Anti-CD20 multivalent HPMA copolymer-Fab' conjugates for the direct induction of apoptosis

A hybrid biomimetic system comprising high-molecular-weight, linear copolymer of N-(2-hydroxypropyl)methacrylamide (HPMA) grafted with multiple Fab' fragments of anti-CD20 monoclonal antibody (mAb) was synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization followed by attachment of Fab' fragments via thioether bonds. Exposure of human non-Hodgkin's lymphoma (NHL) Raji B cells to the multivalent conjugates resulted in crosslinking of CD20 receptors and commencement of apoptosis. Five conjugates with varying molecular weight and valence (amount of Fab' per polymer chain) were synthesized. One of the copolymers contained enzyme degradable peptide sequences (GFLG) in the backbone. The multivalency led to higher avidity and apoptosis induction compared to unconjugated whole mAb. Time-dependent studies showed that the cytotoxicity of conjugates exhibited a slower onset at shorter exposure times than mAb hyper-crosslinked with a secondary Ab; however, at longer time intervals the HPMA copolymer conjugates achieved significantly higher biological efficacies. In addition, study of the relationship between the structure of conjugates and Raji B cell apoptosis revealed that both valency and polymer molecular weight influenced biological activities, while insertion of peptide sequences into the backbone was not a factor in?vitro.     

Iron(III) chelating resins--I. Preparation and properties of Sepharose-desferrioxamine gels.

For the removal of iron(III), Sepharose-desferrioxamine gels were prepared by the coupling of CNBr-activated Sepharose with desferrioxamine (DFO) at pH 7.8-8.3. DFO densities of the gels were 12-23 mumol/ml gel with iron(III) chelating capacities of 8.5-18 mumol/ml gel. The Sepharose-DFO gels with a high affinity for iron(III) were used for the removal of iron(III) from aqueous iron(III) solutions, wine, milk and whey.     

Lymphocyte Activation, Iron Uptake and Release by Human Mononuclear Leukocytes in Tiie Presence of Desferrioxamine

Desferrioxamine (DFO), an iron chelating drug, has been shown to inhibit the proliferative response of leukocytes to mitogen. In the present study we investigated the effect of DFO on different aspects of human mononuclear leukocyte (MNL) function in vitro. DFO, added at the beginning of the culture period, inhibited both tritiated thymidine and radioiron uptake by phytohemagglu tinin-stimulated MNL and the degree of inhibition correlated with the degree of cellular activation, to the extent that in the absence of mitogen a significant stimulatory effect of DFO was observed, especially when iron supplement was present in the culture medium. However DFO was not found to inhibit iron uptake directly, and relatively low concentrations of iron as iron-transferrin totally reversed the inhibitory action of DFO on thymidine uptake. Although the release of iron from preloaded MNL in the presence of DFO was only 15% greater than the spontaneous release of control cultures, we conclude that the site of action of DFO is an intracellular iron pool, that increases in importance when the supply of iron to the cellular iron metabolism become limiting as in optimally activated MNL.     

Extra- and intracellular free iron and the carotid body responses

The hypothesis that chelation of free iron, by decreasing reactive oxygen species (ROS), might mimic hypoxia and stimulate the carotid body was tested. We used the iron chelators, desferrioxamine (DFO, 200-400 microM) initially, and later ciclopirox olamine (CPX, 2.5-5.0 microM), on rat carotid body in vitro and measured chemosensory activity and [Ca2+]i in isolated cultured glomus cell clusters during normoxia and hypoxia. Although acute treatment of DFO might not penetrate the cell, and extracellular DFO would not influence these activities whereas CPX significantly increased chemosensory activities as well as increased [Ca2+]i in normoxia. We concluded that chelation of extracellular free iron did not alter ROS formation and oxygen sensing. Chelation of intracellular free iron and, therefore, a decrease in intracellular ROS appears to influence oxygen sensing in the carotid body.     

Involvement of Hif-1 in desferrioxamine-induced invasion of glioblastoma cells

Glioblastoma multiforme are highly invasive brain tumors. Experimental approaches focus on unravelling the mechanisms of invasion, this being a major reason for the poor prognosis of these tumors. Our previous results hinted towards involvement of the iron metabolism in invasion. In this study, we examined the effect of iron depletion on the invasive phenotype of glioblastoma cells. Transwell Matrigel invasion assays were used to monitor iron-dependent invasion of human glioblastoma cell lines U373MG and DBTRG05MG. Intracellular iron concentrations were modulated by applying desferrioxamine (DFO) and ferric ammonium citrate (FAC). We detected enhanced invasion of glioblastoma cells upon DFO-induced iron depletion. Treatment of cells with FAC strongly inhibited invasion. DFO treatment resulted in hypoxia-inducible factor 1 (Hif-1)-mediated induction of urokinase plasminogen activator receptor and matrix metalloproteinase 2. Further, RNA interference-mediated repression of urokinase plasminogen activator receptor inhibited DFO-induced invasion. Our data demonstrate a direct effect of DFO on Hif-1 expression resulting in activation of factors associated with ECM degradation and invasion of glioma cells. These findings caution on utilization of DFO and other iron chelators in the treatment of tumors with invasive potential.     

Tumor targeting by pH-sensitive,biodegradable, cross-linked N-(2-hydroxypropyl) methacrylamide copolymer micelles

Increasing the molecular weight of N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers by using micellar structures could result in more pronounced enhanced permeability and retention effect, thus increase the tumor accumulation of drug. However, most micellar formulations are relatively unstable and release their drug non-specifically. To improve on these disadvantages, we developed a micellar drug delivery system based on self-assembly of HPMA copolymers. Amphiphilic conjugates were synthesized by conjugating the hydrophobic drug doxorubicin and hydrophobic β-sitosterol to the hydrophilic HPMA polymer backbone via pH-sensitive hydrazone linkages. This linkage is quite stable at physiological pH but hydrolyzes easily at acidic pH. After conjugates self-assembly into micelles, HPMA copolymer side chains were cross-linked through the hydrazone linkages to ensure micelle stability in the blood. Using this approach, cross-linked micelles were obtained with molecular weight of 1030 KD and diameter of 10–20 nm. These micelles remained stable with undetectable doxorubicin release at pH 7.4 or mouse plasma, whereas collapsed quickly with 80% of the drug released at pH 5 which corresponds to the pH of lyso/endosome compartments of tumor cells. Both cross-linked and non-cross-linked micelles displayed similar in vitro anti-tumor activity as linear copolymer conjugates in Hep G2 and A549 cancer cell lines with internalization mechanism by caveolin, clathrin, and giant macropinocytosis. In vivo studies in an H22 mouse xenograft model of hepatocarcinoma showed the tumor accumulation (1633 μCi/L*h) and anti-tumor rate (71.8%) of cross-linked micelles were significantly higher than non-cross-linked ones (698 μCi/L*h, 64.3%). Neither type of micelle showed significant toxicity in heart, lung, liver, spleen or kidney. These results suggest that cross-linked HPMA copolymer micelles with pH-sensitivity and biodegradability show excellent potential as carriers of anti-cancer drugs.     

Immunogenicity of coiled-coil based drug-free macromolecular therapeutics

A two-component CD20 (non-internalizing) receptor crosslinking system based on the biorecognition of complementary coiled-coil forming peptides was evaluated. Exposure of B cells to Fab'-peptide1 conjugate decorates the cell surface with peptide1; further exposure of the decorated cells to P-(peptide2)_x (P is the N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer backbone) results in the formation of coiled-coil heterodimers at the cell surface with concomitant induction of apoptosis. The aim of this study was to determine the potential immunogenicity of this therapeutic system that does not contain low molecular weight drugs. Enantiomeric peptides (l- and d-CCE and l- and d-CCK), HPMA copolymer-peptide conjugates, and Fab' fragment-peptide conjugates were synthesized and the immunological properties of peptide conjugates evaluated in vitro on RAW264.7 macrophages and in vivo on immunocompetent BALB/c mice. HPMA copolymer did not induce immune response in vitro and in vivo. Administration of P-peptide conjugates with strong adjuvant resulted in antibody response directed to the peptide. Fab' was responsible for macrophage activation of Fab'-peptide conjugates and a major factor in the antibody induction following i.v. administration of Fab'-conjugates. There was no substantial difference in the ability of conjugates of d-peptides and conjugates of l-peptides to induce Ab response.     

Effects of the iron-chelating agent deferoxamine on triethylene glycol dimethacrylate, 2-hydroxylethyl methacrylate, hydrogen peroxide-induced cytotoxicity

Triethylene glycol dimethacrylate (TEGDMA) and 2-hydroxylethyl methacrylate (HEMA) are known to deplete glutathione in mammalian cells, generate reactive oxygen species (ROS), and cause oxidative stress. In this study, we investigated whether hydroxyl radicals (·OH), the most lethal and genotoxic ROS, and the Fenton reaction are involved in the cytotoxicity of resin monomers to four different cell types, namely MC3T3-E1 preosteoblasts, human dental pulp cells (HDPCs), human gingival fibroblasts, and L929 mouse fibroblasts. Deferoxamine (DFO), an iron chelating agent, effectively protected MC3T3-E1 cells from resin monomer-induced cytotoxicity, indicating that cytotoxicity was caused primarily by hydroxyl radicals. However, DFO only had a protective effect against relatively high concentrations of TEGDMA and HEMA in HDPCs and human gingival fibroblasts, and resin monomer-induced cytotoxicity in L929 was not attenuated by DFO. A labile iron pool (LIP) was detectable only in MC3T3-E1 cells among the four cell types. This indicates that the generation of hydroxyl radicals induced by resin monomers is likely dependent on LIP levels. In contrast to resin monomers, hydrogen peroxide (H(2)O(2))-induced cytotoxicity was not prevented by DFO in any of the cell types examined, although hydroxyl radicals were detected in MC3T3-E1 cells and HDPCs on exposure to exogenous H(2)O(2). This result suggests that generation of hydroxyl radicals is not always the primary cause of cytotoxicity in H(2)O(2)-treated cells.     

Behavior of blood cells in contact with water-soluble phospholipid polymer.

Omega-Methacryloyloxyalkyl phosphorylcholine (MAPC) polymer, which has various methylene chain lengths between the phosphorylcholine group and the backbone, was synthesized with attention to formation of the biomembrane. The effect of water-soluble poly(MAPC) on the function and activation of blood cells was evaluated to determine the interaction between blood cells and the MAPC polymer. The poly(MAPC) and the MAPC copolymer with a small amount of fluorescent units were synthesized by a conventional radical polymerization technique. Using a fluorescence spectrometer, it was determined that the MAPC polymer was adsorbed on the plasma membrane of platelets when the platelets were suspended in an aqueous solution of the MAPC copolymer. The hemolytic activity of poly(MAPC) was less than that of other water-soluble polymers, such as poly(ethylene glycol) and poly(1-vinyl-2-pyrrolidone) (PVPy). The change in the plasma membrane fluidity of platelets on contact with poly(MAPC) was determined with 1,6-diphenyl-1,3,5,-hexatriene. The plasma membrane fluidity of platelets decreased with an increase in the methylene chain length of the MAPC unit. The aggregation activity of platelets after contact with poly(MAPC) was also evaluated, but no significant difference between that of polymer-contacted platelets and native platelets was observed. Finally, the activity of platelets on contact with poly(MAPC) was determined by measuring the cytoplasmic calcium ion concentration ([Ca2+]i) in platelets. The increase in [Ca2+]i in the platelets after contact with poly(MAPC) was similar to that of native platelets. We conclude that the poly(MAPC) reduced platelet activation even though the poly(MAPC) adsorbed on the membrane surface of the platelets. In particular, poly(10-methacryloyloxydecyl phosphorylcholine) significantly reduced platelet activation compared with PVPy.     

Pharmacological manipulation of ataxia-telangiectasia kinase activity as a treatment for Parkinson's disease

Parkinson's disease (PD) is a major cause of morbidity and mortality among older individuals. Although the causes of Parkinson's disease are multifactorial, considerable evidence indicates that elevated labile iron in the substantia nigra pars compacta plays an important role in producing oxyradicals which subsequently damage nigro-striatal neurons. Based on this several researchers have suggested that blood-brain barrier crossing iron chelators might have clinical efficacy in treating PD. Work demonstrating that iron chelators protect nigro-striatal neurons in the N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine and 6-hydroxydopamine-induced rodent PD models supports this hypothesis. Recently, we found that the ATM gene product (mutated in ataxia-telangiectasia, A-T), is required for cell survival and genomic stability maintenance following exposure to low labile iron concentrations. Iron chelators (desferal, quercetin, and apoferritin) also increase A-T cell genomic stability and viability, and activate ATM-dependent cellular events in normal cells. Additionally Atm-deficient mice exhibit a selective loss of dopaminergic nigro-striatal neurons. Based on this, we propose that iron chelators protect the substantia nigra pars compacta not only by chelating labile iron and reducing oxyradical formation, but also by inducing ATM activity, leading to increased oxidative stress resistance and DNA repair. Support for this hypothesis comes from the recent observation that the iron chelating flavonoid quercetin both directly activates ATM and protects neuronal cells from the toxic effects of the N-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Therefore since; (1) ATM is required for iron toxicity resistance, (2) iron chelators such as quercetin, desferal, and apoferritin induce ATM activity and/or ATM-dependent events, and (3), Atm-deficient mice preferentially lose dopaminergic nigro-striatal neurons, we propose that ATM activity has an important function in PD. Furthermore, pharmacological manipulation of ATM activity via iron chelation might have clinical efficacy in PD treatment.     

Curcumin contributes to in vitro removal of non-transferrin bound iron by deferiprone and desferrioxamine in thalassemic plasma

Non-transferrin-bound iron (NTBI) is detectable in plasma of beta-thalassemia patients with transfusional iron overload. This form of iron may cause oxidative tissue damage and increased iron uptake, into several vital organs. Removal of NTBI species is incomplete and transient using standard intermittent desferrioxamine (DFO) or deferiprone (DFP) monotherapy. Combinations of these or other chelators may improve the protection time from NTBI and increase removal of harmful NTBI species. Curcuminoids from Curcuma longa L. is a naturally occurring phytochemical which shows a wide range of pharmacological properties including anti-oxidative, anti-inflammatory, anti-cancer and iron-chelating activities. In this study, the curcuminoids was investigated for NTBI chelation in thalassemic plasma in vitro and for the potential to improve NTBI removal when used with other chelators. Curcumin bound Fe(3+) to form a Fe(3+)-curcumin complex with a predominant absorption at 500 nm. The chemical binding of curcumin was dose- and time-dependent and more specific for Fe(3+) than Fe(2+). Using a HPLC-based NTBI assay without an aluminium blocking step, curcumin shuttled the iron from Fe(3+)-NTA complex, giving underestimated NTBI values. At equivalent concentrations DFO, DFP and curcumin decreased plasma NTBI with the order of DFP>DFO>curcumin. None of these chelators removed NTBI completely, but curcumin appeared to increase the rate of NTBI removal when added to DFP. It is proposed that the beta-diketo moiety of curcumin participates in the NTBI chelation.     

Increased susceptibility to oxidative stress in scrapie-infected neuroblastoma cells is associated with intracellular iron status

The molecular mechanism of neurodegeneration in prion diseases remains largely uncertain, but one of the features of infected cells is higher sensitivity to induced oxidative stress. In this study, we have investigated the role of iron in hydrogen peroxide (H(2)O(2))-induced toxicity in scrapie-infected mouse neuroblastoma N2a (ScN 2 a) cells. ScN 2 a cells were significantly more susceptible to H(2)O(2) toxicity than N2a cells as revealed by cell viability (MTT) assay. After 2h exposure, significant decrease in cell viability in ScN 2 a cells was observed at low concentrations of extracellular H(2)O(2) (5-10 microM), whereas N2a cells were not affected. The increased H(2)O(2) toxicity in ScN 2 a cells may be related to intracellular iron status since ferrous iron (Fe(2+)) chelator 2,2'-bipyridyl (BIP) prevented H(2)O(2)-induced decrease in cell viability. Further, the level of calcein-sensitive labile iron pool (LIP) was significantly increased in ScN 2 a cells after H(2)O(2) treatment. Finally, the production of reactive oxygen species (ROS) was inhibited by 30% by iron chelators desferrioxamine (DFO) and BIP in ScN 2 a cells, whereas no significant effect of iron chelators on basal ROS production was observed in N2a cells. This study indicates that cellular resistance to oxidative stress in ScN 2 a cells is associated with intracellular status of reactive iron.     

Therapeuetic management of patients with thalassemia major

In industrialised countries, the use of regular blood transfusions and of chelation therapy with Deferoxamine (DFO) has led to the transformation of thalassemia major from a fatal disease in early childhood to a chronic illness associated with prolonged survival. Transfusion regimens maintaining pretransfusion hemoglobin > 9-10 g/dl are effective in suppressing erythroid marrow expansion. Long term DFO therapy using subcutaneous infusions at least 4-6 d a week have clearly demonstrated major effects on iron overload complications. DFO treatment reduces excessive iron and prevents cardiac, hepatic and endocrine diseases. Nonetheless, compliance is difficult for many patients and the cost of DFO limits its use in developing countries. The only oral iron chelating agent that has been investigated extensively is Deferiprone (L1). In France, this oral agent can be administered in patients experiencing toxic side effects under DFO treatment. Since 1981 more than 1500 bone-marrow transplants have been performed word-wide, mostly in Italy. Allogenic BMT is currently able to cure 85% of thalassemic children with an available HLA matched sibling donor.     

Reversion of age-related recognition memory impairment by iron chelation in rats

It is now generally accepted that iron accumulates in the brain during the ageing process. Increasing evidence demonstrate that iron accumulation in selective regions of the brain may generate free radicals, thereby possessing implications for the etiology of neurodegenerative disorders. In a previous study we have reported that aged rats present recognition memory deficits. The aim of the present study was to evaluate the effect of desferoxamine (DFO), an iron chelator agent, on age-induced memory impairment. Aged Wistar rats received intraperitoneal injections of saline or DFO (300mg/kg) for 2 weeks. The animals were submitted to a novel object recognition task 24h after the last injection. DFO-treated rats showed normal recognition memory while the saline group showed long-term recognition memory deficits. The results show that DFO is able to reverse age-induced recognition memory deficits. We also demonstrated that DFO reduced the oxidative damage to proteins in cortex and hippocampus. Thus, the present findings provide the first evidence that iron chelators might prevent age-related memory dysfunction.     

Effect of methylene chain length in phospholipid moiety on blood compatibility of phospholipid polymers

To investigate the effects of the methylene chain length between the phospholipid polar group and the backbone on blood compatibility of a phospholipid polymer, copolymers of ω-methacryloyloxyalkyl phosphorylcholine (MAPC) with n-butyl methacrylate (BMA) were synthesized. The methylene chains were ethylene (n = 2), tetramethylene (n = 4), and hexamethylene (n = 6). Every MAPC copolymer with an MAPC mole fraction in the range of 0.1-0.3 was soluble in ethanol but only swelled in water, and the equilibrium water fraction of the water-swollen MAPC copolymer membrane decreased with the length of the methylene chain. When a rabbit platelet-rich plasma was applied on the MAPC copolymer surface with an 0.1 MAPC mol fraction for 180 min, the number of adhered platelets depended on the length of the methylene chain in the MAPC moiety of the copolymer. The amount of phospholipid adsorbed on the MAPC copolymer from human plasma was larger than that on hydrophobic poly(BMA) and increased with the length of the methylene chain in the MAPC moiety. That is, the reduction of platelet adhesion corresponded to the increase in the amount of phospholipid adsorbed on the MAPC copolymer.     

A smart polymeric platform for multistage nucleus-targeted anticancer drug delivery

Tumor cell nucleus-targeted delivery of antitumor agents is of great interest in cancer therapy, since the nucleus is one of the most frequent targets of drug action. Here we report a smart polymeric conjugate platform, which utilizes stimulus-responsive strategies to achieve multistage nuclear drug delivery upon systemic administration. The conjugates composed of a backbone based on N-(2-hydroxypropyl) methacrylamide (HPMA) copolymer and detachable nucleus transport sub-units that sensitive to lysosomal enzyme. The sub-units possess a biforked structure with one end conjugated with the model drug, H1 peptide, and the other end conjugated with a novel pH-responsive targeting peptide (R8NLS) that combining the strength of cell penetrating peptide and nuclear localization sequence. The conjugates exhibited prolonged circulation time and excellent tumor homing ability. And the activation of R8NLS in acidic tumor microenvironment facilitated tissue penetration and cellular internalization. Once internalized into the cell, the sub-units were unleashed for nuclear transport through nuclear pore complex. The unique features resulted in 50-fold increase of nuclear drug accumulation relative to the original polymer–drug conjugates in vitro, and excellent in vivo nuclear drug delivery efficiency. Our report provides a strategy in systemic nuclear drug delivery by combining the microenvironment-responsive structure and detachable sub-units.     

Synthese und Charakterisierung von Copolymeren aus Azo-Initiatoren und Styrol

Copolymers with azoinitiator functions were prepared from styrene and different arylazoalkylmalonodinitriles by copolymerization in emulsion, using redox-initiators. This type of copolymer can be used for syntheses of graft copolymers. The concentration of azo-groups per polymer chain, the polymerization rate, and the molecular weight of the isolated copolymers were determined as a function of the composition of the starting monomer mixture and the structure of the azo-compounds. It was tried to separate the crude product of the grafting reaction into the ungrafted backbone, the graft-copolymer, and the homopolymer.     

Metalloporphyrins in polymeric matrices and in micelles, 2. Iron porphyrins and cobalt porphyrins in copolymers of styrene and 1-vinylimidazoles

Copolymers from styrene and 1-vinylimidazoles ( 4a – f ), form low spin adducts with iron(II) porphyrins and iron(III) porphyrins, when the content of imidazolyl groups in the polymer is ten mole percent or more. With polymers having a content of imidazolyl groups of less then one mole percent, only high spin adducts are observed. With terpolymers, in which the porphyrin is covalently bound to the copolymer the same phenomenon is observed. Only the high spin Fe(II) porphyrin adducts in the solid state adsorb molecular oxygen like cobalt(II) porphyrins under the same conditions. Adsorptions and desorptions of oxygen are much slower in the cases of iron(II) porphyrins than with the cobalt(II) porphyrins. Electronic and ESR spectra as well as susceptibility measurements were used to characterize the reported spin states.     

A role for hypoxia-inducible factor-1alpha in desferoxamine neuroprotection

The newborn brain has increased vulnerability to hypoxia-ischemia from maturational differences in the oxidative stress response. We hypothesized that desferoxamine (DFO), an iron chelator, would provide protection in an in vitro model of ischemia in part through activation of the hypoxia-inducible gene hypoxia-inducible factor-1alpha (HIF-1alpha). Hippocampal neurons from E16 CD1 mice were exposed to 3 h of oxygen and glucose deprivation with and without pretreatment with 10 mmol/L DFO in the presence and absence of 2 micromol/L antisense oligonucleotides specific for HIF-1alpha (antiHIF-1alpha). DFO pretreatment resulted in 45% reduction in cell death (p = 0.006). This protection was diminished with transfection of antiHIF-1alpha (p = 0.049). Blocking HIF-1alpha reduces DFO protection suggesting that DFO protects through iron chelation and HIF-1alpha induction.