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Strategies for Covalent Attachment of Doxorubicin to Poly(PEG-Lys), a New Water-Soluble Poly(ether urethane
Abstract
Poly(PEG-Lys) is a new, water soluble poly(ether urethane) that has shown promise as an injectable drug carrier. To evaluate the possible use of this drug carrier in chemotherapy, three different approaches for the covalent attachment of doxorubicin to the pendent carboxylic acid groups of poly(PEG- Lys) were developed. In one approach, the pendent carboxylic acid groups of poly(PEG-Lys) were converted to N-hydroxysuccinimide active esters, which spontaneously formed hydrolytically stable amide bonds upon reaction with the amino group located on the daunosamine ring of doxorubicin. The amount of amide-bound doxorubicin was about 7.3 mg/100 mg of conjugate. In a second approach, the degradable hydrazone linkage was formed by reaction of the polymeric hydrazide derivative of poly(PEG-Lys), designated as poly(PEG-Lys hydrazide), with the 13-keto group of doxorubicin. After purification, the amount of carrier-bound doxorubicin was 13.5 mg/100 mg of conjugate. In the third approach, the conjugation of doxorubicin via secondary amine linkages was explored. In this approach, the aldehyde derivative of poly(PEG Lys), desig nated as poly(PEG-Lys-aldehyde), was reacted with doxorubicin, followed by re duction of the intermediate Schiff base with sodium cyanoborohydride. After extensive purification of the carrier, the amount of bound doxorubicin was 10 mg/100 mg of conjugate. All conjugates were characterized by UV/Vis and FTIR spectroscopy and by thin layer chromatography. The conjugates were free of de tectable contamination by unbound drug.
... More extensive research has been focused on the synthesis of PEG block copolymers with the potential, not only of generating linear high Mw polymers but also of increasing the carrying capacity of the polymer by the incorporation of functionalised monomers ( Figure 1.8A). This is illustrated by poly(PEG-lysine), which is not biodegradable but was developed to increase the carrying capacity of PEG derived polymers for the attachment of antimicrobials (Nathan et al, 1993) and doxorubicin (Nathan et al. 1994). ...
... Solvent was removed under a flowing stream of nitrogen and the crude product was redissolved in methanol (3 ml) added prior to purification. APEG-DOX ^ was isolated by repeated (2-3 times) LH20 sephadex column separation (2.5x40 cm) using methanol eluent (Nathan et al, 1994). ...
The synthetic polymers that are used to prepare polymer therapeutics reaching clinical use are predominantly non-biodegradable and this severely limits the molecular weight range that will give certainty of safe elimination. The aim of this thesis was to synthesize water-soluble, biocompatible, functionalised polyacetals that would display pH-dependent degradation. Such degradable polymers would not be subject to the same restrictions of molecular weight as non-degradable polymers and provide a platform for the development of improved polymer therapeutics. Several approaches were examined to produce amino-functionalised polyacetals. A terpolymerization, using the hydrolytically stable diol 9-Fluorenylmethyloxycarbonyl (Fmoc)-serinol, PEG3400 and tri(ethylene glycol) divinyl ether, produced functionalised polyacetals of MW = 20-77,000 g/mol and MW/Mn = 1.8-2.0 which displayed pH-dependent degradation. An enhanced rate of hydrolysis was seen at pH 5.5, (~40 % MW loss in 24 h), compared to pH 7.4 (10 % MW loss in 72 h). The polymers and their degradation products were non-toxic towards B16F10 cells in vitro (IC50 > 5 mg/ml) and non-haemolytic. Varying the ratios of diol monomer gave a family of polymers containing different amounts of pendent group. Preliminary biodistribution studies using 125I-labelled polyacetal (APEG) after intravenous (i.v.) administration to rats 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 results prompted the synthesis and characterisation of a high MW polyacetal-DOX conjugates via a succinyl linker using standard carbodiimide coupling reagents with a range of DOX loading (4-8.5 wt% DOX). In vivo pharmacokinetic studies in B16F10 tumour bearing mice indicated that the polyacetal-DOX conjugate exhibited significantly (p < 0.05) prolonged blood circulation times and enhanced tumour accumulation compared with the HPMA copolymer-DOX conjugate (PK1) which is currently in clinical trials. These novel, degradable, polyacetals have potential for further development as polymer-drug conjugates and potentially in other areas of polymer therapeutics.
... Usually, an increased loading value is needed in therapeutic agents with low biological activity, which would otherwise require the administration of a high dose of conjugate with consequent high viscosity of the solution. To this purpose, PEG diols, PEG dendrons [11,12], or dendrimers [13,14], have been proposed for small drug delivery thanks to the possibility of reaching higher drug to polymer ratios and further approaches have been made to synthesize new multifunctional PEGs [15][16][17][18]. Additionally, PEG-dendrimer hybrids where constructed where the PEG components are attached to the apex [19] or to the peripheral groups [20] of dendrimers. ...
This article reviews the use of multifunctional polymers founded on high-molecular weight poly(ethylene glycol) (PEG). The design of new PEG derivatives assembled in a dendrimer-like multimeric fashion or bearing different functionalities on the same molecule is described. Their use as new drug delivery systems based on the conjugation of multiple copies or diversely active drugs on the same biocompatible support is illustrated.
... Prolonged blood circulation, improved solubility of hydrophobic drugs, lower non-specific toxicity or immunogenicity of polymer-modified proteins and peptides and possible targeting are the major advantages of the polymer-modified drugs and biologically active compounds. Hydrophilic polymers based either on poly(N-(2-hydroxypropyl)methacrylamide) (HPMA) [1][2][3][4][5] or PEG [6][7][8][9][10] have been described as carriers of various low-molecularweight drugs. The HPMA-based copolymers were successfully used as carriers of the anti-cancer drug Dox attached to the polymer backbone via an enzymatically [11] or hydrolytically [12] cleavable bond. ...
The synthesis and physico-chemical characterisation of a biodegradable multiblock polymer drug carrier based on poly(ethylene glycol) (PEG) is described. The blocks of PEG (Mw 2,000) are connected by an enzymatically degradable tripeptide derivative consisting of one lysine and two glutamic acid residues. Doxorubicin (Dox), was attached to the polymer carrier via a hydrazone bond susceptible to acid hydrolysis at pH 5.0. Human immunoglobulin (IgG) was covalently linked to the polymer-Dox conjugate by the reaction of 2-pyridyldisulfanyl groups of the polymer with thiol groups of the antibody modified with 2-iminothiolane. The resulting antibody-polymer-drug conjugates were characterised by size-exclusion chromatography, UV/VIS spectrophotometry, electrophoresis and amino acid analysis. All polymers studied (both with and without IgG) showed high anti-proliferative activity against concanavalin A-stimulated murine splenocytes and various cancer cell lines in vitro. The polymer-Dox conjugate (without IgG) exhibited a significant anti-tumor efficacy against murine EL4 T-cell lymphoma and human colorectal carcinoma SW620 in vivo.
... When the drug is covalently bound to the polymer, the drug diffuses more slowly than the free drug and can be absorbed in specific interfaces. 232,233 Dendrimers could be used to increase entrapment of drugs in liposomes, Purohit et al. 234 recently studied the interaction of ampipathic cationic dendrimers with charged and neutral liposomes. Amine terminated PAMAM dendrimers showed increased encapsulation of a model acidic drug in liposomes; this was because dendrimers were entrapped by charge interaction that created pH and solubility gradients across the bilayer and led to an influx of acidic molecules into the liposome. ...
The use of functional foods for health enhancement is of great public interest. Recognition of the powerful functionality of micronutrients has established a new group of food ingredients, called nutraceuticals, positioned between nutrition and medicine. But despite the growing interest in supplementing foods with nutraceutical ingredients such as antioxidants, phytochemicals, omega-3 fatty acids, proboitic bacteria and others, incorporating these components into existing food formulations presents many challenges due to the high concentration of bioactives required to provide specific health benefits, the disagreeable taste and aroma associated with most nutrients, chemical instability and undesirable interaction with other ingredients in the food system, and the risk of reducing bioavailability due to active inability to reach the target site to provide desirable functionality.Various drug delivery systems such as liposomes, solid lipid nanoparticles, micelles, and polymer micro/nanoparticles have shown much promise in controlled release and targeted drug delivery. Colloidal particles in the size range 10-100 nm are considered nanoparticles. Conversion of coarse particles of nutraceutical compounds to nanoparticles or nanosuspensions can be effective in handling the solubility and bioavailability challenges in the delivery of nutraceuticals.This chapter will review a variety of nanoparticles that are currently under consideration as carriers of therapeutic molecules. These include liposomes, cochelates, coacervates, hydrogels, dendrimers, polymeric micelles, nanoemulsions, and others.
... The conjugates were designed to release Dox inside the tumor Table 1 Inhibition of EL4 T-cell proliferation (IC 50 showed a 20 -40-fold decrease in activity compared with free drug. An interesting poly(ether urethane) carrier based on multiblock PEG with lysine moieties between PEG blocks (poly(PEG-Lys)) was used for attachment of Dox via acid-sensitive hydrazone bonds [105]. The amount of Dox in the conjugate was 13.5 wt.%. ...
Use of macromolecular water-soluble carriers of anti-cancer drugs represents a promising approach to cancer therapy. Release of drugs from the carrier system is a prerequisite for therapeutic activity of most macromolecular anti-cancer conjugates. Incorporation of acid-sensitive spacers between the drug and carrier enables release of an active drug from the carrier in a tumor tissue, either in slightly acidic extracellular fluids or, after endocytosis, in endosomes or lysosomes of cancer cells. This paper reviews advances in development and study of properties of various acid-sensitive macromolecular drug delivery systems, starting from simple polymer-drug conjugates to ending with site-specific antibody-targeted polymer-drug conjugates.
... The same group has also prepared PEG conjugates of paclitaxel (21) by introducing an accessible ketone group through esterification of the parent drug with acetylbenzoyl chloride 22, followed by reaction with a series of maleimide containing acylhydrazides (Fig. 46) [129]. Many other researchers have prepared [130,131,132,133] and used antibody-drug conjugates [134,135,136], liposome conjugates [137,138,139], polymer-drug [140,141,142, [16] and the synthesis of hydrazone derivatives of paclitaxel [129]144] and also antibody-polymer-drug and other [145] conjugates containing hydrazone linkages. Very recently, hydrazones have been used to prepare acid sensitive amphiphiles for use in gene delivery [146]. ...
The targeting of drugs specifically to their sites of action is an important strategy for increasing drug efficacy. Chemists have come up with many elegant schemes that aim to convert drugs into magic bullets. This review focuses on the chemistry that underlies these schemes, with particular emphasis on two types of cleavable covalent bonds that are frequently used to link drugs to their various carriers: disulfide bonds and hydrazone bonds. These linkages have been used to release drugs under specific conditions; in the case of disulfides, cleavage is triggered by the mildly reducing environment found in intracellular fluids, and in the case of hydrazones, the acidic conditions that prevail in endosomes cause release of the drug. The applications of these chemistries in drug delivery are reviewed.
"This book introduces and reviews the chemistry, characteristics and application of diesters of H-phosphonic acid and is an excellent source of ideas to those already working or who plan to work in the field of phosphorus-containing compounds."--BOOK JACKET.
The chapter focuses on the important classes of biologically active compounds, such as aminophosphonates, aminophosphonic acids, bisphosphonates, nucleoside H- phosphonates, and 1,2 and 2,3 epoxyalkylphosphonates. The chapter also discusses the methods for synthesis of these compounds, reactivity of these phosphonates, and their synthetic application in the field of chemistry. Aminophosphonic acids constitute an important class of biologically active compounds and their synthesis has been a focus of considerable attention in synthetic organic chemistry as well as in medicinal chemistry. Most of the described methods for the synthesis of aminophosphonic acids use carbonyl compounds, such as aldehydes, ketones, or carboxylic acids as starting compounds. The chapter also focuses on the biological application of poly(alkylene H-phosphonate)s, which are biopolymers with polyphosphate backbones that belong to the particularly studied areas in chemistry, biochemistry, and biology. They can be synthesized by the polycondensation and polymerization methods that are explained in the chapter. The chapter also describes the various metal derivatives of dialkyl H-phosphonates. The usage of Metal salts of H-phosphonate diesters as reagents, catalysts, stabilizers, and corrosion inhibitors is discussed in the chapter.
Poly(ethylene glycol) (PEG) is the gold standard polymer for biomedical applications. PEG is known for its biocompatibility and antifouling properties and is widely used for bioconjugation. However, like other synthetic polymers in the field, PEG is not biodegradable, limiting its use for parenteral formulations and protein conjugation to a molecular weight range with a specific upper limit (commonly 40–60 kDa) to avoid polyether accumulation in human tissue. For these biomedical applications, but also for other purposes such as cleavable hydrogels and templates for porous membranes, several routes for the insertion of in-chain biocleavable moieties, such as acetals or disulfides, into PEG have been developed. Recently, the synthetic strategies have been extended from step-growth polymerizations of commercially available, telechelic PEGs to more sophisticated routes based on ethylene oxide (co)polymerizations, permitting the incorporation of predetermined breaking points at any position in the PEG chains.
The pH sensitive hydrogels composed of N-isopropylacrylamide (NIPA) and acryloyl phenylalanine (APA) were
prepared by redox polymerization using N,N'-methylenebisacrylamide (MBA) as a crosslinker. Anti-inflammatory and analgesic
agent, Keterolac Tromethamine (KT), was loaded successfully into poly(NIPA-co-APA) copolymeric hydrogels by swelling
equilibrium method. To understand the nature of drug in the polymeric matrix, the newly synthesized drug loaded poly(NIPAco-
APA) copolymeric hydrogels were characterized by using differential scanning calorimetry (DSC) and X-ray diffraction
(XRD) techniques. The scanning electron microscopy (SEM) technique result indicates the spherical smooth surface of the
hydrogels. The drug (KT) releasing nature of the poly(NIPA-co-APA) hydrogels was studied in pH 1.2 and 7.4. Effects of drug
loading, crosslinking agent, pH and the ionic strength of the external medium on swelling of hydrogels were also investigated.
Three well known anticancer drugs, doxorubicin, methotrexate and paclitaxel (taxol), have been modified by the permanent attachment of poly(ethylene glycol)(PEG) and evaluated for in vitro cytotoxic activity against murine leukemias P388 and L1210. The relative potencies of the PEG derivatives suggest that modification of antitumor agents with this type of polymer yields compounds that are highly water soluble but less cytotoxic.
A novel synthesis of poly(ethylene glycol) (PEG)-grafted poly(urethanes) (PURs) is described based on a precursor PUR containing free amino groups in the main chain. Three different poly(urethane) backbones were prepared: a homopoly(urethane) comprised of N-Bocdiethanolamine (BDA) and 4,4′-methylenebis(phenyl isocyanate) (MDI), a copoly(urethane) (COPUR) consisting of BDA, N-benzyldiethanolamine and MDI, and a poly(urethane urea) (PUU) that was prepared from BDA, MDI, and ethylenediamine as the chain extender. The Mn of these poly(urethanes) ranged from 32,000 to 72,000 g/mol. PEG (750, 1,900, and 5,000 g/mol) was grafted onto the boc-deprotected poly(urethanes) via the chloroformate. Films of the polymers were spin cast from dilute solutions, annealed, and the surfaces analyzed by goniometry. Water contact angle data indicates increasing PEG surface coverage of the poly(urethanes) with increasing PEG molecular weight. Reorientation of the polymer films is evidenced by contact angle hysteresis. Polymer thrombogenicity, which was studied using blood perfusion experiments, shows that COPUR-g-PEG5000 and PUU-g-PEG5000 exhibit very little platelet adhesion. © 1999 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 37: 3441–3448, 1999
Poly(aspartic acid) (PAA) being biodegradable is suitable for various industrial medical and agricultural applications to replace many non‐biodegradable polymers in use. Poly(aspartic acid) can be synthesized by different methods with and without catalyst in different forms such as polysuccinimide, either hydrolyzed to acid or salt. The polymer of (aspartic acid) is present in different forms such as α,β and L, D isomers. The conformational analysis of poly(aspartic acid) was done by various analytical methods. Different combinations of these two isomer present in different percentage can be detected by various methods such as Hoffman degradation, IR, and NMR spectroscopic analysis. From the standard test for biodegradability, it was shown that the polymer is fully biodegradable. In this review, synthesis and characterization of homo and copolymer derivatives of PAA, along with the application and biodegradability in comparison with the other polymer in use, is discussed briefly.
A new biodegradable poly(l-aspartic acid-co-poly(ethylene glycol)) having pendant amine functional groups was synthesized by the melt polycondensation reaction of prepolymer prepared from N-(benzyloxycarbonyl)-l-aspartic acid anhydride (N-Z-l-aspartic acid anhydride) and poly(ethylene glycol). The synthesized polymer was characterized by FTi.r., 1H n.m.r., d.s.c., g.p.c. and solubility. The weight-average molecular weight of the prepolymer increased about 11 times via melt polycondensation at 160°C in a vacuum for
Proteins and bioactive substances can be modified by chemically binding a synthetic macromolecule, polyethylene glycol (PEG), to the surface of these biomolecules. This can counter some of the drawbacks of native proteins and bioactive substances and improve their properties, which can be important for their use as protein drugs and catalysts in bioreactors. The most important problem is endowing delicate proteins with new suitable properties by chemical modification without causing any loss of their functions. In the medical applications of PEG-proteins, this review deals with anticancer proteins, antiinflammation proteins, blood proteins, antithrombosis proteins and others. These conjugates have unique properties; a reduction of immunoreactivity and immunogenicity, a prolongation of clearance time and an induction of immune tolerance. In biotechnological applications, this review has focused on the synthesis of unstable and/or chiral compounds using PEG-enzymes which are soluble and active in hydrophobic media such as benzene and chlorinated hydrocarbons. In fact, the reverse reactions of hydrolysis proceeds effectively with PEG-lipase and PEG-proteases to form ester and peptide bonds in organic solvents. Chemical modification has also been applied to liposomes, enzyme cofactors and other species. This approach will open new avenues for the development of useful biomedical, pharmacological and biotechnological processes.
The present review deals with the design and preparation of environment-sensitive hyperbranched polymers, such as pH-, temperature-sensitive and salt-triggered polymers, in order to be employed as drug-delivery system. In particular, using known and well-characterized basic hyperbranched polymers as starting materials, this review discusses the kind of structural modifications that these polymers were subjected to for preparing nanocarriers of low toxicity, high encapsulating capacity, specificity to certain biological cells and transport ability under different environment conditions. Due to the great number of external groups of hyperbranched polymers either functionalization or multifunctionalization can occur, providing products that fulfill one or more of the requirements that an effective drug carrier should exhibit. Thus, hyperbranched polymers will play a significant role in the future development of new biomedical devices for the treatment of human diseases.
The synthesis of star-shaped polymers, which are threaded regularly along a common backbone, is described. Thus, two different approaches for preparation of so-called pearl necklace polymers (synonyms: multigraft copolymers, barbwire or centipedes polymers, multiple dumbbells or star-oligomers) are presented. Poly(ethylene oxide) (PEO) was used as a thread and poly(dimethylaminoethyl methacrylate) (PDMAEMA) stars were used as pearls. In the first approach, a polycondensation reaction of PEO macromonomers with partly protected dipentaerythritol leads first to a multiblock PEO. Afterward, the protected hydroxy groups of dipentaerythritol, which is situated at the junction points of the blocks, can be released for the anchoring of PDMAEMA chains. In the second approach, anionic polymerization of ethylene oxide leads directly to a diblock PEO with an interior dipentaerythritol unit. The chain ends can be modified with further dipentaerythritols, giving access after selective deprotection to either star dimers or star trimers. PDMAEMA was either attached by “grafting from” procedure using atom transfer radical polymerization or “grafting to” approach using click chemistry. The advantages and disadvantages of the different approaches are discussed.
The melt polycondensation reaction of the prepolymer prepared from N-(benzyloxycarbonyl)-L-aspartic acid anhydride (N-CBz-L-aspartic acid anhydride) and low molecular weight poly(ethylene glycol) (PEG) using titanium isopropoxide (TIP) as a catalyst produced the new biodegradable poly(L-aspartic acid-co-PEG). This new copolymer had pendant amine functional groups along the polymer backbone chain. The optimal reaction conditions for the preparation of the prepolymer were obtained by using a 0.12 mol % of p-toluenesulfonic acid with PEG 200 for 48 h. The weight-average molecular weight of the prepolymer increased from 1,290 to 31,700 upon melt polycondensation for 6 h at 130°C under vacuum using 0.5 wt % TIP as a catalyst. The synthesized monomer, prepolymer, and copolymer were characterized by FTIR, 1H- and 13C-NMR, and UV spectrophotometers. Thermal properties of the prepolymer and the protected copolymer were measured by DSC. The glass transition temperature (Tg) of the prepolymer shifted to a significantly higher temperature with increasing molecular weight via melt polycondensation reaction, and no melting temperature was observed. The in vitro hydrolytic degradation of these poly(L-aspartic acid-co-PEG) was measured in terms of molecular weight loss at different times and pHs at 37°C. This pH-dependent molecular weight loss was due to a simple hydrolysis of the backbone ester linkages and was characterized by more rapid rates of hydrolysis at an alkaline pH. These new biodegradable poly(L-aspartic acid-co-PEG)s may have potential applications in the biomedical field. © 1998 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 36: 2949–2959, 1998
The synthesis and physico-chemical characterisation of biodegradable multiblock polymer drug carriers based on poly(ethylene glycol) (PEG) are described. The blocks of PEG (M-w = 2000) are interconnected by an enzymatically degradable tripeptide derivative consisting of one Lys and two Glu residues. An anticancer drug, doxorubicin (Dox), was attached to the polymer carrier by a Gly-Phe-Leu-Gly tetrapeptide spacer, which is also susceptible to degradation by lysosomal enzymes. A targeting polyclonal antibody was covalently linked to the polymer-Dox conjugate by the aminolytic reaction of reactive sulfosuccinimidyl ester groups of the polymer with the protein. The resulting antibody-polymer-drug conjugates were characterised by SEC, UV/VIS spectrophotometry and amino acid analysis. Although the studied polymers show only a moderate antiproliferative activity against concanavalin A-stimulated murine splenocytes and a murine T-cell EL4 lymphoma in vitro, they exhibited significant antitumour efficiency against murine T-cell EL4 lymphoma in vivo.
Reductively degradable multiblock polymers for gene and drug delivery were synthesised by oxidative polycondensation of poly(ethylene glycol)‐cysteine derivatives containing ester or urethane bonds. Hydrolysis of the polymers at pH = 5.5, 7.4 and 8.0 and reductive degradation with glutathione and dithiothreitol were studied. The hydrolysis rate of polymer esters increased with increase in pH; the hydrolysis of polymer urethanes was negligible. Chemical substitution of the pendant COOH or NH 2 groups significantly affected the rate of the polymer degradation. Surface modification of poly( L ‐lysine)‐DNA complexes with PEG‐cystine multiblocks led to the formation of reductively degradable nanoparticles.
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The synthesis and characterization of the poly(N-acryloyl chloride) derivatives containing amino acid, carboxyl groups and pyrene in the side chain of the backbone introduced by a post-modification with suitable compounds are reported. Alkaline hydrolysis of the ester groups in poly(N-acryloyl (S)-phenylalanine benzyl ester-acrylic acid-acryloyloxymethyl-pyrene) (PAcPheBz-Py) or its reducing with H2 in the presence of Pd/C (10%) was carried out to prepare terpolymers with free carboxyl groups (PAcPhe-Py1, PAcPhe-Py2), whose structure and composition are similar. Investigation of such structures by the fluorescence spectroscopy indicates a clear dependence of the fluorescence on the polymer structure, the solvent nature, temperature and pH, with a significant increasing of the monomer emission upon addition of CH3OH to the PAcPhe-Py1 solution in THF. Varying the composition and the pH of the debenzylated polymer solution, a conformational change reflected through increasing and decreasing of the fluorescence intensity of the side chain fluorophore was evidenced. Also, the sensing capability of the above pyrene-polymers determined by measuring the fluorescence quenching suggested that these acrylate analogues could find practical applications for detection of amine molecules.
A hyperbranched, functional, and biocompatible polymer, poly(ϵ-caprolactone)-grafted hyperbranched polyester (HPCL), was synthesized via a facile two-step routine as a prospective nontoxic, biodegradable, and biocompatible drug delivery system. 1H-NMR measurements provided direct evidence that ϵ-caprolactone was grafted by a reaction with the peripheral functional hydroxyl groups of hyperbranched polyesters. pH-responsive HPCL in different synthetic body fluids (SBFs) could controllably incubate aspirin. In the different SBFs, the release rates were diverse. In SBF with a pH of 7.4, the release time was nearly 110 h, whereas in SBF with a pH of 6.4 and SBF with a pH of 7.8, it was 185 and 71 h, respectively. In comparison with previously reported systems, our system had a longer release life. © 2008 Wiley Periodicals, Inc. J Appl Polym Sci, 2008
Polyethylene glycol (PEG) is widely used as a covalent modifier of biological macromolecules and particulates as well as a carrier for low molecular weight drugs. In the first two instances proteins and liposomes are of particular importance. Their conjugates with PEG often possess the ability to avoid quick recognition and clearance in vivo, that their unconjugated counterparts are suffering from. In this review (with 133 references) methods for preparation of PEG conjugates with various biologically active compounds are summarized. Since the bulk of the published work in this field involves proteins, drugs, and lipids, an appropriate emphasis is given to the conjugates of these compounds. While the first two types of PEG conjugates are usually intended for a direct use as therapeutics, PEG-lipids are mainly utilized for formation of long-circulating liposomes. Particular attention is paid to the comparative attributes of various reactive PEG derivatives, properties of the linkages formed, and possible side reactions. The relationships between various conjugation strategies and their influence on the relevant biological properties and/or on in vivo performance of the corresponding conjugates is also discussed.
Dendrimers and hyperbranched polymers are a relatively new class of materials with unique molecular architectures and dimensions in comparison to traditional linear polymers. This review details recent notable advances in the application of these new polymers in terms of the development of new polymeric delivery systems. Although comparatively young, the developing field of hyperbranched drug delivery devices is a rapidly maturing area and the key discoveries in drug-conjugate systems amongst others are highlighted. As a consequence of their ideal hyperbranched architectures, the utilisation of host–guest chemistries in dendrimers has been included within the scope of this review.
Syntheses and characterization of polymer conjugates of 9-[2-(phosphonomethoxy)ethyl] (PME) derivatives of adenine (PMEA), 2,6-diaminopurine (PMEDAP) and guanine (PMEG) are described. The phosphonate group of these acyclic nucleotide analogues was activated by reaction with triphenylphosphine and di(2-pyridyl) disulfide (TPP-PDS). The activated phosphonate reacted with a random copolymer containing N -(2-hydroxypropyl)methacrylamide (HPMA) and N -(3-methacrylamidopropanoyl)ethane-1,2-diamine (Ma-AP-ED) units. The phosphonamide bond between the nucleotide and polymer carrier proved to be relatively stable at physiological pH 7.4 while at pH 5.0 (corresponding to endosomal or lysosomal compartments of cells) underwent slow hydrolysis. The rate of hydrolysis (drug release) was shown to depend on the detailed structure of the heterocyclic base. The polymer-drug conjugates described in the paper represent a new family of antiviral and cytostatic drugs with potentially improved pharmacokinetics, sustained drug release and diminished non-specific toxicity.
The synthesis and characterization of a water-soluble biodegradable multiblock polyurethane based on poly(ethylene glycol) blocks interconnected by a pentapeptide derivative N , N' -bis(aspartylprolyl)lysine is described. The pentapeptide linkages are susceptible both to chemical hydrolysis in neutral and mild acid medium (pH 5) and to enzymatic degradation by lysosomal enzyme cathepsin B. Anti-cancer drug doxorubicin was attached to a hydrazide derivative of the polymer via a hydrolytically labile hydrazone bond. Both the pH-triggered hydrolytic release of the drug and the degradation of the polymer carrier by enzymatic and chemical hydrolysis were studied using liquid chromatography.
Linear polyether polyol (PEP) consisting of glycidol as repeating units is a flexible hydrophilic aliphatic polymer. The polyether main chain is similar to the widely used, biocompatible polymer poly(ethylene glycol) (PEG). While linear PEG has one or two terminal hydroxyl group(s), linear PEP distinguishes itself by the large number of pendant hydroxyl groups along the polyether main chain. We propose that this property of PEP represents a major advantage over PEG, namely, by providing multiple anchorage points and increasing the possibility for introducing different functional groups. As a first step to establishing PEP as a bioconjugation material, we modified the pendant hydroxyl groups on PEP and prepared a series of mono- and heterobifunctional derivatives with the potential to join various drug entities and biomolecules. The synthesis methods and the results of characterization are reported here.
The nature of the groups that reside on the periphery of dendrimers and have contact with the surrounding media is the primary factor that controls the surface-related physico-chemical characteristics of these macromolecules. Therefore, transformation/tailoring of the peripheral functionalities of dendrimers is an economical way to change the overall behaviour of a particular dendrimer class or to impart new properties. In addition, the yields of the completely modified macromolecules could provide valuable information for the accessibility and the back folding of the moieties placed at the dendritic surfaces. The present article reviews the parent toxicity issues associated with cationic dendrimers like PAMAM and PPI, and examines the possibility of addressing this aspect through surface engineering with conjugation of biocompatible molecules.
Synthetic approaches for the preparation of macromolecular conjugates of the antifibrotic agent cis-4-hydroxy-L-proline (cHyp) were explored, and the efficacy of the conjugates in inhibiting collagen accumulation was investigated in vitro and in vivo. In one approach, poly(PEG-Lys), an alternating copolymer of poly(ethylene glycol) (PEG) and lysine, was used as the carrier. To prepare pendent chain systems, cHyp was attached to poly(PEG-Lys) through an amide linkage [poly(PEG-Lys-cHyp amide)] or through an ester linkage [poly(PEG-Lys-cHyp ester)]. In an alternative approach, cHyp was incorporated into the backbone of a linear copolymer consisting of PEG, succinic acid, and cHyp units [poly(PEG-succinate-cHyp)]. Bioactivity in vitro was assessed by the ability of the cHyp conjugates to inhibit growth of cultured smooth muscle cells (SMC) and rat lung fibroblasts (RLF). Cell numbers were compared to control experiments in the presence of biologically inactive trans-4-hydroxy-L-proline (tHyp). After a 5 day period, the presence of 8 micrograms/mL of cHyp delivered by poly(PEG-Lys-cHyp amide) resulted in a 47% reduction in the number of SMC (p < 0.05), the presence of 36 micrograms/mL of cHyp delivered by poly(PEG-Lys-cHyp ester) resulted in a 38% reduction in the number of SMC (p < 0.05), while the presence of 118 micrograms/mL of cHyp delivered by poly(PEG-succinate-cHyp) resulted in a 31% reduction in the number of cells (p < 0.05). An identical trend was observed for the inhibition of RLF growth. In general, poly(PEG-Lys-cHyp amide) was most active, followed by poly(PEG-Lys-cHyp ester) and the backbone system, poly(PEG-succinate-cHyp). Specifically, poly(PEG-Lys-cHyp amide) was over 100-fold more active in inhibiting cell growth than free cHyp. Bioactivity in vivo was evaluated by measuring collagen accumulation in subcutaneously implanted poly(vinyl alcohol) sponges in rats. Among the tested conjugates, poly(PEG-Lys-cHyp amide) was most active, reducing collagen accumulation in the sponge by 33% after 14 days relative to controls (p < 0.05). This result indicates that the covalent attachment of cHyp to poly(PEG-Lys) carries may be a useful strategy for the local inhibition of collagen accumulation in tissues.
The rate of hydrolysis of polyethylene glycol esters of hydrocortisone 21-succinate (H-PEGs) rate by a commercial esterase and by enzymes from ovine cornea was studied. Both the commercial esterase and the corneal enzymes rapidly hydrolysed the H-PEGs. Both lipophilicity and the PEG chain length had an influence on the rate of enzymatic hydrolysis, a significant decrease in enzymatic hydrolysis rate being associated with an increase in the polymer chain length. The in vitro penetration of H-PEGs across ovine cornea and sclera was assessed. In corneal penetration studies no prodrug was detected in the receiver phase while in the sclera penetration study both drug and prodrug were detected. Results from in vitro corneal and scleral absorption studies showed that the use of H-PEG400 increased the rate at which hydrocortisone diffused through excised ovine cornea compared to the use of hydrocortisone itself, particularly when the donor phase was removed after 15 min in order to mimic the situation in vivo. In addition, H-PEG200, H-PEG400, H-PEG600, H-PEG900 and H-PEG2000 gave a significant increase in the rate of diffusion of hydrocortisone+H-PEG through the sclera compared to hydrocortisone itself. Our results on ovine eye show that the rate of diffusion of hydrocortisone across the sclera is about six times higher than that in cornea and for the H-PEGs the scleral diffusion was 10-100 times higher than that observed in the cornea.
Polymer conjugation is of increasing interest in pharmaceutical chemistry for delivering drugs of simple structure or complex compounds such peptides, enzymes and oligonucleotides. For long time drugs, mainly with antitumoral activity, have been coupled to natural or synthetic polymers with the purpose of increasing their blood permanence time, taking advantage of the increased mass that reduces kidney ultrafiltration. However only recently complex constructs were devised that exploit the 'enhanced permeability and retention' (EPR) effect for an efficient tumor targeting, the high molecular weight for adsorption or receptor mediated endocytosis and finally a lysosomotropic targeting, taking advantage of acid labile bonds or cathepsin susceptible polypeptide spacers between polymer and drug. New original, very active conjugates of this type, as those based on poly(hydroxyacrylate) polymers, are already in advanced state of development. Labile oligonucleotides, including antisense drugs, were also successfully coupled to polymers in view of an increased cell penetration and stabilization towards nucleases. However, the most active research activity resides in the field of polypeptides and proteins delivery, mainly for the two following reasons: first of all because a great number of therapeutically interesting compounds are now being produced by genetic engineering in large quantity and, secondly, because these products are difficult to administer to patients for several inherent drawbacks. Proteins are in fact easily digested by many endo- and exo-peptidases present in blood or in other body districts; most of them are immunogenic to some extent and, finally, they are rapidly excreted by kidney ultrafiltration. Covalent polymer conjugation at protein surface was demonstrated to reduce or eliminate these problems, since the bound polymer behaves like a shield hindering the approach of proteolytic enzymes, antibodies, or antigen processing cell. Furthermore, the increase of the molecular weight of the conjugate allows to overcome the kidney elimination threshold. Many successful results were already obtained in peptides and proteins, conjugated mainly to water soluble or amphiphilic polymers like poly(ethylene glycol) (PEG), dextrans, or styrenemaleic acid anhydride. Among the most successful are the conjugates of asparaginase, interleukin-2 or -6 and neocarcinostatin, to remind some antitumor agents, adenosine deaminase employed in a genetic desease treatment, superoxide dismutase as scavenger of toxic radicals, hemoglobin as oxygen carrier and urokinase and streptokinase as proteins with antithrombotic activity. In pharmaceutical chemistry the conjugation with polymers is also of great importance for synthetic applications since many enzymes without loss of catalytic activity become soluble in organic solvents where many drug precursors are. The various and often difficult chemical problems encountered in conjugation of so many different products prompted the development of many synthetic procedures, all characterized by high specificity and mild condition of reaction, now known as 'bioconjugation chemistry'. Bioconjugation developed also the design of new tailor-made polymers with the wanted molecular weight, shape, structure and with the functional groups needed for coupling at the wanted positions in the chain.
The synthesis of a novel water-soluble polymer drug carrier system based on biodegradable poly(ethylene glycol) block copolymer is described in this paper. The copolymer consisting of PEG blocks of molecular weight 2000 linked by means of an oligopeptide with amino end groups was prepared by interfacial polycondensation of the diamine and PEG bis(succinimidyl carbonate). The structure of the oligopeptide diamine consisting of glutamic acid and lysine residues was designed as a substrate for cathepsin B, a lysosomal enzyme, which was assumed to be one of the enzymes responsible for the degradation of the polymer carrier in vivo. Each of the oligopeptide blocks incorporated in the carrier contained three carboxylic groups of which some were used for attachment of an anti-cancer drug, doxorubicin (Dox), via a tetrapeptide spacer Gly-Phe-Leu-Gly. This tetrapeptide spacer is susceptible to enzymatic hydrolysis. In vitro release of Dox and the degradation of the polymer chain by cathepsin B as well as preliminary evaluation of in vivo anti-cancer activity of the conjugate are also demonstrated.
Recently, block copolymers have got tremendous impetus on the ongoing research in the area of drug delivery technology, due to their capability to provide a biomaterial having a broad range of amphiphilic characteristics, as well as targeting the drugs to specific site. This article is an attempt to review applications of block copolymers in surface modification, drug targeting, nano and microparticles, hydrogels, micelles etc. The physicochemical properties of block copolymers and various synthetic routes for block copolymers are also discussed.
A broad spectrum antimicrobial agent, 1-ethyl-6-fluoro-1,4-dihydro-4-oxo-7-(1-piperazinyl)-3-quinolinecarboxylic acid (norfloxacin), has been successfully incorporated as a monomer into a polyurethane backbone structure via a three-step polymerization of norfloxacin, diisocyanatododecane (DDI), and polycaprolactone diol (PCL). The reaction was catalyzed by dibutyltin dilaurate and carried out in dimethyl sulfoxide. The sequential order of monomer feeding had a strong influence on the polymerization behavior and final polymer structure. In the preferred reaction scheme norfloxacin is initially reacted with DDI to form an oligomer. This is followed by a second reaction where PCL is introduced in order to produce a drug polymer chain with higher molecular weight and degradable segments. Cross-linking of urea linkages between the norfloxacin and DDI segments was a particular concern and was minimized by feeding PCL into the reaction system immediately following the completion of the first step. Chain extension by 1,4-butanediol or ethylenediamine was shown to be an effective approach for increasing the molecular weight of the polymers.
We synthesized a library of 50 poly(ethylene glycol) (PEG) derivatives to expand the extent of conjugation with biologically active molecules (biopolymers, peptides, drugs, etc.) and biomaterial substrates. The formation of PEG derivatives was confirmed with HPLC, (1)H and (13)C NMR. PEG derivatives were polymerized into networks in order to study the role of PEG and terminal functional groups in modulating the hydrophilicity of biomaterials and cell-biomaterial interaction. The resulting surface hydrophilicity and the number of adhered fibroblasts were primarily dependent on the PEG concentration with the molecular weight and the terminal functional group of PEG derivatives being less important. One of PEG derivatives, PEG-bis-glutarate, was utilized to link peptide sequences to gelatin backbone in the formation of novel biomedical hydrogels. PEG-peptide conjugates were characterized by mass spectroscopy. PEG-peptide modified gelatins were characterized by gel permeation chromatography.
Alternating amphiphilic multiblock copolymers, consisting of polyoxyethylene (POE) and poly(epsilon-caprolactone) (PCL) of various lengths, were synthesized by a polycondensation reaction between dicarboxylated PEG and dihydroxyl PCL. The polymer formed a physical hydrogel by PCL crystallization. For in vitro hydrolysis in phosphate-buffered saline solution, the change of molecular weight depended on the composing block length of POE. The polymer with longer POE showed a faster decline in molecular weight. The mass remaining at the end of two weeks at 25 degrees C was more than 95 w%. However, when the swollen hydrogels were exposed to temperatures slightly above PCL melting point for 30 min, the degradation rate was accelerated and the mass remaining dropped to less than 10 wt% in one week. In vivo degradation after hydrogel implantation, the polymer degraded as under in vitro. However, the implant irradiated with infrared (IR) accelerated its degradation similar to a treatment with elevated temperature.
A thermoresponsive, genetically engineered, elastin-like polypeptide (ELP) containing a C-terminal cysteine residue was synthesized and purified by inverse transition cycling (ITC) and conjugated to doxorubicin (Dox) molecules through four different pH-sensitive, maleimide-activated, hydrazone linkers. The efficiency of Dox activation, conjugation ratios to ELP and biophysical characterization-hydrodynamic radius (Rh) and the temperature transition kinetics-of the ELP-Dox conjugates and pH-mediated release of Dox were quantified in this study. Conjugation ratios of the maleimide-activated Dox to the thiol group of a unique cysteine in the ELP were close to unity. The Rh of the conjugate increased as the linker length between the ELP backbone and Dox was increased. The linker structure and length had little effect on the Tt of the ELP-Dox conjugates, as all conjugates exhibited Tt's that were similar to the native ELP. However, the ELP-Dox conjugates with longer linkers exhibited slower transition kinetics compared to the ELP-Dox conjugates with shorter linkers. The highest release of the ELP-Dox conjugate by cleavage of the hydrazone bond at pH 4 was nearly 80% over 72 h and was exhibited by the conjugate with the shortest linker.
Poly(ethylene glycol) (PEG) is a versatile biocompatible polymer. Improvement of its limited functionality (two chain termini) may significantly expand its current applications. In this communication, a simple and yet highly efficient strategy for the synthesis of linear multifunctional PEGs with "click" chemistry is reported. A short acetylene-terminated PEG was linked by 2,2-bis(azidomethyl)propane-1,3-diol using Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition in water at room temperature. High-molecular-weight PEGs with pendant hydroxyl groups were obtained and characterized by 1H NMR and size-exclusion chromatography. A prototype bone-targeting polymeric drug delivery system was also successfully synthesized based on this new method. It demonstrates strong biomineral-binding ability and the ease of incorporating therapeutic agents into the delivery system. This simple "click" reaction approach provides a useful tool for the development of novel functional polymers and their conjugates for biomedical applications.
Daunomycin was coupled to dextrans of various molecular sizes. The binding to the dextran carriers augmented the therapeutic efficacy of the antitumor agent in a murine lymphoma line (YAC). When the treatment with the drug or its conjugates was given concomitantly with the tumor cells at separate sites, the unbound drug was able, at its optimally effective doses, to prevent tumors in 40% of the mice, whereas the drug-dextran was efficient in 80% of the mice. The advantage of the drug-dextran over the free drug was also manifested when the treatment was given 6 days after tumor transplantation. However, a further delay of the treatment resulted in a decrease in the potency of the drug-dextran. Similar behavior was observed when increasing tumor loads were transplanted (10(5)-10(8) cells) and when the treatment was administered immediately. The most favorable effect of the drug-dextran was obtained with 10(7) cells, but against 10(8) cells neither the free drug nor the bound one was effective.
The results obtained in this study establish that liposome formulations incorporating a synthetic polyethylene glycol-derivatized phospholipid have a pronounced effect on liposome tissue distribution and can produce a large increase in the pharmacological efficacy of encapsulated antitumor drugs. This effect is substantially greater than that observed previously with conventional liposomes and is associated with a more than 5-fold prolongation of liposome circulation time in blood, a marked decrease in uptake by tissues such as liver and spleen, and a corresponding increased accumulation in implanted tumors. These and other properties described here have expanded considerably the prospects of liposomes as an effective carrier system for a variety of pharmacologically active macromolecules.
DBA2 mice were inoculated i.p. with 10(5)L1210 cells. Animals subsequently treated with daunomycin (single i.p. dose, 0.25-5.0 mg kg-1) all died. The maximum increase in mean survival time observed was approximately 135%. Animals treated with N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers conjugated to daunomycin (DNM) showed a significant increase in mean survival time when the polymer-drug linkage was biodegradable (i.e., Gly-Phe-Leu-Gly). Such treatment also produced a number of long term survivors (greater than 50 days). In contrast, HPMA copolymer conjugated to DNM via a non-degradable linkage (Gly-Gly) produced no increase in survival time relative to untreated control animals. The effect observed with biodegradable HPMA copolymer-DNM conjugates was dependent on the concentration of conjugated drug administered (optimum greater than 5 mg kg-1); the frequency of administration (multiple doses were more effective than single); the timing of administration (single doses given on days 1 and 3 were most effective); and the site of tumour inoculation and route of drug administration. Biodegradable HPMA copolymer-DNM conjugates administered i.p. were active against L1210 inoculated s.c. at higher doses than required to curb a peritoneal tumour. Under certain experimental conditions polymer-DNM conjugates containing fucosylamine or galactosamine proved more active than conjugates without the carbohydrate moeity. The mechanism of drug-conjugate action in vivo is at present unclear. Radioiodination of polymer showed approximately 75% of polymer-drug conjugate to be excreted 24 h after i.p. administration. Synthesis of HPMA conjugates containing [3H]DNM showed that polymer containing Gly-Gly-[3H]DNM was excreted (60% of radioactivity in the urine, 24 h) in macromolecular form. In contrast polymer containing Gly-Phe-Leu-Gly-[3H]DNM was largely excreted in the form of low molecular weight species.
Structural modifications of the daunomycin molecule may lead to a potentiation or complete inhibition of its cytostatic activity. Daunomycin derivatives with substitutions in the anthracycline ring (adriamycin and dihydro daunomycin) or in the aminosugar moeity (N-guanidino-acetamide-daunomycin) and N-acetyl-daunomycin) were tested in vitro on various types of tumors in experimental animals. Tumor suspensions (L-1210 and SV 40) incubated with daunomycin and adriamycin (50 μg/ml) failed to grow in the host animals. Dihydro daunomycin showed a slight activity against L-1210, but a total inhibition against SV 40 induced tumors in hamsters. Daunomycin derivatives with substitutions in the aminosugar moeity were completely ineffective, except N-guanidino-acetamide-daunomycin which showed a significant activity against SV40 induced tumors.Daunomycin is an antibiotic of the anthracycline group isolated from cultures of (Di Marco et al., 1964; Grein et al., 1963) and consists of a pigmented aglycone (daunomycinone) bound by a glycosidic linkage to an aminosugar (daunosamine, Fig. 1) and . The biological activity of daunomycin is dependent on its ability to interact with primer DNA (Di Marco, 1967). The inhibition of DNA polymerases from RNA tumor viruses by some structural analogues of daunomycin has been recently reported by and . They found that substitutions in the amino-sugar moeity lead to an inactivation of daunomycin molecule. The present communication describes the correlation between chemical structure and cytostatic activity of some daunomycin derivatives against various types of tumors in experimental animals.
Water-soluble poly(ether urethanes) (M(w) up to 170 000) have been prepared by the copolymerization of bis(succinimidyl) carbonate derivatives of poly(ethylene glycol) (PEG) with L-lysine (Lys) m a strictly alternating fashion. The resulting copolymers have physical properties similar to those of PEG while the carboxylic acid groups Of L-lysine provide multiple pendent groups along the polymer backbone for further functionalization. By using PEG chains of different molecular weights, a homologous series of PEG-Lys copolymers was prepared containing one lysine unit every 1000, 2000, 4000, and 8000 Da. The pendent carboxylic acid groups were utilized in cross-linking reactions leading to amide cross-links, new acyl semicarbazide cross-links, and hydroxyethyl acrylate and hydroxyethyl methacrylate derived cross-links. The resulting hydrogels formed transparent, highly swollen films. At an equilibrium water content of about 80%, these films exhibited a relatively high degree of tensile strength ranging from 0.26 to 1.09 MPa. Due to their favorable physicomechanical properties and the nontoxicity of the components, PEG-Lys-derived hydrogels may find applications as biomaterials.
Doxorubicin is one member of the anthracycline group of antibiotics and is produced by a variant of the organism that produces daunorubicin (2) (trivial name, daunomycin), the latter drug being preferred in the treatment of acute leukemia, as it is equiactive and lower in cost. Despite its good antitumor activity, doxorubicin is far from an ideal drug as it has both pharmaceutical and toxicological limitations. This chapter focuses on synthetic routes to doxorubicin and other anthracyclines. It discusses the biochemical effects of doxorubicin and analogues of doxorubicin. Most of the work on synthetic routes to anthracyclines has concentrated on doxorubicin (1) and the related compounds, daunorubicin (2) and carminomycin (12). The stimulus has been the low yield of doxorubicin (1) from the fermentation process and the challenge of synthesis of a compound of such complexity. Only recently has attention really widened to synthesis of other natural anthracyclines. The major problem in design of a route is to obtain regiochemical and stereochemical control. New potentially clinically useful analogues have arrived by screening of natural products or by chemically based intuitive semisynthesis and synthesis, rather than by truly rational design.
Daunomycin and adriamycin, two potent anthracycline antibiotics, have been covalently bound to the biocompatible polysaccharides sodium hyaluronate and carboxymethylcellulose. The water-soluble conjugates have been investigated by means of spectroscopic and hydrodynamic techniques. The conformation properties of the polysaccharide chains are not dramatically affected upon derivatization. The acidic dissociation constants of the polymer-supported anthracyclines are decreased as a result of polyelectrolyte effects from the negatively charged backbone. The polymeric drugs are able to bind efficiently biologically relevant metal ions, in particular Cu++. The binding stoichiometry, with reference to the parent anthracyclines, is somewhat changed under similar experimental conditions. The equilibrium constants for Cu++ binding show on the other hand characteristics well related to the corresponding properties of the original compounds. In addition, the redox potentials of the drugs remain practically unchanged following covalent binding to the polymers. These results are promising in view of a therapeutic application of the polymer-supported antineoplastic derivatives.
Soluble macromolecular conjugates for the controlled delivery of the strongly hydrophobic antitumor drug adriamycin (ADR) were prepared. The association behaviour of two types of these amphiphilic conjugates consisting of ADR bound to poly(L-glutamic acid) (PGA) in aqueous solution was investigated using absorbance and fluorescence spectroscopy as well as GPC and lowangle laser light scattering (LALLS) measurements. ADR was bound via the aminoribosyl moiety either directly with PGA side-chain groups or via oligopeptide spacer groups. UV/VIS, GPC and LALLS data showed that in buffer solution both ADR conjugates associate intermolecularly. The data of the directly bound ADR conjugates are consistent with the presence of multimers of different degrees of association in equilibrium with single polymer chains. In contrast, spacer-containing ADR conjugates associate to give stable uniform multimers. UV/VIS and fluorescence spectroscopy performed at very low conjugate concentration show that polymer-bound ADR residues associate intramolecularly as well. The degree of intra- and intermolecular association of the conjugate-bound ADR molecules depends on the type of conjugate, ADR load and conjugate concentration as well as on ionic strength of the solvent, the presence of organic cosolvents and temperature. The data indicate that hydrophobic domains in spacer-containing conjugates are more stable compared with directly bound conjugates, probably due to enhanced flexibility and the presence of hydrophobic leucine residues. It is concluded that spacer-containing conjugates of ADR may have a potential for effective drug delivery under in vivo conditions due to their solution properties in addition to the biodegradability of the drug-polymer bond.
N-(2-Hydroxypropyl)methyocrylamide (HPMA) copolymers were synthesized to contain adriamycin (ADR) and in certain cases fucosylamine or galactosamine residues. Drug was attached to polymer via biodegradable (-Gly-Phe-Leu-Gly) or non-biodegradable (-Gly-Gly) oligopeptide side-chains. Fucosylamine and galactosamine were included to promote conjugate targeting to L1210 cells and hepatocytes, respectively. Although free ADR (5 mg/kg) can increase the mean life span of DBA2 mice bearing L1210 leukaemia (up to 24%), animals do not survive beyond this time. Treatment with P-Gly-Phe-Leu-Gly-ADR (5 mg/kg) consistently increased mean survival time, and in addition produced survivors at 50 days (up to 80% surviving). Polymers containing in addition galactosamine or fucosylamine were equally effective. Degradation of the drug-polymer linkage was shown to be a prerequisite for pharmacological activity, P-Gly-Gly-ADR was totally ineffective. Conjugation of ADR limited toxicity, a > 10 fold increase in dose could be given in the polymer-bound form without obvious ill effect. Measurement of the pharmacokinetics of 125I-labelled HPMA copolymer-ADR conjugates showed a marked alteration from the pattern of distribution reported previously for free ADR, and the levels of radioactivity detected in the heart were extremely low. The latter observation supports the observed decrease in toxicity seen for conjugated drug.
Antibody-morpholinodoxorubicin conjugates were prepared for targeted immunotherapy of human melanoma. Spacer molecules that differ in hydrolytic stability were employed between the C-13 of the drug and amino residue of lysine on the monoclonal antibody. Antibody-drug conjugates were made with five structurally different morpholinodoxorubicin derivatives including oxime, phenylhydrazone, (sulfonylphenyl)hydrazone, and acylhydrazone moieties. Hydrolytic stability of the antibody conjugates directly correlated with their in vitro cytotoxicity against melanoma cells. Derivatives or conjugates with the greatest hydrolytic stability showed the least cytotoxicity.
The effect of daunomycin upon DNA condensed states induced by poly(ethylene glycol) (PEG) was studied by circular dichroism (CD) and circular intensity differential scattering (CIDS). The CD spectra of these aggregates showed psi-type anomalies and intensities 10-100 times greater than those obtained with the dispersed DNA solutions in the absence of PEG. Increasing concentrations of daunomycin, added to the DNA solution prior to its aggregation, led, in the presence of PEG, to CD and CIDS signals which gradually decreased in magnitude and eventually inverted sign. The coincidence of the transition point of both signals and a careful characterization of the CD spectrum at the transition point clearly indicated that the inversion observed corresponds to an inversion of the handedness of the aggregates. The latter result suggests that the structure of the aggregates at the inversion point should resemble that of a nematic liquid-crystalline structure. The characteristic B-DNA spectrum obtained in this case further suggests that the packing process does not affect the secondary structure of the DNA molecules and that small changes in their local structure can induce dramatic changes in their long-range tertiary packing. The results obtained in this study represent a confirmation of a recent theory of psi-type CD in which the anomalous signals are interpreted as a manifestation of the long-range chirality of the aggregates.
Poly(PEG-Lys), a new, water-soluble poly(ether urethane), derived from L-lysine and poly(ethylene glycol) was investigated as a precursor for the preparation of polymeric drug conjugates. To facilitate a wide variety of coupling chemistries, the pendent carboxyl groups of poly(PEG-Lys) were converted to other reactive functional groups (amino, hydroxyl, active ester, and aldehyde) in high yield. These reactive pendent chains were then used as anchors for the covalent attachment of penicillin V and cephradine, two clinically used antimicrobial agents. Coupling to the carrier was achieved in good yields and the chemical versatility of this system was demonstrated by the preparation of conjugates having antibiotic ligands linked via biostable or biodegradable linkages to the carrier, either directly or via a spacer. Conjugate 4, poly(PEG-Lys-penicillin V ester), was obtained by linking penicillin V to the polymer backbone via hydrolytically labile ester bonds. This conjugate exhibited activity similar to that of the parent drug against three clinically important strains of bacteria. Drug activity coincided with the release of the drug from the carrier. Hydrolytically stable cephradine-containing conjugates were prepared by three different coupling methods but showed no antibiotic activity. 14C-labeled poly(PEG-Lys) was injected into mice and its biodistribution was monitored for 48 h. The carrier showed no preferential uptake by liver, spleen, or kidney. No signs of acute toxicity were evident in mice or rats when poly(PEG-Lys) was administered iv and ip at doses up to 10 g/kg. These results indicate that poly(PEG-Lys) is a promising precursor for the preparation of soluble drug conjugates.