitonin (1.2 mg/mg of protein)-solubilized membranes displayed the same rank-order of, but was twice as sensitive to inhibition by metolazone, indacrinone, indapamide and HCTZ.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polymer-drug conjugates, PDEPT and PELT: basic principles for design and transfer from the laboratory to clinic.

There are now at least seven polymer-drug conjugates that have entered phase I/II clinical trial as anticancer agents. These include N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (PK1, FCE28068), HPMA copolymer-paclitaxel (PNU 166945), HPMA copolymer-camptothecin, PEG-camptothecin, polyglutamic acid-paclitaxel, an HPMA copolymer-platinate (AP5280) and also an HPMA copolymer-doxorubicin conjugate bearing additionally galactosamine (PK2, FCE28069). The galactosamine is used as a means to target the conjugate to liver for the treatment of primary and secondary liver cancer. Promising early clinical results with lysosomotropic conjugates has stimulated significant interest in this field. Ongoing research is developing (1) conjugates containing drugs that could otherwise not progress due to poor solubility or uncontrollable toxicity; (2) conjugates of agents directed against novel targets; and (3) two-step combinations such as polymer-directed enzyme prodrug therapy (PDEPT) and polymer-enzyme liposome therapy (PELT) that can cause explosive liberation of drug from either polymeric prodrugs or liposomes within the tumour interstitium. Moreover, bioresponsive polymer-based constructs able to promote endosomal escape and thus intracytoplasmic delivery of macromolecular drugs (peptides, proteins and oligonucleotides) are also under study.     

Targetable water-soluble polymer-drug conjugates for the treatment of visceral leishmaniasis.

The present work describes the synthesis, characterization, and biological evaluation of targetable N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-anti-leishmanial drug conjugates for the treatment of visceral leishmaniasis (VL). Conjugates of HPMA copolymer with NPC1161, an 8-aminoquinoline analog with anti-leishmanial activity, containing N-acetylmannosamine (ManN) in the side chains were synthesized and characterized. In vitro anti-leishmanial efficacy of the conjugates was determined in mouse peritoneal macrophages infected with Leishmania donovani amastigotes. The conjugates were tested against mammalian KB cells for cytotoxicity. The effect of ManN content on uptake was evaluated in RAW 264 murine macrophages. In vivo anti-leishmanial efficacy was evaluated at 1 mg/kg intravenous dose in BALB/c mice. HPMA copolymer-NPC1161 conjugates with 5 mole% or higher ManN content were significantly (p<0.0001) more active (ED50<15 microg/ml) than nontargeted conjugates (ED50>30 microg/ml). All conjugates were relatively nontoxic towards the mammalian cells. Significantly (p<0.003) higher uptake was observed for targeted conjugates compared to nontargeted conjugates. The targeted conjugates were significantly more effective in vivo (67-80% inhibition, p<0.0001) than nontargeted conjugate (47% inhibition). HPMA copolymers containing ManN in the side chains can potentially reduce the toxicity and increase efficacy of anti-leishmanial drugs for the treatment of VL.     

Cytotoxic and cytostatic effects of anti-Thy 1.2 targeted doxorubicin and cyclosporin A

Recently we have developed a new generation of antibody-targeted immunosuppressive (cyclosporin A, CyA) and cytostatic (doxorubicin, Dox) drugs effective in vitro and in vivo. The drugs and the targeting antibody (polyclonal and monoclonal anti-Thy 1.2) are conjugated to the oligopeptidic side chains of a water-soluble synthetic carrier, copolymer of N-(2-hydroxypropyl)methacrylamide (HPMA). In this study we investigated the effects of such drugs on cell proliferation and cytokine release by EL-4 mouse thymoma and X63Ag8-653 (IL-2) and X63Ag8-653 (IL-3) mouse myeloma transfected with IL-2 and IL-3 gene, respectively. The Thy 1.2⁺ EL-4 mouse thymoma cell line strongly reacted with the anti-Thy 1.2-targeted polymer bound CyA and Dox as assayed by cell fluorometry, whereas Thy 1.2⁻ X63Ag8-653 (IL-2) and X63Ag8-653 (IL-3) mouse myelomas bound anti-Thy 1.2 targeted drugs to a considerably lower extent. Anti-Thy 1.2 targeted doxorubicin and cyclosporin A effectively inhibit proliferation and IL-2 release by EL-4 mouse thymoma. More importantly, low sensitivity of EL-4 mouse thymoma to the anti-proliferative effect of free doxorubicin is partly overcome when anti-Thy 1.2 targeted form of the drug is used. Proliferation of both X63Ag8-653 mouse myelomas and their lymphokine (IL-2 or IL-3) release is inhibited only in the presence of a very high concentration of antibody-targeted conjugates with no difference between the efficacy of non-targeted and anti-Thy 1.2 targeted form. Preincubation of mouse T-cell lymphoma EL-4 with free anti-Thy 1.2 antibody considerably decreases the cytotoxic effects of anti-Thy 1.2 targeted doxorubicin.     

N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers for targeted delivery of 8-aminoquinoline antileishmanial drugs.

A challenge to successful chemotherapy of visceral leishmaniasis is the dose-limiting toxicity of antileishmanial agents. One approach to increase the efficacy and reduce the toxicity of these agents is to direct the drug to the phagolysosomes of the reticuloendothelial system (RES) where the leishmanial parasites reside. In this work a series of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-antileishmanial drug conjugates containing lysosomally degradable side chains and with or without sugar targeting moieties were synthesized, characterized and investigated for their in vivo efficacy in mice infected with Leishmania. An 8-aminoquinoline analog, namely 8-[(4-amino-1-methylbutyl)amino]-5-[3,4-dichlorophenoxy]-6-methoxy-4-methylquinoline (NPC1161) was used as a model antileishmanial agent. At 5 mg/kg body weight drug equivalent dose, all HPMA copolymer-drug conjugates which contained lysosomally degradable side chains showed significant in vivo antileishmanial activity (>99% inhibition), comparable to the activity of the free drug. At 2 mg dose, the same conjugates were significantly more effective (84-90% inhibition) than the free drug (67% inhibition). These results indicate the potential of lysosomotropic HPMA copolymers for the targeted delivery of antileishmanial compounds in the treatment of visceral leishmaniasis.     

The effects of subcellular localization of N-(2-hydroxypropyl)methacrylamide copolymer-Mce(6) conjugates in a human ovarian carcinoma.

Photosensitizers, light-sensitive compounds, become activated upon illumination with a specific wavelength of light generating cytotoxic oxygen species. Due to the short half-life of singlet oxygen, the subcellular site of localization and excitation affects the type of cellular damage produced as well as cellular responses to different types of photodamage created within the cell. Here, we investigated the effects of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-mesochlorin e(6) monoethylenediamine (Mce(6)) conjugates localized to different subcellular compartments. Temperature was utilized to achieve subcellular localization of conjugates and subcellular fractionation was performed to confirm localization patterns of HPMA copolymer-Mce(6) conjugates. Cytotoxicity studies suggest plasma membrane and late endosomes were more sensitive to photodamage than lysosomal compartments as observed by an approximate 2-fold decrease in the IC(50) compared to lysosomally accumulated conjugate. Releasing Mce(6) from the polymer backbone within lysosomal compartments significantly lowered the IC(50) when compared to HPMA copolymer conjugates with Mce6 bound via a nondegradable linkage. These differences will prove useful in the future design of HPMA copolymer-Mce(6) conjugates for the treatment of ovarian cancer.