Milada Šírová

New HPMA copolymer-based drug carriers with covalently bound hydrophobic substituents for solid tumour targeting

Various conjugates of anticancer drug doxorubicin (Dox) covalently bound by the hydrolytically degradable hydrazone bond to the drug carrier based on N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers were synthesised. Structure of the conjugates differed in the type and the content of hydrophobic substituent (dodecyl, oleic acid and cholesterol moieties) introduced into the polymer structure. In aqueous solutions the conjugates self-assembled into high-molecular-weight supramolecular structures, such as polymeric micelles or stable hydrophilic nanoparticles 13-37 nm in diameter, depending on the type and the content of hydrophobic substituents. Treatment of mice bearing EL-4 T cell lymphoma with the conjugates in the therapeutic regime of drug administration (i.v.) resulted in significant tumour regression with up to 100% of long-term survivors, depending on the dose and the detailed structure of the carrier. The nanoparticles formed by the conjugate bearing cholesterol moiety exhibited prolonged blood circulation and enhanced tumour accumulation indicating an important role of the EPR effect in excellent anticancer activity of the conjugate.

HPMA Copolymer Conjugates of Paclitaxel and Docetaxel with pH-Controlled Drug Release

In this paper we describe the synthesis, physicochemical characteristics and data on the biological activity of polymer prodrugs based on the anticancer drugs paclitaxel (PTX) and docetaxel (DTX) conjugated with a water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer drug carrier. The drugs were derivatized and then attached to the polymer backbone via a spacer that is stable under physiological conditions (pH 7.4) and hydrolytically degradable in mild acidic environments (e.g., endosomes, pH approximately 5). Polymer-drug conjugates were designed to achieve prolonged blood circulation and release of the active compound in target cells. Six types of conjugates differing in the structure of the keto acid (levulic, 3-(acetyl)acrylic acid) and 4-(2-oxopropyl)benzoic acid-containing spacer or in the amount of drug bound to the HPMA copolymer were synthesized. In all the conjugates, the linkage susceptible to hydrolytic cleavage was formed by the reaction of the carbonyl group of a drug derivative with the hydrazide group-terminated side chains of the polymer. In vitro incubation of the conjugates in buffers resulted in much faster release of drugs or their derivatives from the polymer at pH 5 than at pH 7.4 with the rate depending on the detailed structure of the spacer. Conjugates containing drugs acylated with levulic acid were tested for their anticancer activity in vivo using two murine models. The PTX-containing conjugate showed better antitumor efficacy in the 4T1 model of mammary carcinoma than the parent drug and its derivative. The DTX-containing conjugate demonstrated high activity in treating EL4 T cell lymphoma. The treatment with the polymer conjugates was devoid of side toxicity. In both models, we achieved complete regression of established tumors accompanied by a durable tumor resistance in most of the cured animals.

Conjugates of doxorubicin with graft HPMA copolymers for passive tumor targeting

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.

HPMA copolymer-doxorubicin conjugates: The effects of molecular weight and architecture on biodistribution and in vivo activity.

The molecular weight and molecular architecture of soluble polymer drug carriers significantly influence the biodistribution and anti-tumour activities of their doxorubicin (DOX) conjugates in tumour-bearing mice. Biodistribution of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-DOX conjugates of linear and star architectures were compared in EL4 T-cell lymphoma-bearing mice. Biodistribution, including tumour accumulation, and anti-tumour activity of the conjugates strongly depended on conjugate molecular weight (MW), polydispersity, hydrodynamic radius (R(h)) and molecular architecture. With increasing MW, renal clearance decreased, and the conjugates displayed extended blood circulation and enhanced tumour accumulation. The linear conjugates with flexible polymer chains were eliminated by kidney clearance more quickly than the highly branched star conjugates with comparable MWs. Interestingly, the data suggested different mechanisms of renal filtration for star and linear conjugates. Only star conjugates with MWs below 50,000g.mo(-1) were removed by kidney filtration, while linear polymer conjugates with MWs near 70,000g.mol(-1), exceeding the generally accepted limit for renal elimination, were detected in the urine 36-96h after injection. Additionally, survival of tumour-bearing mice was strongly dependent on molecular weight and polymer conjugate architecture. Treatment of mice with the lower MW conjugate at a dose of 10mg DOX eq./kg resulted in 12% long-term surviving animals, while treatment with the corresponding star conjugate enabled 75% survival of animals.

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 hosts 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.

Preclinical Evaluation of Linear HPMA-Doxorubicin Conjugates with pH-Sensitive Drug Release: Efficacy, Safety, and Immunomodulating Activity in Murine Model

ABSTRACTPurposeIn vivo efficacy and safety of HPMA-based copolymers armed with doxorubicin via a spacer containing pH-sensitive linkage that can be prepared within a broad range of attached drug contents (1) was tested in murine tumor models.MethodsMice bearing T cell lymphoma EL4 or B cell lymphoma 38C13 were treated with a single dose of the conjugate (15, 25, and 75 mg Dox eq./kg i.v.) in a therapeutic regime. Anti-tumor resistance of the cured animals was proved by a second challenge with a lethal dose of tumor cells without additional treatment.ResultsThe content of drug bound to the polymer is an important parameter in relation to the conjugate therapeutic efficacy. The best anti-tumor effects were produced by conjugates with 10 – 13 wt% of bound doxorubicin. Free doxorubicin up to 4.6% relative to total drug content had no impact on the treatment efficacy and acute toxicity. The conjugates induced a complete cure of mice and regular treatment-dependent development of specific anti-tumor resistance. No myelosuppression or organ damage was observed.ConclusionsA well-defined HPMA copolymer-doxorubicin conjugate with pH-sensitive drug release is a good candidate for clinical trials as it has remarkable anti-tumor efficacy and a favorable safety profile.

Immunoprotective therapy with targeted anticancer drugs

Doxorubicin or mitomycin C bonded to poly[N 5 -(2-hydroxyethyl)-L-glutamine]-graft-poly(ethylene glycol) or poly[N-(2-hydroxypropyl)methacryl-amide], non-targeted or targeted with monoclonal antibodies, do not induce expression of FasL on selected cancer cells (human colorectal cancer cell line SW 620) thus protecting effector cells of the immune system against Fas-counterattack. The pre-treatment with PHPMA-bound DOX does not only protect but in fact mobilizes defense mechanisms of the tumor-bearing hosts. The treatment with selected monoclonal antibody-targeted PHPMA-bound DOX causes a rapid and complete rejection of established tumors (mouse B cell leukemia BCL1, mouse B cell lymphoma 38C13 and mouse T cell lymphoma EL4) and generates prolonged systemic anti-tumor immunity.

Implantation of ultrathin, biofunctionalized polyimide membranes into the subretinal space of rats.

Subretinal implants aim to replace the photoreceptor function in patients suffering from degenerative retinal disease by topically applying electrical stimuli in the subretinal space. Critical obstacles in the design of high-resolution subretinal implants include the proximity of stimulating electrodes to the target cells and enabling nutrient flow between the retina and the choroid. The present work evaluates the adhesion, migration and survival of retinal cells on an ultrathin (5 μm), highly porous (Ø 1 μm spaced 3 μm), gelatin-coated polyimide (PI) membrane. The biocompatibility was examined in mice indicating a good tolerance upon subcutaneous implantation with only a mild inflammatory response. In addition, organotypic cultures of rat retina evidenced that the porous membrane allowed the necessary nutrient flow for the retinal cell survival and maintenance. A transscleral implantation technique was applied to position the membrane into the subretinal space of rats. The effect on the obtained retinal integration was investigated in vivo using scanning laser ophthalmoscopy (SLO) and optical coherence tomography (OCT). In 12 out of 18 rat eyes, the implant was successfully placed subretinally. SLO and OCT demonstrated complete retinal attachment and fluorescein angiography showed no retinal vascular abnormalities over and around the implant, immediately after and up to four weeks after the implantation. Histological examination of the eyes showed a close attachment of a thin fibrocyte layer to the implant, the occlusion of the pores by living cells and the survival of some photoreceptors at the implantation site.

Surface Modification of Polyimide Sheets for Regenerative Medicine Applications

In the present work, two strategies were elaborated to surface-functionalize implantable polyimide sheets. In the first methodology, cross-linkable vinyl groups were introduced on the polyimide surface using aminopropylmethacrylamide. In the second approach, a reactive succinimidyl ester was introduced on the surface of PI. Using the former approach, the aim is to apply a vinyl functionalized biopolymer coating. In the latter approach, any amine containing biopolymer can be immobilized. The foils developed were characterized in depth using a variety of characterization techniques including atomic force microscopy, static contact angle measurements, and X-ray photoelectron spectroscopy. The results indicated that both modification strategies were successful. The subcutaneous implantation in mice indicated that both modification strategies resulted in biocompatible materials, inducing only limited cellular infiltration to the surrounding tissue.