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Francesco M Veronese,
Oddone Schiavon,
Gianfranco Pasut,
Raniero Mendichi,
Lars Andersson,
Anders Tsirk,
Jayne Ford,
Gefei Wu,
Samantha Kneller,
John Davies, Ruth Duncan
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ABSTRACT: Polymer-drug conjugates (polymer therapeutics) are finding increasing use as novel anticancer agents. Here a series of poly(ethylene glycol) PEG-doxorubicin (Dox) conjugates were synthesized using polymers of linear or branched architecture (molecular weight 5000-20000 g/mol) and with different peptidyl linkers (GFLG, GLFG, GLG, GGRR, and RGLG). The resultant conjugates had a drug loading of 2.7-8.0 wt % Dox and contained <2.0% free drug (% total drug). All conjugates containing a GFLG linker showed approximately 30% release of Dox at 5 h irrespective of PEG molecular weight or architecture. The GLFG linker was degraded more quickly (approximately 57% Dox release at 5 h), and the other linkers more slowly (<16% release at 5 h), by lysosomal enzymes in vitro. In vitro there was no clear relationship between cytotoxicity toward B16F10 cells and the observed Dox release rate. All PEG conjugates were more than 10-fold less toxic (IC50 values > 2 microg/mL) than free Dox (IC50 value = 0.24 microg/mL). Biodistribution in mice bearing sc B16F10 tumors was assessed after administration of PEGs (5000, 10000, or 20000 g/mol) radioiodinated using the Bolton and Hunter reagent or PEG-Dox conjugates by HPLC. The 125I-labeled PEGs showed a clear relationship between Mw and blood clearance and tumor accumulation. The highest Mw PEG had the longest plasma residence time and consequently the greatest tumor targeting. The PEG-Dox conjugates showed a markedly prolonged plasma clearance and greater tumor targeting compared to free Dox, but there was no clear molecular weight-dependence on biodistribution. This was consistent with the observation that the PEG-Dox conjugates formed micelles in aqueous solution comprising 2-20 PEG-Dox molecules depending on polymer Mw and architecture. Although PEG-Dox showed greater tumor targeting than free Dox, PEG conjugation led to significantly lower anthracycline levels in heart. Preliminary experiments to assess antitumor activity against sc B16F10 in vivo showed the PEG5000linear (L)-GFLG-Dox and PEG10000branched (B)-GLFG-Dox (both 5 mg/kg Dox-equiv) to be the most active with T/C values of 146 and 143%, respectively. Free Dox did not show significant activity in this model (T/C = 121%). Dose escalation of PEG5000(L)-GFLG-Dox to 10 mg/kg Dox-equiv prolonged further animal survival (T/C = 161%). Using the Dox-sensitive model ip L1210 (where Dox displayed a T/C = 150% after single ip dose), the PEG5000(L)-GFLG-Dox displayed a maximum T/C of 141% (10 mg/kg Dox-equiv) using a once a day (x3) schedule. Further studies are warranted with PEG5000(L)-GFLG-Dox to determine its spectrum of antitumor activity and also the optimum dosing schedule before clinical testing.
Bioconjugate Chemistry 05/2005; 16(4):775-84. · 4.93 Impact Factor
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Lars Andersson,
John Davies, Ruth Duncan,
Paolo Ferruti,
Jayne Ford,
Samantha Kneller,
Raniero Mendichi,
Gianfranco Pasut,
Oddone Schiavon,
Clive Summerford,
Anders Tirk,
Francesco M Veronese,
Veronica Vincenzi,
Gefei Wu
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ABSTRACT: Water soluble polymer anticancer conjugates can improve the pharmacokinetics of covalently bound drugs by limiting cellular uptake to the endocytic route, thus prolonging plasma circulation time and consequently facilitating tumor targeting by the enhanced permeability and retention (EPR) effect. Many of the first generation antitumor polymer conjugates used nonbiodegradable polymeric carriers which limits the molecular weight that can be safely used to <40,000 g/mol. The aim of this ambitious study was to synthesize and evaluate a novel, prototype biodegradable polymeric system based on high molecular weight, water-soluble functionalized polyesters. The main polymeric platform was prepared from bis(4-hydroxy)butyl maleate (DBM) and poly(ethylene glycol) (PEG4000) blocks to give the polymer DBM2-PEG4000 containing biodegradable carbonate bonds and having a M(w) of 100,000-190,000 g/mol; M(n) of 37,000-53,000 g/mol, and M(w)/M(n) of 3.0-3.7. Using thioether linkages, this polymer was then grafted with HS-PEG3000-Gly-Phe-Lue-Gly doxorubicin (HS-PEG3000-GFLG-Dox) pendant side chains ( approximately 30 per DBM2-PEG chain). The final construct, DBM2-PEG4000-S-PEG3000-GFLG-Dox had a total Dox content of 3-4 wt % and a free Dox content of < or = 0.7% total Dox. During incubation with isolated lysosomal enzymes, the rate of Dox release from the polymer backbone was relatively slow (<5% release over 5 h) compared to that seen for PEG5000-GFLG-Dox alone (>20% over 5 h). The in vitro cytotoxicity was assessed using B16F10 murine melanoma (MTT assay). DBM2-PEG4000-S-PEG3000-GFLG-Dox was 10-20-fold less toxic than free Dox. In vivo antitumor activity of the DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates was assessed using a subcutaneous (s.c.) B16F10 murine melanoma model, and an intraperitoneal (i.p.) L1210 leukaemia model. The increased toxicity (attributed to poor solubility) and low antitumor activity of DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates compared to PEG5000-GFLG-Dox and HPMA copolymer-Dox conjugates was attributed to the slow rate of Dox release. The DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates were considered unfavorable as candidates for further development. However, the successful scale-up synthesis of DBM2-PEG4000-S-PEG3000 constructs suggest that they are worthy of further investigation as carriers for controlled release and targeting of less hydrophobic agents.
Biomacromolecules 01/2005; 6(2):914-26. · 5.48 Impact Factor
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ABSTRACT: The poly(amidoamine)s (PAAs) ISA 1 and ISA 23 display pH-dependent conformational change and pH-dependent membrane perturbation. These properties confer potential for use as endosomolytic polymers for intracytoplasmic delivery of toxins and genes. Both polymers are relatively non-toxic, and moreover ISA 23 has the beneficial property in vivo, of being non hepatotropic when administered intravenously. Although ISA 23 and ISA 1 demonstrate ability to transfect cells, ISA 1 is also able to promote intracellular delivery of non-permeant toxins. The aim of this study was to synthesise random and block copolymers of ISA 1 and ISA 23 and investigate whether these second generation hybrids would allow optimisation of PAA biological characteristics. Random and block copolymers of ISA 1 and ISA 23 were synthesised by hydrogen transfer polyaddition to generate a library of PAAs with an ISA 23:ISA 1 molar ratios of 2:1 to 4:1. The resultant polymers have a pI slightly below 7.4 and a M(w) of 19,900-49,000 g/mol and a M(n) of 13,100-24,100 g/mol. Whereas none of the random or block copolymers were haemolytic at pH 7.4 all demonstrated pH-dependent membrane activity. At pH 5.5 they caused 50-60% haemoglobin (Hb) release over 1 h. This was slightly less than that seen for ISA 23 (80% Hb release). None of the copolymers were cytotoxic against B16F10 cells during a 72 h incubation (IC(50) > 2 mg/ml; MTT assay). The ability of the random and block copolymer PAAs to deliver the toxin gelonin was also examined, but only ISA 1 and the block copolymer B2 (ISA 23:ISA 1 at a 2:1 molar ratio) were able to promote intracellular delivery, as measured by cytotoxic activity. It would be interesting to study the body distribution of B2 and determine whether this toxin-delivering PAA is able to escape liver capture.
Macromolecular Bioscience 11/2004; 4(10):922-9. · 3.89 Impact Factor
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Sunil Shaunak,
Sharyn Thomas,
Elisabetta Gianasi,
Antony Godwin,
Emma Jones,
Ian Teo,
Kamiar Mireskandari,
Philip Luthert, Ruth Duncan,
Steve Patterson,
Peng Khaw,
Steve Brocchini
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ABSTRACT: Dendrimers are hyperbranched macromolecules that can be chemically synthesized to have precise structural characteristics. We used anionic, polyamidoamine, generation 3.5 dendrimers to make novel water-soluble conjugates of D(+)-glucosamine and D(+)-glucosamine 6-sulfate with immuno-modulatory and antiangiogenic properties respectively. Dendrimer glucosamine inhibited Toll-like receptor 4-mediated lipopolysaccharide induced synthesis of pro-inflammatory chemokines (MIP-1 alpha, MIP-1 beta, IL-8) and cytokines (TNF-alpha, IL-1 beta, IL-6) from human dendritic cells and macrophages but allowed upregulation of the costimulatory molecules CD25, CD80, CD83 and CD86. Dendrimer glucosamine 6-sulfate blocked fibroblast growth factor-2 mediated endothelial cell proliferation and neoangiogenesis in human Matrigel and placental angiogenesis assays. When dendrimer glucosamine and dendrimer glucosamine 6-sulfate were used together in a validated and clinically relevant rabbit model of scar tissue formation after glaucoma filtration surgery, they increased the long-term success of the surgery from 30% to 80% (P = 0.029). We conclude that synthetically engineered macromolecules such as the dendrimers described here can be tailored to have defined immuno-modulatory and antiangiogenic properties, and they can be used synergistically to prevent scar tissue formation.
Nature Biotechnology 09/2004; 22(8):977-84. · 23.27 Impact Factor
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ABSTRACT: An efficient total synthesis of cis-solamin (1) has been achieved in 21% overall yield and with a longest linear sequence of just 11 steps from aldehyde 8. A key feature of the approach was the use of asymmetric permanganate-promoted oxidative cyclization to introduce four of the five required stereocenters in a single step. The use of robust and chemoselective methodology meant that the use of protecting groups could be avoided during the assembly of cis-solamin (1) from the three fragments 23, 6, and 4. The methodology was also applied to the synthesis of three further cis-solamin isomers 2, ent-1, and ent-2. Cytotoxicity and hemolytic properties of cis-solamin isomers and synthetic intermediates are reported.
The Journal of Organic Chemistry 06/2004; 69(10):3368-74. · 4.45 Impact Factor
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ABSTRACT: On exposure to an acidic pH, linear poly(amidoamine)s (PAAs) cause membrane perturbation and consequently have potential as endosomolytic polymers for the intracellular delivery of genes and toxins. Previous studies used PAAs in the hydrochloride form only. The aim of this study was to investigate systematically the effect of the PAA counterion on pH-dependent membrane activity, general cytotoxicity, and PAA solution properties to help guide optimization of PAA structure for further development of PAA-protein conjugates. PAAs (ISA 1, 4, 22, and 23; M(w) 10000-50000 g/mol) were synthesized to provide a library of PAAs having different counterions including the acetate, citrate, hydrochloride, lactate, phosphate, and sulfate salts. pH-Dependent membrane activity was assessed using a rat red blood cell haemolysis assay (conducted at a starting pH of 7.4, 6.5, or 5.5; 1 mg/mL; 1 h), and general cytotoxicity was investigated using a murine melanoma cell line (B16F10) and a human bladder endothelial-like cell line (ECV-304). Whereas poly(ethyleneimine) was haemolytic at the starting pH of 7.4 at 1 h [ approximately 50% haemoglobin (Hb) release], none of the PAA salts were haemolytic at a starting pH of 7.4 or 6.5. Although PAA acetate, citrate, and lactate were also non-haemolytic at the starting pH of 5.5, the sulfate and hydrochloride forms caused significant haemolysis (up to 80% Hb release) and ISA 22 and 23 phosphate were also markedly haemolytic ( approximately 70% Hb release). These counterion-specific differences were also clearly visible using scanning electron microscopy, which was used to visualize the red blood cell morphology. All PAAs were relatively nontoxic (IC(50) >or= 300-5000 microg/mL) compared to poly-l-lysine (IC(50) = 2-10 microg/mL), the PAA hydrochloride salts produced the greatest cytotoxicity, and the B16F10 cells were more sensitive than the ECV-304 cells. Small-angle neutron scattering suggested that ISA 23 hydrochloride had a larger hydrodynamic radius (5.1 +/- 0.2 nm) than the citrate salt (3.1 +/- 0.2 nm). These results provide indirect evidence for the salt- and pH-dependent changes in the conformation of the polymer coil. This study clearly demonstrates the importance of optimization of the counterion form when developing endosomolytic polymers designed to mediate pH-dependent membrane permeabilization.
Biomacromolecules 03/2004; 5(3):1102-9. · 5.48 Impact Factor
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ABSTRACT: Those polymer anticancer-drug conjugates currently undergoing clinical evaluation have a tripartite structure; a water-soluble polymer, an anticancer agent and a pendant linker. To simplify the construct it would be attractive to develop anticancer polymer therapeutics that contain the bioactive agent as an integral part of the polymer backbone. The aim of this study was to utilise the reaction between a divinyl ethers and diols, to synthesise polyacetals incorporating a drug with bis-hydroxyl functionality into the polymer backbone. Degradation of the polymer backbone in the acidic environment of the lysosome or the extracellular fluid of some tumours would then trigger drug release eliminating the need for a biodegradable linker. A tert-polymerisation approach was used to incorporate non-steroidal oestrogen diethylstilboestrol (DES) into the mainchain of water-soluble polyacetals synthesised using as co-monomer PEG of Mw 2900 or 3400 g/mol. When PEG2900 was used the resultant polymer had a Mw of 18,900 g/mol, a Mw/Mn of 1.9 and a DES loading 4.3 wt.%. With PEG3400 the polymer Mw was 43,000 g/mol, Mw/Mn=1.8 and it had a DES loading 4.7 wt.%. 1H-NMR confirmed the presence of two distinct sets of acetal peaks, which correspond to the two possible mainchain acetals; from PEG at 1.25-1.3(d) and 4.7-4.8(q) ppm and from DES at 1.55-1.6(d) and 5.4-5.5(q) ppm. These were consistent with the acetal signals observed for the non-water-soluble co-polymer DES: tri(ethylene glycol) divinyl ether (TEGDVE) (1 : 2, Mw=6859 g/mol, Mw/Mn=1.3). When evaluated in vitro, the DES-polyacetal displayed greater cytotoxicity than DES against human and murine tumour cell lines (IC50=48 and 420 microg/ml against MCF-7 human breast cancer cells and IC50=97 and 560 microg/ml against B16F10 murine melanoma cells, respectively). These polymers showed no significant haemolysis at concentrations up to 20 mg/ml confirming suitability for further in vivo evaluation. An enhanced rate of hydrolytic degradation of the polymer backbone was seen at pH 5.5, (65% trans-DES released in 96 h), compared to pH 7.4 (4% trans-DES released in 96 h). These bioresponsive DES-polyacetals tert-polymers are the first water-soluble anticancer polymeric drugs designed for acidic pH-triggered release of a drug incorporated into the polymer mainchain. Their in vitro characteristics suggest further in vivo evaluation is warranted.
Journal of Drug Targeting 02/2004; 12(8):491-501. · 2.70 Impact Factor
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ABSTRACT: 1,5-diazaanthraquinones (DAQs) are promising anticancer drugs, however, their clinical potential is limited due to poor solubility. Conjugation of anticancer agents to hydrophilic water-soluble polymers can overcome this problem and has already been used to generate conjugates with demonstrated clinical benefit. Here a library of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates containing a novel amino-functionalised 1,5-diazaanthraquinone derivative (amino-DAQ) have been synthesised. The conjugates were fully characterised by UV, HPLC, SEC, FT-Raman and NMR spectroscopy. Conjugation to HPMA copolymers improved amino-DAQ aqueous solubility (>7-fold). The HPMA copolymer-amino-DAQ conjugates were slightly less haemolytic than the parent compound (2% Hb released in 1 h for conjugate HPMA copolymer-GFLG (5 mol%)-amino-DAQ conjugate compared to 13% obtained with amino-DAQ). When conjugates were incubated with isolated rat liver lysosomal enzymes (Tritosomes) the rate of amino-DAQ release was influenced by both drug loading and the composition of the peptidyl side chain used to link the drug to the carrier. The higher the drug loading the lower the rate of drug release. Whereas the GG linker did not release amino-DAQ, up to 26% of the amino-DAQ was released from a GFLG linker over 24 h. The in vitro cytotoxicity of these conjugates was evaluated against two different cell lines, B16F10 murine melanoma and MCF-7 human breast cancer cells. HPMA copolymer-amino-DAQ conjugates, which are internalised by cells by the endocytic pathway, showed much lower in vitro cytotoxicity (IC50 for HPMA copolymer-GFLG (5 mol%)-amino-DAQ conjugate>397 microM drug-equiv.) than the free drug (the IC50 for amino-DAQ was 12.6 and 2.8 microM against the B16F10 murine melanoma and the MCF-7 breast cancer cell line, respectively). Nonetheless, the observed lysosomal activation of the HPMA copolymer-GFLG-amino-DAQ conjugates, suggests that evaluation of the antitumour potential in vivo is warranted.
Journal of Drug Targeting 02/2004; 12(8):503-15. · 2.70 Impact Factor
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Advanced Drug Delivery Reviews 12/2003; 55(11):1353-7. · 11.50 Impact Factor
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ABSTRACT: Polymer conjugates are becoming established as a new approach towards improved cancer therapy. These water-soluble, hybrid constructs fall into two main categories: polymer-protein conjugates (already available as licensed products), and polymer-drug conjugates (currently in clinical development). Polyethyleneglycol conjugation of proteins is accepted as a means to reduce immunogenicity, prolong plasma half-life and enhance protein stability. Polymer-drug conjugation promotes tumor targeting by the 'enhanced permeation and retention' effect, and at the cellular level, allows lysosomotropic drug delivery. Eleven polymer-drug conjugates have entered clinical development and activity has already been observed in chemotherapy refractory patients. Certain compounds have also demonstrated a marked reduction in drug toxicity.
Current opinion in investigational drugs (London, England: 2000) 07/2003; 4(6):701-9. · 3.31 Impact Factor
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Ruth Duncan
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ABSTRACT: As we enter the twenty-first century, research at the interface of polymer chemistry and the biomedical sciences has given rise to the first nano-sized (5-100 nm) polymer-based pharmaceuticals, the 'polymer therapeutics'. Polymer therapeutics include rationally designed macromolecular drugs, polymer-drug and polymer-protein conjugates, polymeric micelles containing covalently bound drug, and polyplexes for DNA delivery. The successful clinical application of polymer-protein conjugates, and promising clinical results arising from trials with polymer-anticancer-drug conjugates, bode well for the future design and development of the ever more sophisticated bio-nanotechnologies that are needed to realize the full potential of the post-genomic age.
dressNature Reviews Drug Discovery 06/2003; 2(5):347-60. · 29.01 Impact Factor
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Macromolecular Bioscience 02/2003; 3(1):59 - 66. · 3.89 Impact Factor
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ABSTRACT: N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer platinates were prepared from polymeric intermediates containing Gly-Phe-Leu-Gly side chains terminating in either malonate or aspartate dicarboxylato ligands. Platinum(II) was bound by reaction of the dicarboxylato ligands with cis-[Pt(NH3)2(H2O)2]2+. The HPMA copolymer platinates obtained had a Mw of 29,000-31,000 Da and a platinum loading of approximately 10wt% (by AAS). This is close to the theoretical maximum value. The release rate of platinum species in vitro at pH 7.4 correlated with the expected stability of the 6 and 7 membered chelate rings; 14%/24 h platinum released in the case of the malonate and 68%/24 h platinum released in the case of the aspartate. Cisplatin and the aspartate conjugate displayed similar toxicity in vitro against B16F10 and COR-L23 cells while the malonate was at least 8-fold less toxic. The malonate conjugate showed significantly improved activity (T/C = 1.27-1.5) when compared with cisplatin (T/C = 1.18) that was not active when administered intravenously to treat a subcutaneous B16F10 tumour. The conjugate was at least 20-fold less toxic than cisplatin in vivo. After i.v. administration, the platinum accumulation in B16F10 tumour tissue showed a 19-fold increase in Pt AUC for the malonate conjugate when compared to cisplatin administered equi-dose at its maximum tolerated dose (MTD) (1 mg/kg).
Journal of Drug Targeting 12/2002; 10(7):549-56. · 2.70 Impact Factor
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ABSTRACT: The synthetic polymers that are used to prepare polymer therapeutics reaching clinical use are predominantly nonbiodegradable, and this severely limits the molecular weight range that will give certainty of safe elimination. The aim of this study was to synthesize water-soluble, biocompatible, amino-functionalized polyacetals that would display pH-dependent degradation and, moreover, be suitable for drug conjugation. To test the feasibility of the synthetic procedure, polyacetals were first prepared by the reaction of a diol (e.g., poly(ethylene glycol) (PEG)) and a divinyl ether (e.g., tri(ethylene glycol) divinyl ether) using an acid catalyst. Using PEG3400, these polyacetals had a Mw of 36 000−43 000 g/mol (Mw/Mn = 1.6−1.8) and displayed pH-dependent degradation. An enhanced rate of hydrolysis was seen at pH 5.5 (41% Mw loss in 25 h) compared to pH 7.4 (10% Mw loss in 73 h). The polymers and their degradation products were nontoxic toward B16F10 cells in vitro (IC50 > 5 mg/mL), and they were also nonhemolytic (rat red blood cells). Several approaches were examined to produce amino-functionalized polyacetals. It was found that modification of either the divinyl ether or PEG monomer was not the best strategy. However, terpolymerization, for example using the hydrolytically stable diol 9-fluorenylmethyloxycarbonyl (Fmoc)-serinol, PEG3400, and tri(ethylene glycol) divinyl ether, did produce functionalized polyacetals of Mw 20 000−77 000 g/mol and Mw/Mn = 1.8−2.0. Varying the ratios of diol monomer gave a family of polymers containing different amounts of pendent group. One of these amino-polyacetals was used to prepare a polymer containing 125I-labeled Bolton−Hunter reagent (74 μCi/mg), introduced to facilitate a preliminary biodistribution study after intravenous administration to rats. The polyacetals showed no preferential accumulation in the major organs (at 1 h; liver (4.2 % dose), lung (0.7%), and kidney (1.1%)), and the log blood clearance with time was linear over 24 h. These novel, biodegradable polyacetals have potential for further development as polymer therapeutics and more generally as a new family of biodegradable polymers.
12/2001;
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ABSTRACT: Five poly(amido-amine)s (PAAs) carrying two ter-amino groups and one carboxyl group per repeating unit were prepared by hydrogen-transfer polyaddition of 2-methylpiperazine (ISA 23), 1,2-bis(N-methylamino)ethane (DMEDA-BAC), 1,2-bis(N-ethylamino)ethane (DEEDA-BAC, 1,3-bis(N-methylamino)propane (DMEPDA-BAC), or 1,6-bis(N-methylamino)hexane (DMEXA-BAC), in each case to 2,2-bis(acrylamido)acetic acid (BAC). The resultant PAAs had an Mn in the range 7 985−24 980 g/mole and an Mw in the range 11 420−42 710 g/mole. Considerable differences were observed in the basicity of the amino groups present (log K°1 = 7.5−9.5; log K°2 = 3.2−8.4), whereas the log K°3 value (2−3) of the carboxyl groups was consistent with that of a fairly strong acid. DEEDA-BAC, DMEDA-BAC, and ISA 23 were nontoxic (IC50 > 5 mg/mL). Those PAAs with the highest log K°2 values were more cytotoxic (IC50 = 3.55 mg/mL for DMEPDA-BAC, and IC50 = 0.23 m/mL for DMEXA-BAC). All the PAAs displayed pH-dependent haemolysis (most lytic at pH 5.5), consistent with their proposed use as endosomolytic polymers.
09/2000;
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ABSTRACT: Polyamidoamine polymers were prepared by hydrogen-transfer polyaddition of 2-methylpiperazine to 2,2′-bis(acrylamido)acetic acid sodium salt to yield PAA-1, polyaddition of amino-β-cyclodextrin and 2-methylpiperazine to 2,2′-bis(acrylamido)acetic acid to give PAA-2 and polyaddition of the same amino-β-cyclodextrin and 2-methylpiperazine to 1,4-bis(acryloyl)piperazine to produce PAA-3. These polymers were reacted with cisplatin to give products containing between 8–70 wt.-% platinum. The amount of platinum released from the conjugates during incubation at pH 5.5 and pH 7.4 varied between 0–20%/72 h. PAA-3-Pt showed pH-dependent platinum release. The PAA-platinates were generally less toxic towards lung tumour cell lines in vitro. The IC50 for cisplatin being 2–5 μg/mL and for the PAA-platinates 1–130 μg/mL, this was only to be expected due to their very different cellular pharmacokinetics. In vivo experiments showed that the PAA-1-Pt and PAA-2-Pt were equi-active compared with cisplatin against an i.p. L1210 leukaemia model, confirming their ability to liberate biologically active platinum species. Whereas PAA-1-Pt was significantly less toxic than cisplatin, PAA-2-Pt did show toxicity on repeated dosing, suggesting further investigations are needed to establish the biocompatibility of PAAs containing pendant β-cyclodextrin. PAA-1-Pt is suitable for further in vivo preclinical study in a range of solid tumour models.
Macromolecular Chemistry and Physics 06/1999; 200(7):1644 - 1654. · 2.36 Impact Factor
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ABSTRACT: Previously it has been shown that bioerodible poly (ortho ester) matrices can be synthesised to contain the anticancer drug 5-fluorouracil (5-FU) and, with adjustment of their suberic acid excipient content, designed to produce approximately zero order release of 5-FU in vitro over 15 days. This short study describes the first evaluation of such matrices against a human colorectal carcinoma model in vivo (a LS174T xenograft), colorectal cancer being the ultimate therapeutic target. First, in order to assess the potential general toxicity of the polymer, between one and four drug-free polymer matrices were implanted in the peritoneal cavity of DBA2 mice, and the animals subsequently monitored for 60 days. Following implantation of one to three matrices (a polymer dose equivalent to 8.4 g/kg) animals showed no weight loss, and no overt signs of toxicity. However, implantation of four matrices did produce visible signs of toxicity, even though these animals displayed no significant weight loss. To evaluate their antitumour activity, 5-FU-containing matrices (one to four matrices equivalent to 5-FU doses of 280, 560, 840 and 1120 mg/kg, respectively) were implanted into the peritoneal cavity of nude mice bearing established subcutaneous LS174T xenografts, and the antitumour activity compared with that seen following bolus administration of free 5-FU at a dose of 200 or 500 mg/kg). An increased survival relative to untreated controls was observed following implantation of one, two and three matrices, the values being 122, 178, and 155%, respectively. The animals receiving three 5-FU-containing matrices were eventually withdrawn due to drug toxicity rather than tumour growth, and those receiving four matrices showed very early signs of drug-related toxicity . Although 5-FU did suppress tumour growth, mice treated with free drug showed either no increase in survival (200 mg/kg), or a decreased lifespan due to drug-related toxicity (500 mg/kg). Dose-dependent control of xenograft growth, and prolongation of animal survival was seen following implantation of 5-FU-containing matrices.
Journal of Controlled Release.
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ABSTRACT: Macromolecular therapeutics and nano-sized drug delivery systems often require localisation to specific intracellular compartments. In particular, efficient endosomal escape, retrograde trafficking, or late endocytic/lysosomal activation are often prerequisites for pharmacological activity. The aim of this study was to define a fluorescence microscopy technique able to confirm the localisation of water-soluble polymeric carriers to late endocytic intracellular compartments. Three polymeric carriers of different molecular weight and character were studied: dextrin (Mw ~ 50,000 g/mol), a N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer (Mw ~ 35,000 g/mol) and polyethylene glycol (PEG) (Mw 5000 g/mol). They were labelled with Oregon Green (OG) (0.3–3 wt.%; < 3% free OG in respect of total). A panel of relevant target cells were used: THP-1, ARPE-19, and MCF-7 cells, and primary bovine chondrocytes (currently being used to evaluate novel polymer therapeutics) as well as NRK and Vero cells as reference controls. Specific intracellular compartments were marked using either endocytosed physiological standards, Marine Blue (MB) or Texas-red (TxR)-Wheat germ agglutinin (WGA), TxR-Bovine Serum Albumin (BSA), TxR-dextran, ricin holotoxin, C6-7-nitro-2,1,3-benzoxadiazol-4-yl (NBD)-labelled ceramide and TxR-shiga toxin B chain, or post-fixation immuno-staining for early endosomal antigen 1 (EEA1), lysosomal-associated membrane proteins (LAMP-1, Lgp-120 or CD63) or the Golgi marker GM130. Co-localisation with polymer–OG conjugates confirmed transfer to discreet, late endocytic (including lysosomal) compartments in all cells types. The technique described here is a particularly powerful tool as it circumvents fixation artefacts ensuring the retention of water-soluble polymers within the vesicles they occupy.
Journal of Controlled Release.
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ABSTRACT: A doxorubicin N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugate (PK1) designed for intracellular lysosomal cleavage following fluid phase pinocytic uptake is currently in early clinical development. This macromolecular prodrug has been encapsulated in niosomes prepared from Span 60/cholesterol/Solulan C24 (a cholesteryl poly-24-oxyethylene ether) (45:45:10). Lipid/surfactant films hydrated with a 3 mg ml−1 solution of PKI using a modification of the dehydration rehydration vesicle (DRV) method gave an encapsulation efficiency of 49.0 ± 1.54%. Vesicle size was 583 ± 191 nm. Span 60 PK1 niosomes were photographed by optical and transmission electron microscopy and found to have a bright fluorescence on the outside of the vesicles and diminished fluorescence in the vesicle core. This is thought to be due to fluorescence quenching of the higher concentration of PK1 inside the niosomes. Span 60 PK1 niosomes stored freeze dried at -40°C, 4°C and 25°C were completely stable and when stored as a liquid suspension at 4°C and 25°C retain 75% of their encapsulated material even after 28 days. Addition of excipients, e.g. polyethylene glycol 8000 and polyvinylpyrrolidone at the rehydration step increased the PK1 encapsulation efficiency to 62% and 65%, respectively. Incubation of PK1 niosomes with plasma resulted in less than 0.02% doxorubicin release after 72 h. When PK1 niosomes were incubated with a lysosomal enzyme preparation doxorubicin release increased to 7% after 72 h. PK1 niosomes have potential for use in targeted cancer chemotherapy.
International Journal of Pharmaceutics.
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Journal of Drug Targeting 15(7-8):456. · 2.70 Impact Factor
