In this study, we compared the enhanced permeability and retention (EPR) effect, toxicity, and therapeutic effect of the conjugate of the linear polymer poly(N-(2-hydroxypropyl)methacrylamide) (HPMA) with pirarubicin with an Mw below the renal threshold (39g/mol) (named LINEAR) and the disulfide-linked tandem-polymeric dimer of the poly(HPMA)-pirarubicin conjugate with an Mw above the renal threshold (93g/mol) (named DIBLOCK). The DIBLOCK conjugate, which was susceptible to reductive degradation, showed both a better EPR effect (tumor delivery) (2.5 times greater at 24h) and a prolonged plasma half-life. In addition, DIBLOCK had a better antitumor effect, as judged by percent survival, than did LINEAR (80% vs 65% at 150days), without any apparent toxicity in an S180 tumor model. However, the LD50 value of LINEAR was slightly higher than that of DIBLOCK (50mg/kg vs 37.5mg/kg, respectively). DIBLOCK required a longer time than LINEAR to reach maximum accumulation in the tumor. DIBLOCK also showed a greater time-dependent increase in the concentration in the tumor compared with the plasma concentration.
- MeSH
- Antineoplastic Agents adverse effects chemistry pharmacology MeSH
- Biological Availability MeSH
- Biological Transport MeSH
- Doxorubicin adverse effects analogs & derivatives chemistry pharmacology MeSH
- Polymethacrylic Acids chemical synthesis MeSH
- Humans MeSH
- Molecular Weight MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- Drug Carriers chemical synthesis MeSH
- Half-Life MeSH
- Renal Reabsorption MeSH
- Tissue Distribution MeSH
- Drug Liberation MeSH
- Chromatography, High Pressure Liquid methods MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Comparative Study MeSH
Obstructed blood flow and erratic blood supply in the tumor region attenuate the distribution and accumulation of nanomedicines in the tumor. Therefore, improvement of these conditions is crucial for efficient drug delivery. In this study, we designed and synthesized a novel N-(2-hydroxypropyl)methacrylamide (HPMA)-based copolymer conjugate of BK, which possessed adequate systemic stability and tumor-selective action required to improve the accumulation of nanomedicines in the tumor. Levulinoyl-BK (Lev-BK) was conjugated to an HPMA-based polymer via an acid-cleavable hydrazone bond (P-BK). An acid-responsive release of Lev-BK from P-BK was observed, and P-BK alone after intradermal application showed below 10% of the BK activity, thus proving a reduction in the vascular permeability activity of BK when attached to the polymer carrier. P-BK pre-treatment improved blood flow in the tumor tissue by 1.4-1.7-fold, which was maintained for more than 4 h. In addition, P-BK pre-treatment increased the tumor accumulation of pegylated liposomal doxorubicin (PLD) by approximately 3-fold. Furthermore, P-BK pre-treatment led to superior antitumor activity of PLD and significantly improved the survival of tumor-bearing mice. The release of BK from P-BK in the acidic milieu of the tumor was a prerequisite for P-BK to exert its effect, as the vascular permeability enhancing activity of P-BK was negligible. Collectively, P-BK pre-treatment improved intratumoral blood flow and augmented tumor accumulation of nanomedicine, thereby resulting in a significant suppression of tumor growth. Therefore, these findings demonstrate that P-BK is a potential concomitant drug for improving the tumor delivery of nanomedicines.
- MeSH
- Antineoplastic Agents * therapeutic use MeSH
- Bradykinin therapeutic use MeSH
- Doxorubicin therapeutic use MeSH
- Methacrylates MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- Neoplasms * drug therapy MeSH
- Nanomedicine MeSH
- Drug Carriers therapeutic use MeSH
- Polymers therapeutic use MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
We have developed a tumor environment-responsive polymeric anticancer prodrug containing pirarubicin (THP) conjugated to N-(2-hydroxypropyl) methacrylamide copolymer (PHPMA), [P-THP], through a spacer containing pH-sensitive hydrazone bond, that showed remarkable therapeutic effect against various tumor models and in a human pilot study. Toward clinical development, here we report THP release profile from its HPMA copolymer conjugate, the conjugate stability, protein and cell-binding and solubility of P-THP. Size exclusion chromatography of P-THP (molecular weight 38 kDa) showed similar hydrodynamic volume as bovine serum albumin (BSA) in aqueous solution, with no apparent interactions with BSA, nor aggregation by itself. pH-responsive release of free THP was reconfirmed at pHs 6.5 and lower. The drug release was significantly affected by a type of used buffer. Phosphate buffer seems to facilitate faster hydrazone bond cleavage at pH 7.4 whereas higher stability was achieved in L-arginine solution which yielded only little cleavage and THP release, approx. 15% within 2 weeks at the same pH at 25 °C. Furthermore, ex vivo study using sera of different animal species showed very high stability of P-THP. Incubation with blood showed high stability of P-THP during circulation, without binding to blood cells. These findings revealed that L-arginine solution provides appropriate media for formulation of P-THP infusion solution as tumor-targeted polymeric anticancer drug based on EPR effect.
- MeSH
- Antineoplastic Agents chemistry MeSH
- Arginine chemistry MeSH
- Doxorubicin analogs & derivatives chemistry MeSH
- Rabbits MeSH
- Rats MeSH
- Humans MeSH
- Methacrylates chemistry MeSH
- Mice MeSH
- Drug Carriers chemistry MeSH
- Pilot Projects MeSH
- Polymers chemistry MeSH
- Solubility drug effects MeSH
- Serum Albumin, Bovine chemistry MeSH
- Drug Liberation drug effects MeSH
- Animals MeSH
- Check Tag
- Rabbits MeSH
- Rats MeSH
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
Multidrug resistance (MDR) is a common cause of failure in chemotherapy for malignant diseases. MDR is either acquired as a result of previous repeated exposure to cytostatic drugs (P388/MDR cells) or naturally, as some tumors are congenitally resistant to chemotherapy (CT26 cells). One of the most common mechanisms of MDR is upregulation of P-glycoprotein (P-gp) expression. Here, we used HPMA copolymer conjugates, whereby the cytostatic drug doxorubicin (Dox) or the derivative of the P-gp inhibitor reversin 121 (R121) or both were covalently bound through a degradable pH-sensitive hydrazone bond. We proved that R121, when bound to a polymeric carrier, is capable of inhibiting P-gp in P388/MDR cells and sensitizing them in relation to the cytostatic activity of Dox. Conjugate bearing both Dox and R121 was found to be far more potent in P388/MDR cells than conjugate bearing Dox alone or a mixture of conjugates bearing either Dox or R121 when cytostatic activity in vitro, cell cycle arrest, accumulation of Dox in cells and induction of apoptosis were determined. Importantly, conjugate bearing R121 is also effective in vivo as it inhibits P-gp in P388/MDR tumors after intraperitoneal administration, while both the conjugate bearing Dox and R121 induces apoptosis in P388/MDR tumors more effectively than conjugate bearing Dox alone. Only conjugate bearing Dox and R121 significantly inhibited P388/MDR tumor growth and led to the prolonged survival of treated mice. However, the most dramatic antitumor activity of this conjugate was found in the CT26 tumor model where it completely cured six out of eight experimental mice, while conjugate bearing Dox alone cured no mice.
- MeSH
- Drug Resistance, Neoplasm MeSH
- Cytostatic Agents administration & dosage MeSH
- Doxorubicin administration & dosage MeSH
- Neoplasms, Experimental drug therapy pathology MeSH
- Mice, Inbred Strains MeSH
- Methacrylates chemistry MeSH
- Drug Resistance, Multiple MeSH
- Mice, Nude MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- Nanocapsules administration & dosage chemistry MeSH
- Nanoconjugates administration & dosage chemistry MeSH
- Oligopeptides administration & dosage MeSH
- ATP Binding Cassette Transporter, Subfamily B, Member 1 antagonists & inhibitors MeSH
- Antineoplastic Combined Chemotherapy Protocols administration & dosage MeSH
- Treatment Outcome MeSH
- Animals MeSH
- Check Tag
- Mice MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
Synthesis and preliminary anticancer activity of new star-shaped immunoglobulin-containing polymer-doxorubicin (DOX) conjugates were investigated. The polymer precursors used for the synthesis of immunoglobulin-polymer-drug conjugates are based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers, the anticancer drug DOX is attached to the immunoglobulin-modified polymer via a pH-sensitive hydrazone linkage. Such polymer-DOX conjugates are stable in aqueous solution at pH 7.4 (pH of blood plasma) and the drug is released in mildly acid environment at pH 5-5.5 (pH in endosomes or lysosomes of target cells). Semitelechelic copolymer chains are linked to the immunoglobulin via one-point attachment to avoid branching of the conjugate observed in our earlier studied systems. The cytostatic activity of the conjugates tested on several cancer cell lines was similar to that of free DOX.HCl and correlated with the sensitivity of a particular cell line to DOX. The star-shaped conjugates containing immunoglobulin showed a significantly higher antitumor activity in vivo than immunoglobulin-free non-targeted polymer conjugates when tested in mice bearing EL4 T-cell lymphoma.
- MeSH
- Antibiotics, Antineoplastic administration & dosage pharmacology chemistry MeSH
- Doxorubicin analogs & derivatives administration & dosage pharmacology chemistry MeSH
- Financing, Organized MeSH
- Immunoglobulins administration & dosage chemistry MeSH
- Immunoconjugates administration & dosage chemistry MeSH
- Polymethacrylic Acids administration & dosage pharmacology chemistry MeSH
- Drug Screening Assays, Antitumor MeSH
- Humans MeSH
- Lymphoma, T-Cell drug therapy MeSH
- Mice, Inbred C57BL MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- Drug Carriers administration & dosage chemical synthesis chemistry MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Mice MeSH
- Animals MeSH
Synthesis and physico-chemical behavior of new polymer-drug conjugates intended for the treatment of cancer were investigated. In the polymer conjugate with the expected dual therapeutic activity, two drugs, a cytostatic agent doxorubicin (DOX) and anti-inflammatory drug dexamethason (DEX) were covalently attached to the same polymer backbone via hydrolytically labile pH-sensitive hydrazone bonds. The precursor, a copolymer of N-(2-hydroxypropyl)methacrylamide (HPMA) bearing hydrazide groups randomly distributed along the polymer chain, was conjugated with DOX (through its C13 keto group) or with a keto ester (DEX). Two derivatives of DEX, 4-oxopentanoate and 4-(2-oxopropyl)benzoate esters, were synthesized and employed for conjugation reaction. As a control, also a few polymer conjugates containing only a single drug (DOX or DEX) attached to the polymer carrier were synthesized. Physico-chemical properties of the polymer conjugates strongly depend on the attached drug, spacer structure and the drug content. Polymer-drug conjugates incubated in buffers modeling intracellular environment released the drug (DOX) or a drug derivatives (DEX) at the rate significantly exceeding the release rate observed under conditions mimicking situation in the blood stream. Incubation of the DEX conjugates in a buffer containing carboxyesterase resulted in complete ester hydrolysis thus demonstrating susceptibility of the system to release free active drug in the two-step release profile.
- MeSH
- Antibiotics, Antineoplastic administration & dosage chemistry MeSH
- Doxorubicin administration & dosage chemistry MeSH
- Drug Combinations MeSH
- Carboxylesterase chemistry MeSH
- Magnetic Resonance Spectroscopy MeSH
- Methacrylates chemical synthesis chemistry MeSH
- Polymers MeSH
- Excipients MeSH
- Spectrophotometry, Ultraviolet MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
Linkage of doxorubicin (Dox) to a water-soluble synthetic N-(2-hydroxypropyl)methacrylamide copolymer (PHPMA) eliminates most of the systemic toxicity of the free drug. In EL-4 lymphoma-bearing C57BL/6 mice, a complete regression of pre-established tumours has been achieved upon treatment with Dox-PHPMA-HuIg conjugate. The treatment was effective using a range of regimens and dosages, ranging from 62.5 to 100% cured mice treated with a single dose of 10-20 mg of Dox eq./kg, respectively. Fractionated dosages producing lower levels of the conjugate for a prolonged time period had substantial curative capacity as well. The cured mice developed anti-tumour protection as they rejected subsequently re-transplanted original tumour. The proportion of tumour-protected mice inversely reflected the effectiveness of the primary treatment. The treatment protocol leading to 50% of cured mice produced only protected mice, while no mice treated with early treatment regimen (i.e. starting on day 1 after tumour transplantation) rejected the re-transplanted tumour. Exposure of the host to the cancer cells was a prerequisite for developing protection. The anti-tumour memory was long lasting and specific against the original tumour, as the cured mice did not reject another syngeneic tumour, melanoma B16-F10. The immunity was transferable to naive recipients in in vivo neutralization assay by spleen cells or CD8(+) lymphocytes derived from cured animals. We propose an effective treatment strategy which eradicates tumours without harming the protective immune anti-cancer responses.
- MeSH
- Antibiotics, Antineoplastic therapeutic use MeSH
- Doxorubicin analogs & derivatives therapeutic use MeSH
- Financing, Organized MeSH
- Immunoglobulins therapeutic use MeSH
- Immune Tolerance MeSH
- Polymethacrylic Acids therapeutic use MeSH
- Humans MeSH
- Lymphoma, T-Cell drug therapy immunology prevention & control MeSH
- Melanoma, Experimental drug therapy immunology metabolism MeSH
- Survival Rate MeSH
- Mice, Inbred C57BL MeSH
- Mice, Nude MeSH
- Mice MeSH
- Tumor Cells, Cultured transplantation MeSH
- Skin Neoplasms drug therapy immunology metabolism MeSH
- Drug Carriers MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Mice MeSH
- Female MeSH
- Animals MeSH
Synthesis, physicochemical behavior, tumor accumulation and preliminary anticancer activity of a new biodegradable graft copolymer-doxorubicin (DOX) conjugates designed for passive tumor targeting were investigated. In the graft high-molecular-weight conjugates the multivalent N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer was grafted with a similar but semitelechelic HPMA copolymer; both types of polymer chains were bearing doxorubicin attached by hydrazone bonds enabling intracellular pH-controlled drug release. The polymer grafts were attached to the main chain through spacers, degradable enzymatically or reductively, facilitating, after the drug release, intracellular degradation of the graft polymer carrier to short fragments excretable from the organism by glomerular filtration. The graft polymer-DOX conjugate exhibited prolonged blood circulation and enhanced tumor accumulation in tumor-bearing mice indicating the important role of the EPR effect in the anticancer activity. The graft polymer-DOX conjugates showed a significantly higher antitumor activity in vivo than DOX.HCl or the linear polymer conjugate when tested in mice bearing 38C13 B-cell or EL4 T-cell lymphoma, with a significant number of long-term-surviving (LTS) mice with EL4 T-cell lymphoma treated with a single dose 15 mg DOX equiv./kg on day 10.
- MeSH
- Acrylamides administration & dosage pharmacokinetics pharmacology chemical synthesis MeSH
- Antibiotics, Antineoplastic administration & dosage pharmacokinetics pharmacology chemical synthesis MeSH
- Biological Transport MeSH
- Cell Line MeSH
- Chemistry, Pharmaceutical MeSH
- Doxorubicin administration & dosage pharmacokinetics pharmacology chemical synthesis MeSH
- Hydrolysis MeSH
- Injections, Intravenous MeSH
- Hydrogen-Ion Concentration MeSH
- Delayed-Action Preparations MeSH
- Lymphoma drug therapy metabolism pathology MeSH
- Molecular Weight MeSH
- Mice, Inbred C3H MeSH
- Mice, Inbred C57BL MeSH
- Mice MeSH
- Drug Carriers MeSH
- Xenograft Model Antitumor Assays MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
Synthesis and characterization of N-(2-hydroxypropyl)methacrylamide (HPMA)-copolymer-based drug carriers targeted on specific receptors in the membrane of endothelial cells by oligopeptides (GRGDG, cyclo(RGDfK), and PHSCN) are described in this study. The copolymers containing targeting oligopeptides bound to the polymer via dodeca(ethylene glycol) spacer showed a receptor-specific time-dependent uptake with selected endothelial cell lines. The polymers were labeled with a fluorescent dye to enable monitoring of the interaction of the polymer conjugate with cells using fluorescence microscopy. Cellular uptake and apoptosis induction have been studied in vitro using various cell lines (EA.hy926, 3T3, SW620, and EL4). In vivo accumulation of the conjugate specifically targeted with cyclo(RGDfK) within the tumor vasculature was detected using fluorescence intravital microscopy in mice. The conjugate targeted by cyclo(RGDfK) was accumulated preferentially in the periphery of the growing tumor suggesting that the cyclo(RGDfK) peptide targets the polymer conjugate to the site of neoangiogenesis, rather than to the tumor mass.
- MeSH
- Acrylamides chemistry MeSH
- Apoptosis drug effects MeSH
- Cell Line MeSH
- Time Factors MeSH
- Endothelium, Vascular metabolism pathology MeSH
- Microscopy, Fluorescence MeSH
- Humans MeSH
- Mice, Inbred C57BL MeSH
- Mice MeSH
- Cell Line, Tumor MeSH
- Neoplasms drug therapy pathology MeSH
- Oligopeptides administration & dosage pharmacokinetics chemical synthesis MeSH
- Neovascularization, Pathologic MeSH
- Drug Delivery Systems MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Research Support, Non-U.S. Gov't MeSH
